沉默Id-1基因对人口腔癌细胞增殖、侵袭、凋亡的影响
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摘要
第一部分Id-1基因在口腔癌细胞中的表达
目的
口腔癌是临床较常见疾病,近年来发病率有增加趋势,其发病年龄以40～60岁为发病高峰年龄段,但60～70岁年龄段发病率明显增加,占总数17.8%,发病平均年龄54.1岁,表明口腔癌发病有老龄化趋向。口腔癌大部分发生于口腔(唇部除外),占总数78.4%,其他依次为大唾液腺、上颌窦、颈、咽、唇部。口腔中好发部位按序为舌、上下牙龈、颊、腭、上下颌骨、口底,舌癌已跃居首位。口腔癌的病理类型以鳞癌和腺样囊性癌居多,这两种类型恶性程度也比较大,容易复发和转移,尤其是腺样囊性癌,极容易发生远处转移,而且具有嗜神经性。因此,我们选择了最具代表性的鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2这五种典型的口腔癌细胞系进行筛选。DNA结合抑制蛋白(Inhibitor of DNA binding，Id),属于螺旋-环-螺旋蛋白质超家族,相对分子质量为13000～20000,它是一组缺乏碱性DNA连接结构域的HLH因子。Id-1是人类迄今为止发现的4个Id蛋白家族成员(Id-1-4)中研究最多,也是最重要的一个。研究发现：Id-1在上皮来源的肿瘤中呈高表达,它通过抑制细胞分化、推进细胞周期进程、诱导细胞增殖、抑制衰老、诱导侵袭、参与肿瘤性血管新生而介导肿瘤的发生和发展；在大肠癌、食管癌、乳腺癌、前列腺癌、卵巢癌、宫颈癌的发生发展过程中都发挥着重要的作用。然而在口腔癌中,Id-1的表达量如何,其对于口腔癌细胞的生长和侵袭能力的影响尚不得而知。本研究将检测口腔癌这五种典型细胞系的Id-1表达情况。
方法
口腔鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2解冻复苏,于含10%胎牛血清的DMEM培养基、37℃、含5%CO2的细胞培养箱中密闭培养。取对数生长的细胞,用Trizol裂解液提取总RNA,将总RNA逆转录为cDNA，然后用Id-1引物进行(Real-time fluorescence quantitative PCR, RFQ-PCR)扩增,扩增体中加入SYBR Green I,进行RFQ-PCR实验,检测五种细胞系的Id-1基因的1mRNA表达情况。再取对数生长的细胞,常规方法提取细胞总蛋白,一抗和二抗孵育,进行Western Blot实验,检测五种细胞系的Id-1基因的蛋白表达情况。
结果
实验结果显示,鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2五种细胞系Id-1均呈现高表达,其中,腺癌细胞系Id-1表达量最高的是ACCM,鳞癌细胞系Id-1表达量最高的是SAS。RFQ-PCR结果显示：腺癌细胞系Id-1mRNA表达量最高的是ACCM(P<0.05),鳞癌细胞系Id-1mRNA表达量最高的是SAS(P<0.05)c Western Blot结果显示：其中,腺癌细胞系Id-1表达量最高的是ACCM(P<0.05),鳞癌细胞系Id-1表达量最高的是SAS(P<0.05)。
结论
实验结果提示,鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2五种细胞系Id-1mRNA和Id-1蛋白均呈现高表达,其中,腺癌细胞系Id-1表达量最高的是ACCM,鳞癌细胞系Id-1表达量最高的是SAS。我们筛选出了腺癌细胞系Id-1表达量最高的ACCM和鳞癌细胞系Id-1表达量最高的SAS两个细胞系。
第二部分沉默Id-1基因对腺样囊性癌细胞增殖、侵袭、凋亡的影响
目的
腺样囊性癌(adenoid cystic carcinoma, ACC),是发生于头颈部涎腺最常见的恶性肿瘤,约占所有恶性肿瘤20%,具有高度的侵袭和转移特性。由于对ACC生物学性质认识上的局限性,目前尚无很有效的治疗措施,即使进行了广泛的手术切除及放射治疗,大多数患者最终出现复发和转移。临床上急待寻求治疗ACC的新方法。
Id蛋白属于螺旋-环-螺旋蛋白质超家族,相对分子质量为13000～20000,它是一组缺乏碱性DNA连接结构域的HLH因子。bHLH蛋白二聚体的形成是DNA与“E盒”(CANNTG)或“N盒”(CACNAG)识别序列结合的必要条件,Id蛋白通过与bHLH转录因子形成异型二聚体,干扰其与DNA的结合,阻断其对下游分子的转录激活作用,抑制基因的表达,从而抑制细胞的正常分化,促进细胞增殖。Id-1是人类迄今为止发现的4个Id蛋白家族成员(Id-1-4)中研究最多,也是最重要的一个。研究发现：Id-1在上皮来源的肿瘤中呈高表达,它通过抑制细胞分化、推进细胞周期进程、诱导细胞增殖、抑制衰老、诱导侵袭、参与肿瘤性血管新生而介导肿瘤的发生和发展；在大肠癌、食管癌、乳腺癌、前列腺癌、卵巢癌、宫颈癌的发生发展过程中都发挥着重要的作用。然而在ACC中,Id-1的表达量如何,其对于ACC的生长和侵袭能力的影响尚有待于研究证实。
以往研究发现的与肿瘤相关的Id-1信号通路有很多,有Akt-mediated Wnt信号通路,p53 and NF-kB信号通,PI3K/Akt/NFkB信号通路,ERK/MAPK信号通路等等。我们前期研究发现,正常涎腺组织中Id-1的表达量非常低(甚至难以检测到),但是在涎腺腺样囊性癌中却呈现高表达,这说明Id-1表达水平的升高可能打破了正常细胞内癌基因与抑癌基因相互制约的平衡关系,进而激活某条或多条信号传导通路推进细胞周期进程、抑制细胞分化、促进细胞增殖,从而诱导正常细胞向肿瘤细胞转化,增加通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。
我们选用设计好的Id-1siRNA序列,应用lipofectamine2000进行转染,沉默掉ACCM细胞系的Id-1基因,然后检测ACCM细胞系增殖、侵袭和凋亡的改变及ki67、c-myc和p21蛋白含量变化。
方法
于含10%胎牛血清的DMEM培养基、37℃、含5%C02的细胞培养箱中密闭培养腺样囊性癌高转移细胞系ACCM。用设计好的Id-1siRNA序列,应用lipofectamine 2000进行转染,沉默ACCM细胞系的Id-1基因。RFQ-PCR、Westernblot检测、免疫荧光确认干扰成功。MTT比色法于570 nm处测定光密度值(OD),描记干扰前后细胞的生长曲线。Transwell小室检测干扰前后细胞侵袭性能的改变。流式细胞仪检测干扰前后细胞的凋亡情况。Western blot检测ki67、c-myc和p21蛋白含量变化。
结果
实验结果显示RFQ-PCR、Western blot检测、免疫荧光确认干扰成功。MTT比色法描记的干扰前后细胞的生长曲线结果显示：Id-1siRNA干扰后ACCM增殖能力下降,与未干扰对照组有显著差异(0.5806±0.0063vs0.7469±0.0424,P<0.01；0.6378±0.0040vsl.0110±0.0010，P<0.01；0.7679±0.0036vs1.2774±0.0036,P<0.01)。Transwell小室检测的结果显示：Id-1siRNA干扰后ACCM侵袭能力下降,与未干扰对照组有显著差异((10.6667±2.0656)vs(29.8333±1.7224),P<0.01)。流式细胞仪检测的凋亡结果显示：Id-lsiRNA干扰后ACCM凋亡增多,与未干扰对照组有显著差异((23.2767±1.2872)%vs(2.2467±0.0924)%,P<0.01)。Western blot检测ki67、c-myc和p21蛋白含量变化显不：Id-1siRNA干扰后ACCM中ki67含量减少、c-myc含量减少和p21含量增加,与未干扰对照组有显著差异。
结论
结果提示通过RNA干扰沉默Id-1基因后,ACCM生长受到抑制、侵袭能力均受到抑制,细胞凋亡增多；ACCM中ki67含量减少、c-myc含量减少和p21含量增加。其机制可能是通过沉默Id-1基因,腺样囊性癌细胞系ACCM本来呈现高表达的Id-1出现低表达或者无表达,本来被打破了的癌基因与抑癌基因相互制约的平衡关系恢复了正常,激活了的某(些)条信号传导通路得到抑制或者关闭,抑制细胞周期进程、促进细胞分化、抑制细胞增殖,抑制了通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。但是Id-1对ACC生长和侵袭起调节作用的精确信号传导通路,还有待进一步的研究与探讨。
第三部分沉默Id-1基因对口腔鳞状细胞癌细胞增殖、侵袭、凋亡的影响
目的
口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)占口腔癌90%以上2005年WHO头颈肿瘤病理学和遗传学分类中将口腔鳞状细胞癌定义为“一种具有不同程度分化的侵袭性肿瘤,倾向于早期、广泛的淋巴结转移,主要发生于40-70岁的烟酒嗜好者。口腔癌是恶性程度较高的肿瘤,虽然经肿瘤学家、外科医师的不断努力,在过去20年里口腔癌的死亡率略有下降,但其5年生存率仍只有41%-79.5%,这促使人们努力寻找与肿瘤预后相关的因素,以采取更有针对性的措施进行预防和治疗。
Id蛋白属于螺旋-环-螺旋蛋白质超家族,相对分子质量为13000～20000,它是一组缺乏碱性DNA连接结构域的HLH因子。bHLH蛋白二聚体的形成是DNA与“E盒”(CANNTG)或“N盒”(CACNAG)识别序列结合的必要条件,Id蛋白通过与bHLH转录因子形成异型二聚体,干扰其与DNA的结合,阻断其对下游分子的转录激活作用,抑制基因的表达,从而抑制细胞的正常分化,促进细胞增殖。Id-1是人类迄今为止发现的4个Id蛋白家族成员(Id-1-4)中研究最多,也是最重要的一个。研究发现：Id-1在上皮来源的肿瘤中呈高表达,它通过抑制细胞分化、推进细胞周期进程、诱导细胞增殖、抑制衰老、诱导侵袭、参与肿瘤性血管新生而介导肿瘤的发生和发展；在大肠癌、食管癌、乳腺癌、前列腺癌、卵巢癌、宫颈癌的发生发展过程中都发挥着重要的作用。然而在OSCC中,Id-1的表达量如何,其对于OSCC的生长和侵袭能力的影响尚有待于研究证实。
以往研究发现的与肿瘤相关的Id-1信号通路有很多,有Akt-mediated Wnt信号通路,p53 and NF-kB信号通,PI3K/Akt/NFKB信号通路, ERK/MAPK信号通路等等。我们前期研究发现,正常涎腺组织中Id-1的表达量非常低(甚至难以检测到),但是在涎腺腺样囊性癌中却呈现高表达,这说明Id-1表达水平的升高可能打破了正常细胞内癌基因与抑癌基因相互制约的平衡关系,进而激活某条或多条信号传导通路推进细胞周期进程、抑制细胞分化、促进细胞增殖,从而诱导正常细胞向肿瘤细胞转化,增加通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。
我们选用设计好的Id-1siRNA序列,应用lipofectamine 2000进行转染,沉默掉OSCC细胞系SAS的Id-1基因,然后检测SAS细胞系增殖、侵袭和凋亡的改变。
方法
于含10%胎牛血清的DMEM培养基、37℃、含5%CO2的细胞培养箱中密闭培养口腔鳞状细胞癌细胞系SAS。用设计好的Id-1siRNA序列,应用lipofectamine2000进行转染,沉默SAS细胞系的Id-1基因。RFQ-PCR、Western blot检测、免疫荧光确认干扰成功。MTT比色法于570 nm处测定光密度值(OD),描记干扰前后细胞的生长曲线。Transwell小室检测干扰前后细胞侵袭性能的改变。流式细胞仪检测干扰前后细胞的凋亡情况。
结果
实验结果显示RFQ-PCR、Western blot检测、免疫荧光确认干扰成功。MTT比色法描记的干扰前后细胞的生长曲线结果显示：Id-1siRNA干扰后SAS增殖能力下降,与未干扰对照组有显著差异(0.68055±0.0457vs0.8469±0.0878,P<0.01；0.73775±0.0866vsl.11098±0.0899,P<0.01；0.8679±0.0442vsl.37743±0.0471.P<0.01)。Transwell小室检测的结果显示：Id-1siRNA干扰后SAS侵袭能力下降,与未干扰对照组有显著差异(10.1666667±0.012vsl7.8±0.008,P<0.01)。流式细胞仪检测的凋亡结果显示：Id-1 siRNA干扰后SAS凋亡增多,与未干扰对照组有显著差异((5.15±3.02)%vs(20.77±2.56)%,P<0.01)。
结论
结果提示通过RNA干扰沉默Id-1基因后,SAS生长受到抑制、侵袭能力均受到抑制,细胞凋亡增多。其机制可能是通过沉默Id-1基因,鳞状细胞癌细胞系SAS本来呈现高表达的Id-1出现低表达或者无表达,本来被打破了的癌基因与抑癌基因相互制约的平衡关系恢复了正常,激活了的某(些)条信号传导通路得到抑制或者关闭,抑制细胞周期进程、促进细胞分化、抑制细胞增殖,抑制了通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。但是Id-1对OSCC生长和侵袭起调节作用的精确信号传导通路,还有待进一步的研究与探讨。
SectionⅠExpression of Id-1 gene in oral cancer cells
Objective
Oral cancer is more common clinical disease, the incidence of an increasing trend in recent years, their age of onset of 40 to 60 years as the peak onset age, but 60 to 70-year-old age group a marked increase in the incidence, accounting for 17.8%, the average age of onset 54.1 years old, showed that the incidence of oral cancer has an aging trend. Most oral cancers occur in the oral cavity (excluding lip), accounting for 78.4% of the total, followed by the major salivary glands, maxillary sinus, neck, throat, lips. Mouth predilection sites in sequence as the tongue, upper and lower gums, cheek, palate, upper and lower jaw, floor of the mouth, tongue cancer has leapt to the top. Pathological types of oral cancer squamous cell carcinoma and adenoid cystic carcinoma of the majority, and that the degree of malignancy is relatively large, easy to recurrence and metastasis, especially adenoid cystic carcinoma is highly prone to distant metastasis, but also has neurotropic nature. Therefore, we chose five most representative oral cancer cell lines:the squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2 for screening. DNA binding protein (Inhibitor of DNA binding, Id), are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role.However, in oral cancer, Id-1 expression levels of how their oral cancer cells for growth and invasion capacity of the unknown. This study will detect the expression of Id-1 in this five typical oral cancer cell lines.
Methods
Oral squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2 thaw recovery, containing 10% fetal bovine serum in DMEM medium,37℃, the cells containing 5% CO2 incubator closed-culture. Logarithmic growth of cells, using Trizol lysate extracted total RNA, the total RNA reverse transcription of cDNA, and then Id-1 primers for real-time fluorescence quantitative PCR (RFQ-PCR), amplification of the body by adding SYBR Green I, for RFQ-PCR experiment, detection the expression of Id-1mRNA in the five cell lines. And then take on the exponential growth of cells, conventional methods of extrACTINg cells, total protein, first anti-and second anti-incubated for Western Blot test, test Id-1 protein expression in five kinds of cell lines.
Results
Experimental results show that it showed a high Id-1 expression in this five cell lines:squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2, which, Id-1 expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines,and Id-1 expression in SAS was significantly higher in squamous cell carcinoma cell lines. RFQ-PCR results showed that:Id-1 mRNA expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines(P<0.05), and Id-1 mRNA expression in SAS was significantly higher in squamous cell carcinoma cell lines(P<0.05). Western Blot results showed that:Id-1 protein expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines(P<0.05), and Id-1 protein expression in SAS was significantly higher in squamous cell carcinoma cell lines(P<0.05).
Conclusion
These results suggest that, Id-1mRNA and Id-1 protein showed a high expression in this five cell lines:squamous cell carcinoma cell line SAS, Tca8113 Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2, which, Id-1 expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines, and Id-1 expression in SAS was significantly higher in squamous cell carcinoma cell lines. We selected two high expression of Id-1 gene in oral cancer cell lines:adenoid cystic carcinoma cell line ACCM and squamous cell carcinoma SAS.
Section II The effects of silencing Id-1 gene on the proliferation, invasion and apoptosis in adenoid cystic carcinoma cells
Objective
Adenoid cystic carcinoma (ACC), occurred in the head and neck, the most common salivary gland malignancy, accounting for 20% of all malignancies, with a high degree of invasion and metastasis characteristics. Because of the understanding of the limitations to the biological nature of ACC, there is not a very effective treatment measures, even if carried out an extensive surgical excision and radiation therapy, most patients will eventually become a recurrence and metastasis. It is urgent to seek treatment for clinically ACC.
Id proteins are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. bHLH protein dimer formation of DNA with the "E box" (CANNTG) or "N Box" (CACNAG) recognition sequence with a necessary condition, Id proteins with bHLH transcription factors form a heterodimer, interference with DNA, combination of blocking its transcriptional activation of downstream molecules, inhibit gene expression, thus inhibiting normal cell differentiation and promote cell proliferation. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role. However, in the ACC, what ahout Id-1 expression levels and how it effects ACC of the growth and invasive ability is unknown.
Found in previous studies with tumor-related Id-1 signaling pathway there are many, there is Akt-mediated Wnt signaling pathway, p53 and NF-kB signal-pass, PI3K/Akt/NFKB signaling pathway, ERK/MAPK signaling pathway and so on. Our preliminary study found that expression levels of Id-1 in normal salivary gland tissue is very low (or even difficult to detect), but having a high expression in salivary adenoid cystic carcinoma, indicating the level of Id-1 expression increased normal cells may be breaking the oncogene and tumor suppressor genes balanced relationship of checks and balances, thereby activating one or more of a certain signal transduction pathways to promote cell cycle progression, inhibit cell differentiation, promote cell proliferation, thereby inducing normal cells to tumor cell transformation, to increase through the lymphatic channels and blood Road to the surrounding lymph nodes and distant organs, invasion and metastasis of opportunities.
We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in ACCM cell lines, and then test proliferation, invasion and apoptosis changes in ACCM cell.
Methods
The high metastasis adenoid cystic carcinoma cell lines ACCM were contained in DMEM medium containing 10% fetal bovine serum,37℃,5% CO2 incubator in the closed-culture. We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in ACCM cell lines. RFQ-PCR, Western blot and immunofluorescence assay were used to test to confirm the successful disruption. MTT assay was measured at 570 nm optical density (OD), cell growth curve was recorded before and after interference. Transwell chamber was used to test the changes in cell invasiveness before and after the interference. Flow cytometry test the changes in cell apoptosis before and after the interference. Western blot were used to test the protein expression of ki67,c-myc and p21.
Results
The results showed that RFQ-PCR, Western blot test and immunofluorescence assay to confirm the successful interference. MTT assay recording cell growth curve before and after the interference showed that:after silencing Id-1 gene, the proliferation of ACCM was decreased, and was significantly different with control (0.5806±0.0063 vs 0.7469±0.0424,P<0.01; 0.6378±0.0040 vs 1.0110±0.0010, P<0.01; 0.7679±0.0036 vsl.2774±0.0036, P<0.01). Transwell chamber test results showed that: after silencing Id-1gene, the invasiveness of ACCM was decreased, and was significantly different with control (10.6667±2.0656vs29.8333±1.7224, P<0.01). Flow cytometry showed that:after silencing Id-1 gene, the cell number of apoptosis of ACCM was increased, and was significantly different with control ((23.2767±1.2872) % vs(2.2467±0.0924)%, P<0.01). Western blot test results showed that:after silencing Id-1 gene, and was significantly different with control, the expression of ki67and c-myc were reduction, however, the expression of p21 was upregulation.
Conclusion
The results suggested that after RNA interference silencing by Id-1 gene, ACCM growth was inhibited,invasion ability were inhibited, apoptosis increased; after silencing Id-1 gene, the expression of ki67and c-myc were reduction,however, the expression of p21 was upregulation.lts possible mechanism is through silence, Id-1 gene, adenoid cystic carcinoma cell lines showed high expression of ACCM,had Id-1 expression at low or no expression of what might be breaking the cancer gene and tumor suppressor genes balanced relationship of checks and balances returned to normal, the activation of a certain of the signal transduction pathway to be inhibited or shut down, inhibit cell cycle progression and promoting cell differentiation, inhibit cell proliferation, inhibition of blood through the lymphatic channels and the Road to the surrounding lymph nodes and distant organs, invasion and metastasis opportunities. However, the precise signal transduction pathway of Id-1 in ACC to regulate the growth, invasion and of, remains to be further research and discussion.
SectionⅢEffects of silencing Id-1 gene on the proliferation, invasion and apoptosis in oral squamous cell carcinoma cells
Objective
Oral squamous cell carcinoma (oral squamous cell carcinoma, OSCC) accounts for more than 90% of oral cancer, in 2005 WHO classification of pathology and genetics head and neck cancer will be oral squamous cell carcinoma is defined as "a different degree of differentiation of invasive tumors, tend to the early, extensive lymph node metastasis, mainly in the 40 to 70 years of age who smoke or alcohol abuse. oral cancer is highly malignant tumor, although the oncologist, the surgeon's continuous efforts in the past 20 years, Village of oral cancer mortality rate declined slightly, but its five-year survival rate was still only 41%～79.5%, which prompted efforts to find the factors associated with tumor prognosis in order to take a more targeted measures for the prevention and treatment.
Id proteins are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. bHLH protein dimer formation of DNA with the "E box" (CANNTG) or "N Box" (CACNAG) recognition sequence with a necessary condition, Id proteins with bHLH transcription factors form a heterodimer, interference with DNA, combination of blocking its transcriptional activation of downstream molecules, inhibit gene expression, thus inhibiting normal cell differentiation and promote cell proliferation. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role. However, in OSCC, Id-1 expression levels of how their growth and invasion of OSCC for the ability of research yet to be confirmed.
Found in previous studies with tumor-related Id-1 signaling pathway there are many, there is Akt-mediated Wnt signaling pathway, p53 and NF-kB signal-pass, PI3K/Akt/NFkB signaling pathway, ERK/MAPK signaling pathway and so on. Our preliminary study found that normal salivary gland tissue expression levels of Id-1 is very low (or even difficult to detect), but in salivary adenoid cystic carcinoma is having a high expression, indicating the level of Id-1 expression increased possible to break the normal cell oncogene and tumor suppressor genes within the constraints of a balanced relationship with each other, thereby activating a certain or more signal transduction pathways to promote cell cycle progression, inhibit cell differentiation, promote cell proliferation, thereby inducing normal cells to tumor cell transformation, to increase through the lymphatic channels and blood Road to the surrounding lymph nodes and distant organs, invasion and metastasis of opportunities.
We use designed Id-1siRNA sequence, the application lipofectamine 2000 transfection, silence Id-1 gene in OSCC cell lines SAS, and then test proliferation, invasion and apoptosis changes in SAS. Methods The high metastasis adenoid cystic carcinoma cell lines SAS were contained in DMEM medium containing 10% fetal bovine serum,37℃,5% CO2 incubator in the closed-culture. We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in SAS cell lines. RFQ-PCR, Western blot and immunofluorescence assay were used to test to confirm the successful disruption. MTT assay was measured at 570 nm optical density (OD), cell growth curve was recorded before and after interference. Transwell chamber was used to test the changes in cell invasiveness before and after the interference. Flow cytometry test the changes in cell apoptosis before and after the interference.
Results
The results showed that RFQ-PCR, Western blot test and immunofluorescence assay to confirm the successful interference. MTT assay recording cell growth curve before and after the interference showed that:after silencing Id-1gene, the proliferation of SAS was decreased (0.68055±0.0457vs0.8469±0.0878,P<0.01; 0.73775±0.0866vsl.11098±0.0899,P<0.01;0.8679±0.0442vsl.37743±0.0471,P<0.01). Transwell chamber test results showed that:after silencing Id-1gene, the invasiveness of SAS was decreased (10.1666667±0.012vsl7.8±0.008, P<0.01). Flow cytometry showed that:after silencing Id-1gene, the cell number of apoptosis of SAS was increased ((5.15±3.02)%vs (20.77±2.56)%,P<0.01).
Conclusion
The results suggested that after RNA interference silencing by Id-1 gene, SAS growth was inhibited,invasion ability were inhibited, apoptosis increased.Its possible mechanism is through silence, Id-1 gene, oral squamous cell carcinoma cell lines showed high expression of SAS,had Id-1 expression at low or no expression of what might be breaking the cancer gene and tumor suppressor genes balanced relationship of checks and balances returned to normal, the activation of a certain of the signal transduction pathway to be inhibited or shut down,inhibit cell cycle progression and promoting cell differentiation,inhibit cell proliferation,inhibition of blood through the lymphatic channels and the Road to the Surrounding lymph nodes and distant organs, invasion and metastasis opportunities. However, the precise signal transduction pathway of Id-1 in OSCC to regulate the growth,invasion and of, remains to be further research and discussion. 18国家自然基金面上项目(30672339 and 30772269)和SRF for ROCS提供资金资助
目的
口腔癌是临床较常见疾病,近年来发病率有增加趋势,其发病年龄以40～60岁为发病高峰年龄段,但60～70岁年龄段发病率明显增加,占总数17.8%,发病平均年龄54.1岁,表明口腔癌发病有老龄化趋向。口腔癌大部分发生于口腔(唇部除外),占总数78.4%,其他依次为大唾液腺、上颌窦、颈、咽、唇部。口腔中好发部位按序为舌、上下牙龈、颊、腭、上下颌骨、口底,舌癌已跃居首位。口腔癌的病理类型以鳞癌和腺样囊性癌居多,这两种类型恶性程度也比较大,容易复发和转移,尤其是腺样囊性癌,极容易发生远处转移,而且具有嗜神经性。因此,我们选择了最具代表性的鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2这五种典型的口腔癌细胞系进行筛选。DNA结合抑制蛋白(Inhibitor of DNA binding，Id),属于螺旋-环-螺旋蛋白质超家族,相对分子质量为13000～20000,它是一组缺乏碱性DNA连接结构域的HLH因子。Id-1是人类迄今为止发现的4个Id蛋白家族成员(Id-1-4)中研究最多,也是最重要的一个。研究发现：Id-1在上皮来源的肿瘤中呈高表达,它通过抑制细胞分化、推进细胞周期进程、诱导细胞增殖、抑制衰老、诱导侵袭、参与肿瘤性血管新生而介导肿瘤的发生和发展；在大肠癌、食管癌、乳腺癌、前列腺癌、卵巢癌、宫颈癌的发生发展过程中都发挥着重要的作用。然而在口腔癌中,Id-1的表达量如何,其对于口腔癌细胞的生长和侵袭能力的影响尚不得而知。本研究将检测口腔癌这五种典型细胞系的Id-1表达情况。
方法
口腔鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2解冻复苏,于含10%胎牛血清的DMEM培养基、37℃、含5%CO2的细胞培养箱中密闭培养。取对数生长的细胞,用Trizol裂解液提取总RNA,将总RNA逆转录为cDNA，然后用Id-1引物进行(Real-time fluorescence quantitative PCR, RFQ-PCR)扩增,扩增体中加入SYBR Green I,进行RFQ-PCR实验,检测五种细胞系的Id-1基因的1mRNA表达情况。再取对数生长的细胞,常规方法提取细胞总蛋白,一抗和二抗孵育,进行Western Blot实验,检测五种细胞系的Id-1基因的蛋白表达情况。
结果
实验结果显示,鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2五种细胞系Id-1均呈现高表达,其中,腺癌细胞系Id-1表达量最高的是ACCM,鳞癌细胞系Id-1表达量最高的是SAS。RFQ-PCR结果显示：腺癌细胞系Id-1mRNA表达量最高的是ACCM(P<0.05),鳞癌细胞系Id-1mRNA表达量最高的是SAS(P<0.05)c Western Blot结果显示：其中,腺癌细胞系Id-1表达量最高的是ACCM(P<0.05),鳞癌细胞系Id-1表达量最高的是SAS(P<0.05)。
结论
实验结果提示,鳞癌细胞系SAS、Tca8113、Bucc1885和腺样囊性癌的细胞系ACCM、ACC-2五种细胞系Id-1mRNA和Id-1蛋白均呈现高表达,其中,腺癌细胞系Id-1表达量最高的是ACCM,鳞癌细胞系Id-1表达量最高的是SAS。我们筛选出了腺癌细胞系Id-1表达量最高的ACCM和鳞癌细胞系Id-1表达量最高的SAS两个细胞系。
第二部分沉默Id-1基因对腺样囊性癌细胞增殖、侵袭、凋亡的影响
目的
腺样囊性癌(adenoid cystic carcinoma, ACC),是发生于头颈部涎腺最常见的恶性肿瘤,约占所有恶性肿瘤20%,具有高度的侵袭和转移特性。由于对ACC生物学性质认识上的局限性,目前尚无很有效的治疗措施,即使进行了广泛的手术切除及放射治疗,大多数患者最终出现复发和转移。临床上急待寻求治疗ACC的新方法。
Id蛋白属于螺旋-环-螺旋蛋白质超家族,相对分子质量为13000～20000,它是一组缺乏碱性DNA连接结构域的HLH因子。bHLH蛋白二聚体的形成是DNA与“E盒”(CANNTG)或“N盒”(CACNAG)识别序列结合的必要条件,Id蛋白通过与bHLH转录因子形成异型二聚体,干扰其与DNA的结合,阻断其对下游分子的转录激活作用,抑制基因的表达,从而抑制细胞的正常分化,促进细胞增殖。Id-1是人类迄今为止发现的4个Id蛋白家族成员(Id-1-4)中研究最多,也是最重要的一个。研究发现：Id-1在上皮来源的肿瘤中呈高表达,它通过抑制细胞分化、推进细胞周期进程、诱导细胞增殖、抑制衰老、诱导侵袭、参与肿瘤性血管新生而介导肿瘤的发生和发展；在大肠癌、食管癌、乳腺癌、前列腺癌、卵巢癌、宫颈癌的发生发展过程中都发挥着重要的作用。然而在ACC中,Id-1的表达量如何,其对于ACC的生长和侵袭能力的影响尚有待于研究证实。
以往研究发现的与肿瘤相关的Id-1信号通路有很多,有Akt-mediated Wnt信号通路,p53 and NF-kB信号通,PI3K/Akt/NFkB信号通路,ERK/MAPK信号通路等等。我们前期研究发现,正常涎腺组织中Id-1的表达量非常低(甚至难以检测到),但是在涎腺腺样囊性癌中却呈现高表达,这说明Id-1表达水平的升高可能打破了正常细胞内癌基因与抑癌基因相互制约的平衡关系,进而激活某条或多条信号传导通路推进细胞周期进程、抑制细胞分化、促进细胞增殖,从而诱导正常细胞向肿瘤细胞转化,增加通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。
我们选用设计好的Id-1siRNA序列,应用lipofectamine2000进行转染,沉默掉ACCM细胞系的Id-1基因,然后检测ACCM细胞系增殖、侵袭和凋亡的改变及ki67、c-myc和p21蛋白含量变化。
方法
于含10%胎牛血清的DMEM培养基、37℃、含5%C02的细胞培养箱中密闭培养腺样囊性癌高转移细胞系ACCM。用设计好的Id-1siRNA序列,应用lipofectamine 2000进行转染,沉默ACCM细胞系的Id-1基因。RFQ-PCR、Westernblot检测、免疫荧光确认干扰成功。MTT比色法于570 nm处测定光密度值(OD),描记干扰前后细胞的生长曲线。Transwell小室检测干扰前后细胞侵袭性能的改变。流式细胞仪检测干扰前后细胞的凋亡情况。Western blot检测ki67、c-myc和p21蛋白含量变化。
结果
实验结果显示RFQ-PCR、Western blot检测、免疫荧光确认干扰成功。MTT比色法描记的干扰前后细胞的生长曲线结果显示：Id-1siRNA干扰后ACCM增殖能力下降,与未干扰对照组有显著差异(0.5806±0.0063vs0.7469±0.0424,P<0.01；0.6378±0.0040vsl.0110±0.0010，P<0.01；0.7679±0.0036vs1.2774±0.0036,P<0.01)。Transwell小室检测的结果显示：Id-1siRNA干扰后ACCM侵袭能力下降,与未干扰对照组有显著差异((10.6667±2.0656)vs(29.8333±1.7224),P<0.01)。流式细胞仪检测的凋亡结果显示：Id-lsiRNA干扰后ACCM凋亡增多,与未干扰对照组有显著差异((23.2767±1.2872)%vs(2.2467±0.0924)%,P<0.01)。Western blot检测ki67、c-myc和p21蛋白含量变化显不：Id-1siRNA干扰后ACCM中ki67含量减少、c-myc含量减少和p21含量增加,与未干扰对照组有显著差异。
结论
结果提示通过RNA干扰沉默Id-1基因后,ACCM生长受到抑制、侵袭能力均受到抑制,细胞凋亡增多；ACCM中ki67含量减少、c-myc含量减少和p21含量增加。其机制可能是通过沉默Id-1基因,腺样囊性癌细胞系ACCM本来呈现高表达的Id-1出现低表达或者无表达,本来被打破了的癌基因与抑癌基因相互制约的平衡关系恢复了正常,激活了的某(些)条信号传导通路得到抑制或者关闭,抑制细胞周期进程、促进细胞分化、抑制细胞增殖,抑制了通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。但是Id-1对ACC生长和侵袭起调节作用的精确信号传导通路,还有待进一步的研究与探讨。
第三部分沉默Id-1基因对口腔鳞状细胞癌细胞增殖、侵袭、凋亡的影响
目的
口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)占口腔癌90%以上2005年WHO头颈肿瘤病理学和遗传学分类中将口腔鳞状细胞癌定义为“一种具有不同程度分化的侵袭性肿瘤,倾向于早期、广泛的淋巴结转移,主要发生于40-70岁的烟酒嗜好者。口腔癌是恶性程度较高的肿瘤,虽然经肿瘤学家、外科医师的不断努力,在过去20年里口腔癌的死亡率略有下降,但其5年生存率仍只有41%-79.5%,这促使人们努力寻找与肿瘤预后相关的因素,以采取更有针对性的措施进行预防和治疗。
Id蛋白属于螺旋-环-螺旋蛋白质超家族,相对分子质量为13000～20000,它是一组缺乏碱性DNA连接结构域的HLH因子。bHLH蛋白二聚体的形成是DNA与“E盒”(CANNTG)或“N盒”(CACNAG)识别序列结合的必要条件,Id蛋白通过与bHLH转录因子形成异型二聚体,干扰其与DNA的结合,阻断其对下游分子的转录激活作用,抑制基因的表达,从而抑制细胞的正常分化,促进细胞增殖。Id-1是人类迄今为止发现的4个Id蛋白家族成员(Id-1-4)中研究最多,也是最重要的一个。研究发现：Id-1在上皮来源的肿瘤中呈高表达,它通过抑制细胞分化、推进细胞周期进程、诱导细胞增殖、抑制衰老、诱导侵袭、参与肿瘤性血管新生而介导肿瘤的发生和发展；在大肠癌、食管癌、乳腺癌、前列腺癌、卵巢癌、宫颈癌的发生发展过程中都发挥着重要的作用。然而在OSCC中,Id-1的表达量如何,其对于OSCC的生长和侵袭能力的影响尚有待于研究证实。
以往研究发现的与肿瘤相关的Id-1信号通路有很多,有Akt-mediated Wnt信号通路,p53 and NF-kB信号通,PI3K/Akt/NFKB信号通路, ERK/MAPK信号通路等等。我们前期研究发现,正常涎腺组织中Id-1的表达量非常低(甚至难以检测到),但是在涎腺腺样囊性癌中却呈现高表达,这说明Id-1表达水平的升高可能打破了正常细胞内癌基因与抑癌基因相互制约的平衡关系,进而激活某条或多条信号传导通路推进细胞周期进程、抑制细胞分化、促进细胞增殖,从而诱导正常细胞向肿瘤细胞转化,增加通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。
我们选用设计好的Id-1siRNA序列,应用lipofectamine 2000进行转染,沉默掉OSCC细胞系SAS的Id-1基因,然后检测SAS细胞系增殖、侵袭和凋亡的改变。
方法
于含10%胎牛血清的DMEM培养基、37℃、含5%CO2的细胞培养箱中密闭培养口腔鳞状细胞癌细胞系SAS。用设计好的Id-1siRNA序列,应用lipofectamine2000进行转染,沉默SAS细胞系的Id-1基因。RFQ-PCR、Western blot检测、免疫荧光确认干扰成功。MTT比色法于570 nm处测定光密度值(OD),描记干扰前后细胞的生长曲线。Transwell小室检测干扰前后细胞侵袭性能的改变。流式细胞仪检测干扰前后细胞的凋亡情况。
结果
实验结果显示RFQ-PCR、Western blot检测、免疫荧光确认干扰成功。MTT比色法描记的干扰前后细胞的生长曲线结果显示：Id-1siRNA干扰后SAS增殖能力下降,与未干扰对照组有显著差异(0.68055±0.0457vs0.8469±0.0878,P<0.01；0.73775±0.0866vsl.11098±0.0899,P<0.01；0.8679±0.0442vsl.37743±0.0471.P<0.01)。Transwell小室检测的结果显示：Id-1siRNA干扰后SAS侵袭能力下降,与未干扰对照组有显著差异(10.1666667±0.012vsl7.8±0.008,P<0.01)。流式细胞仪检测的凋亡结果显示：Id-1 siRNA干扰后SAS凋亡增多,与未干扰对照组有显著差异((5.15±3.02)%vs(20.77±2.56)%,P<0.01)。
结论
结果提示通过RNA干扰沉默Id-1基因后,SAS生长受到抑制、侵袭能力均受到抑制,细胞凋亡增多。其机制可能是通过沉默Id-1基因,鳞状细胞癌细胞系SAS本来呈现高表达的Id-1出现低表达或者无表达,本来被打破了的癌基因与抑癌基因相互制约的平衡关系恢复了正常,激活了的某(些)条信号传导通路得到抑制或者关闭,抑制细胞周期进程、促进细胞分化、抑制细胞增殖,抑制了通过淋巴管道和血道向周围淋巴结和远处器官侵袭和转移的机会。但是Id-1对OSCC生长和侵袭起调节作用的精确信号传导通路,还有待进一步的研究与探讨。
SectionⅠExpression of Id-1 gene in oral cancer cells
Objective
Oral cancer is more common clinical disease, the incidence of an increasing trend in recent years, their age of onset of 40 to 60 years as the peak onset age, but 60 to 70-year-old age group a marked increase in the incidence, accounting for 17.8%, the average age of onset 54.1 years old, showed that the incidence of oral cancer has an aging trend. Most oral cancers occur in the oral cavity (excluding lip), accounting for 78.4% of the total, followed by the major salivary glands, maxillary sinus, neck, throat, lips. Mouth predilection sites in sequence as the tongue, upper and lower gums, cheek, palate, upper and lower jaw, floor of the mouth, tongue cancer has leapt to the top. Pathological types of oral cancer squamous cell carcinoma and adenoid cystic carcinoma of the majority, and that the degree of malignancy is relatively large, easy to recurrence and metastasis, especially adenoid cystic carcinoma is highly prone to distant metastasis, but also has neurotropic nature. Therefore, we chose five most representative oral cancer cell lines:the squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2 for screening. DNA binding protein (Inhibitor of DNA binding, Id), are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role.However, in oral cancer, Id-1 expression levels of how their oral cancer cells for growth and invasion capacity of the unknown. This study will detect the expression of Id-1 in this five typical oral cancer cell lines.
Methods
Oral squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2 thaw recovery, containing 10% fetal bovine serum in DMEM medium,37℃, the cells containing 5% CO2 incubator closed-culture. Logarithmic growth of cells, using Trizol lysate extracted total RNA, the total RNA reverse transcription of cDNA, and then Id-1 primers for real-time fluorescence quantitative PCR (RFQ-PCR), amplification of the body by adding SYBR Green I, for RFQ-PCR experiment, detection the expression of Id-1mRNA in the five cell lines. And then take on the exponential growth of cells, conventional methods of extrACTINg cells, total protein, first anti-and second anti-incubated for Western Blot test, test Id-1 protein expression in five kinds of cell lines.
Results
Experimental results show that it showed a high Id-1 expression in this five cell lines:squamous cell carcinoma cell line SAS, Tca8113, Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2, which, Id-1 expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines,and Id-1 expression in SAS was significantly higher in squamous cell carcinoma cell lines. RFQ-PCR results showed that:Id-1 mRNA expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines(P<0.05), and Id-1 mRNA expression in SAS was significantly higher in squamous cell carcinoma cell lines(P<0.05). Western Blot results showed that:Id-1 protein expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines(P<0.05), and Id-1 protein expression in SAS was significantly higher in squamous cell carcinoma cell lines(P<0.05).
Conclusion
These results suggest that, Id-1mRNA and Id-1 protein showed a high expression in this five cell lines:squamous cell carcinoma cell line SAS, Tca8113 Buccl885, and adenoid cystic carcinoma cell line ACCM, ACC-2, which, Id-1 expression in ACCM was significantly higher in adenoid cystic carcinoma cell lines, and Id-1 expression in SAS was significantly higher in squamous cell carcinoma cell lines. We selected two high expression of Id-1 gene in oral cancer cell lines:adenoid cystic carcinoma cell line ACCM and squamous cell carcinoma SAS.
Section II The effects of silencing Id-1 gene on the proliferation, invasion and apoptosis in adenoid cystic carcinoma cells
Objective
Adenoid cystic carcinoma (ACC), occurred in the head and neck, the most common salivary gland malignancy, accounting for 20% of all malignancies, with a high degree of invasion and metastasis characteristics. Because of the understanding of the limitations to the biological nature of ACC, there is not a very effective treatment measures, even if carried out an extensive surgical excision and radiation therapy, most patients will eventually become a recurrence and metastasis. It is urgent to seek treatment for clinically ACC.
Id proteins are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. bHLH protein dimer formation of DNA with the "E box" (CANNTG) or "N Box" (CACNAG) recognition sequence with a necessary condition, Id proteins with bHLH transcription factors form a heterodimer, interference with DNA, combination of blocking its transcriptional activation of downstream molecules, inhibit gene expression, thus inhibiting normal cell differentiation and promote cell proliferation. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role. However, in the ACC, what ahout Id-1 expression levels and how it effects ACC of the growth and invasive ability is unknown.
Found in previous studies with tumor-related Id-1 signaling pathway there are many, there is Akt-mediated Wnt signaling pathway, p53 and NF-kB signal-pass, PI3K/Akt/NFKB signaling pathway, ERK/MAPK signaling pathway and so on. Our preliminary study found that expression levels of Id-1 in normal salivary gland tissue is very low (or even difficult to detect), but having a high expression in salivary adenoid cystic carcinoma, indicating the level of Id-1 expression increased normal cells may be breaking the oncogene and tumor suppressor genes balanced relationship of checks and balances, thereby activating one or more of a certain signal transduction pathways to promote cell cycle progression, inhibit cell differentiation, promote cell proliferation, thereby inducing normal cells to tumor cell transformation, to increase through the lymphatic channels and blood Road to the surrounding lymph nodes and distant organs, invasion and metastasis of opportunities.
We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in ACCM cell lines, and then test proliferation, invasion and apoptosis changes in ACCM cell.
Methods
The high metastasis adenoid cystic carcinoma cell lines ACCM were contained in DMEM medium containing 10% fetal bovine serum,37℃,5% CO2 incubator in the closed-culture. We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in ACCM cell lines. RFQ-PCR, Western blot and immunofluorescence assay were used to test to confirm the successful disruption. MTT assay was measured at 570 nm optical density (OD), cell growth curve was recorded before and after interference. Transwell chamber was used to test the changes in cell invasiveness before and after the interference. Flow cytometry test the changes in cell apoptosis before and after the interference. Western blot were used to test the protein expression of ki67,c-myc and p21.
Results
The results showed that RFQ-PCR, Western blot test and immunofluorescence assay to confirm the successful interference. MTT assay recording cell growth curve before and after the interference showed that:after silencing Id-1 gene, the proliferation of ACCM was decreased, and was significantly different with control (0.5806±0.0063 vs 0.7469±0.0424,P<0.01; 0.6378±0.0040 vs 1.0110±0.0010, P<0.01; 0.7679±0.0036 vsl.2774±0.0036, P<0.01). Transwell chamber test results showed that: after silencing Id-1gene, the invasiveness of ACCM was decreased, and was significantly different with control (10.6667±2.0656vs29.8333±1.7224, P<0.01). Flow cytometry showed that:after silencing Id-1 gene, the cell number of apoptosis of ACCM was increased, and was significantly different with control ((23.2767±1.2872) % vs(2.2467±0.0924)%, P<0.01). Western blot test results showed that:after silencing Id-1 gene, and was significantly different with control, the expression of ki67and c-myc were reduction, however, the expression of p21 was upregulation.
Conclusion
The results suggested that after RNA interference silencing by Id-1 gene, ACCM growth was inhibited,invasion ability were inhibited, apoptosis increased; after silencing Id-1 gene, the expression of ki67and c-myc were reduction,however, the expression of p21 was upregulation.lts possible mechanism is through silence, Id-1 gene, adenoid cystic carcinoma cell lines showed high expression of ACCM,had Id-1 expression at low or no expression of what might be breaking the cancer gene and tumor suppressor genes balanced relationship of checks and balances returned to normal, the activation of a certain of the signal transduction pathway to be inhibited or shut down, inhibit cell cycle progression and promoting cell differentiation, inhibit cell proliferation, inhibition of blood through the lymphatic channels and the Road to the surrounding lymph nodes and distant organs, invasion and metastasis opportunities. However, the precise signal transduction pathway of Id-1 in ACC to regulate the growth, invasion and of, remains to be further research and discussion.
SectionⅢEffects of silencing Id-1 gene on the proliferation, invasion and apoptosis in oral squamous cell carcinoma cells
Objective
Oral squamous cell carcinoma (oral squamous cell carcinoma, OSCC) accounts for more than 90% of oral cancer, in 2005 WHO classification of pathology and genetics head and neck cancer will be oral squamous cell carcinoma is defined as "a different degree of differentiation of invasive tumors, tend to the early, extensive lymph node metastasis, mainly in the 40 to 70 years of age who smoke or alcohol abuse. oral cancer is highly malignant tumor, although the oncologist, the surgeon's continuous efforts in the past 20 years, Village of oral cancer mortality rate declined slightly, but its five-year survival rate was still only 41%～79.5%, which prompted efforts to find the factors associated with tumor prognosis in order to take a more targeted measures for the prevention and treatment.
Id proteins are helix-loop-helix protein super-family, relative molecular mass of 13000～20000, which is a group of the lack of basic HLH domain of DNA connecting factor. bHLH protein dimer formation of DNA with the "E box" (CANNTG) or "N Box" (CACNAG) recognition sequence with a necessary condition, Id proteins with bHLH transcription factors form a heterodimer, interference with DNA, combination of blocking its transcriptional activation of downstream molecules, inhibit gene expression, thus inhibiting normal cell differentiation and promote cell proliferation. Id-1 in human so far found four members of the Id proteins (Id-1～4) studied the largest and most important one. The study found:Id-1 in epithelial origin tumors showed high expression, which by inhibiting cell differentiation, promote cell cycle progression and induce cell proliferation and suppress senescence, induced invasion, involved in tumor-mediated angiogenesis and tumor occurrence and development of; in colorectal cancer, esophageal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer occurrence and development have played an important role. However, in OSCC, Id-1 expression levels of how their growth and invasion of OSCC for the ability of research yet to be confirmed.
Found in previous studies with tumor-related Id-1 signaling pathway there are many, there is Akt-mediated Wnt signaling pathway, p53 and NF-kB signal-pass, PI3K/Akt/NFkB signaling pathway, ERK/MAPK signaling pathway and so on. Our preliminary study found that normal salivary gland tissue expression levels of Id-1 is very low (or even difficult to detect), but in salivary adenoid cystic carcinoma is having a high expression, indicating the level of Id-1 expression increased possible to break the normal cell oncogene and tumor suppressor genes within the constraints of a balanced relationship with each other, thereby activating a certain or more signal transduction pathways to promote cell cycle progression, inhibit cell differentiation, promote cell proliferation, thereby inducing normal cells to tumor cell transformation, to increase through the lymphatic channels and blood Road to the surrounding lymph nodes and distant organs, invasion and metastasis of opportunities.
We use designed Id-1siRNA sequence, the application lipofectamine 2000 transfection, silence Id-1 gene in OSCC cell lines SAS, and then test proliferation, invasion and apoptosis changes in SAS. Methods The high metastasis adenoid cystic carcinoma cell lines SAS were contained in DMEM medium containing 10% fetal bovine serum,37℃,5% CO2 incubator in the closed-culture. We use a good screening Id-1siRNA sequence, the application lipofectamine 2000 transfection, to silence Id-1 gene in SAS cell lines. RFQ-PCR, Western blot and immunofluorescence assay were used to test to confirm the successful disruption. MTT assay was measured at 570 nm optical density (OD), cell growth curve was recorded before and after interference. Transwell chamber was used to test the changes in cell invasiveness before and after the interference. Flow cytometry test the changes in cell apoptosis before and after the interference.
Results
The results showed that RFQ-PCR, Western blot test and immunofluorescence assay to confirm the successful interference. MTT assay recording cell growth curve before and after the interference showed that:after silencing Id-1gene, the proliferation of SAS was decreased (0.68055±0.0457vs0.8469±0.0878,P<0.01; 0.73775±0.0866vsl.11098±0.0899,P<0.01;0.8679±0.0442vsl.37743±0.0471,P<0.01). Transwell chamber test results showed that:after silencing Id-1gene, the invasiveness of SAS was decreased (10.1666667±0.012vsl7.8±0.008, P<0.01). Flow cytometry showed that:after silencing Id-1gene, the cell number of apoptosis of SAS was increased ((5.15±3.02)%vs (20.77±2.56)%,P<0.01).
Conclusion
The results suggested that after RNA interference silencing by Id-1 gene, SAS growth was inhibited,invasion ability were inhibited, apoptosis increased.Its possible mechanism is through silence, Id-1 gene, oral squamous cell carcinoma cell lines showed high expression of SAS,had Id-1 expression at low or no expression of what might be breaking the cancer gene and tumor suppressor genes balanced relationship of checks and balances returned to normal, the activation of a certain of the signal transduction pathway to be inhibited or shut down,inhibit cell cycle progression and promoting cell differentiation,inhibit cell proliferation,inhibition of blood through the lymphatic channels and the Road to the Surrounding lymph nodes and distant organs, invasion and metastasis opportunities. However, the precise signal transduction pathway of Id-1 in OSCC to regulate the growth,invasion and of, remains to be further research and discussion. 18国家自然基金面上项目(30672339 and 30772269)和SRF for ROCS提供资金资助
引文
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29 Young T,Mei F,Liu J,et al.Proteomics analysis of H-RAS.Mediated oncogenic transformation in a genetically defined human ovarian cancer model. Oncogene,2005, 24:6174-6184.
30 Yoo J, Robinson RA.H-ras gene mutations in salivary gland mucoepidermoid carcinomas.Cancer,2000,88:518-523.
31 Simon AR,Severgnini M,Takahashi S,et al.5-HT induction of c-fos gene expression requires reactive oxygen species and Rac 1 and Ras GTPases. Cell Biochem Biophys,2005,42:263-276.
32 Plaza-Menacho I,van der Sluis T,Hollema H,et al.Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation.J Biol Chem,2007,282:6415-6424.
33 Jayachandran G,Sazaki J,Nishizaki M,et al.Fragile histidine triad-mediated tumor suppression of lung cancer by targeting multiple components of the Ras/Rho GTPase molecular switch.Cancer Res,2007,67:10379-10388.
34 Wang Yong-xiang, Gao Liang, Ji Zongzheng. For the K-ras gene point mutations in antisense oligonucleotide on human pancreatic cancer cell line PC-2 rats. Surgery,2005,43 (21):1387-1390.
35 Zhang YA,Nemunaitis J,Scanlon KJ,et al.Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice. Gene Ther.2000 Dec;7(23):2041-50.
36 Feldkamp MM,Lau N,Roncari L,et al.Isotype-specific Ras-GTP-levels predict the efficacy of famesyl transferase inhibitors against human astrocytomas regardless of Ras mutmional status. Cancer Res,2001,61:4425-4431.
37 Shuai Xiaoming. Mechanism of antisense research. Foreign Medical Sciences. MOLECULAR BIOLOGY,2001,23 (4):237-240.
38 Hou Y.Chromosomal localization of transforming oncogene BGC-Ha-ras in gastric cancer cell line.Zhonghua Zhong Liu Za Zhi,1990,12:95-97.
39 Brummelkamp TR,Bemards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell,2002:243-247.
40 Rubin LL, Philpott KL,Brooks SF.Apoptosis:the cell cycle and cell death.Curr Biol,1993,3:391-394.
41 Maciej Wiznerowicz, Didier Trono.Conditional suppression of cellular genes:lentivirus vector-mediated drug-inducible RNA interference.Journal of virology 2003,77(16):8957-8961.
42 Richard J Fish,Egbert KO Kruithof. Short—term cytotoxic effects and long—term instability of RNAi delivered using lentiviral vectors. BMC Molecular Biology 2004,5:9.
43 Li L, Liu X,Staver M,et al, Evaluating hypoxia-inducible factor-1 alpha as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res,2005; 65(16):7249-7258.
44 Lee TK, Man K, Ling MT, Wang XH, Wong YC, Lo CM, et al. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway. Carcinogenesis 2003; 24(11):1729-36.
45 Lyden D, Young AZ, Zagzaf D, et al. Id-1 and Id-3 are required for neurogenesis, angiofenesis and vascularization of tumour xenografts. Nature,1999, 401:670-677.
46 Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia:role of the HIF system. Nat Med,2003; 9:677-684.
47 Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism. Cancer Res,1996; 56(23):5309-5318.
48 Virials F, Reiriz J, Ambrosio S, Bartrons R, Rosa JL, Ventura F. BMP-2 decreases Mashl stability by increasing Idl expression. EMBO J 2004; 23(17):3527-37.
49 Ouyang XS,Wang X,Ling MT,el al.Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p I 6(INK4a)/Prb pathway.Carcinogenesis,2002,23(5):721-725.
50 Ling MT,Wang X,Ouyang XS,et al.Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth.Oncogene,2002,21(55):8498-8505.
51 Ling MT, Wang X, Ouyang XS, et al.ld-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells.Oncogene, 2003,22(29):4498-4508.
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[4]Ruzinova MB, Benezra R.Id proteins in development, cell cycle and cancer. Trends Cell Biol.2003; 13 (8):410-8.
[5]Wong YC, Wang X, Ling MT. Id-1 expression and cell survival. Apoptosis. 2004; 9 (3):279-89.
[6]Coppe JP, Itahana Y, Moore DH, et al. Id-1 and Id-2 proteins as molecular markers for human prostate cancer progression [J].Clin Cancer Res,2004,10 (6): 2044-2051.
[7]Desprez PY, Lin CQ, Thomasset N, et al.A novel pathway for mammary epithelial cell invasion induced by the helix-loop- helix protein Id- 1 [J]. Mol Cell Biol, 1998,18 (8):4577-4588.
[8]Fong S, Itahana Y, Sumida T, et al. Idl as a molecular target in therapy for breast cancer cell invasion and metastasis[J]. ProcNatl Acad Sci U S A,2003,100 (23):13543-13548.
[9]Lyden D, Young AZ, Zagzag D, et al. Idl and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts[J]. Nature,1999, 401 (6754):670-677.
[10]Zhao ZR, Zhang ZY, Zhang H, Jiang L, Wang MW, Sun XF. Overexpression of Id-1 protein is a marker in colorectal cancer progression. Oncol Rep.2008; 19 (2):419-24.
[11]Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, and etc. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway. Int J Cancer.2009; 125 (11):2576-85.
[12]Xu B, Sun Y, Tang G, Xu C, Wang L, Zhang Y, and etc. Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor. Cancer Lett.2009; 278 (2):220-9.
[13]McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells. Mol Cancer Ther.2007; 6(11):2921-7.
[14]Li J, Jia H, Xie L, Wang X, He H, Lin Y, Hu L. Correlation of inhibitor of differentiation 1 expression to tumor progression, poor differentiation and aggressive behaviors in cervical carcinoma. Gynecol Oncol.2009;114(1):89-93.
[15]Guan XF, Qiu WL, He RG, and etc.Lung highly metastatic adenoid cystic carcinoma cell line screening [J]. Chinese Journal of Oral Medicine,1996,31 (2):74-77.
[16]Lee JY, Kang MB, Jang SH, Qian T, Kim HJ, Kim CH, and etc. Id-1 activates Akt-mediated Wnt signaling and p27(Kipl) phosphorylation through PTEN inhibition. Oncogene.2009; 28 (6):824-31.
[17]Kim H, Chung H, Kim HJ, Lee JY, Oh MY, Kim Y, and etc. Id-1 regulates Bcl-2 and Bax expression through p53 and NF-kB in MCF-7 breast cancer cells. Breast Cancer Res Treat.2008; 112 (2):287-96.
[18]Li B, Cheung PY, Wang X, Tsao SW, Ling MT, Wong YC, and etc.Id-1 activation of PI3K/Akt/NFikB signaling pathway and its significance in promoting survival of esophageal cancer cells. Carcinogenesis.2007; 28 (11):2313-20.
[19]Jorda M, Vinyals A, Marazuela A, Cubillo E, Olmeda D, Valero E, and etc. Id-1 is induced in MDCK epithelial cells by activated ERK/MAPK pathway in response to expression of the Snail and E47 transcription factors.Exp Cell Res.2007; 313(11):2389-403.
1 Lister J,Forrester WC. Inhibition ofanerythroid differentiation switch by the helix-loop-helix protein Idl. J Biol Chem,1995,270; 17939-17946.
2 Katagiri T,Imada M,Yanai T.et al,Identification of a BMP-responsive element in Id-1.the gene for inhibition of myogenesis. Genes CellS,2002,7:949-960.
3 Ruzinova M B, Benezra R.Id proteins in development, cell cycle and cancer. Trends Cell Biol 2003,13:410-418.
4 Norton J. D.,Deed R. W.,Craggs G and Sablitzky F. (1998) Id helix-loop-helix proteins in cell growth and differentiation.Trends Cell Biol.,8,58-65.
5 Ishigum A, Spifin K, Shiohara M, et al. Expression of Id2 and Id3 mRNA Inhuman lymphocytes [J]. Leuk Res 1995:19 (12):989-996
6 Zhao ZR, Zhang ZY, Zhang H, Jiang L, Wang MW, Sun XF. Overexpression of Id-1 protein is a marker in colorectal cancer progression. Oncol Rep.2008; 19 (2):419-24.
7 Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, and etc. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway. Int J Cancer.2009; 125(11):2576-85.
8 Xu B, Sun Y, Tang G, Xu C, Wang L, Zhang Y, and etc. Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor. Cancer Lett.2009; 278(2):220-9.
9 McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY. Cannabidiol as a novel inhibitor of Id-I gene expression in aggressive breast cancer cells. Mol Cancer Ther.2007; 6(11):2921-7.
10 Li J, Jia H, Xie L, Wang X, He H, Lin Y, Hu L. Correlation of inhibitor of differentiation 1 expression to tumor progression, poor differentiation and aggressive behaviors in cervical carcinoma. Gynecol Oncol.2009; 114(1):89-93.
11 Alani R M, Young A Z, and Shifflett C B. Idl regulation of cellular senescence through transcriptional repression of P16ink4a Proc.Natl. Acad. Sci. USA,2001,98: 7812-7816
12 Ling M T, Wang X, Ouyang X S,et al.Id-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells. Oncogene 2003,22 (29):4498-4508
13 Barns L, Everson JW, Reichart P, et al. Pathology and genetics of head and neck tumors[M]. Lyon:IARC Press,2005:168-175.
14 Garzino-demo P, Dell'Acqua A, Dalmasso P, et al. Clinicopathological parameters and outcome of 245 patients operated for oral squamous cell carcinoma [J]. J Craniomaxillofac Surg,2006,34:344-350.
15 Nishimine M, Nakamura M, Mishima K, Kishi M, Kirita T, Sugimura M, Konishi N. Id proteins are overexpressed in human oral squamous cell carcinomas.J Oral Pathol Med.2003 Jul; 32(6):350-7.
16 KyatsPA.,GeleffS.,Batistatou A,et al. Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma[J].J Pathol, 2005,206(2):170-177.
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18 Tao Peng,Zhang Jun,Tang Ni,et al.Potent specific inhibition of SARS-CoV antigen expression by RNA interference.Chinese Medical Journal 2005,118(9): 714-719.
19 Yin-cheng Zhang, Xiao-Ming Jin. Oncogenes and tumor suppressor genes in salivary gland tumors. Of Oral Medicine,2000,14:60-62.
20 DAI Yu-cheng, Chen Mao Hang.Apoptosis in the tumor and treatment significance. Cancer,1995,15:279-282.
21 Leicht DT,Balan V,Kaplun A,et al.Raf kinases:function,regulation and role in human cancer.Biochim Biophys Acta,2007,1773:1196-1212.
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23 Hannon GJ. RNA interference. Nature,2002; 418 (6894):244-251.
24 Davenport RJ. Gene silencing:a faster way to shut down genes. Science,2001; 292(5521):1469-1471.
25 Lan Chun-Hui, Zhang Xiao Yu, Xiao-Ling. Gastric cancer and precancerous lesions of epidermal growth factor receptor and p21ras protein expression. Chinese Medical Journal,2000,10 (7):17.
26 Ming Xue-Zhi, Hao-Ran Yin, Zheng-gang, et al. Gastric H. ras, and VEGF expression and angiogenesis and its clinical significance. Chinese Journal of Cancer, 2006,13 (5):357-360.
27 Miura S,Matsuo Y,Saku K.Simvastatin suppresses coronary artery endothelial tube formation by disrupting Ras/Raf/ERK signaling.Atherosclerosis,2004,1 75(2):235-243.
28 Zhao L,Lobo S,Dong X,et al.Erf4p and Erf2p form an endoplasmic reticulum—associated complex involved in the plasma membrane localization ofyeast Ras proteins.J Biol Chem,2002,277:49352-49359.
29 Young T,Mei F,Liu J,et al.Proteomics analysis of H-RAS.Mediated oncogenic transformation in a genetically defined human ovarian cancer model. Oncogene,2005, 24:6174-6184.
30 Yoo J, Robinson RA.H-ras gene mutations in salivary gland mucoepidermoid carcinomas.Cancer,2000,88:518-523.
31 Simon AR,Severgnini M,Takahashi S,et al.5-HT induction of c-fos gene expression requires reactive oxygen species and Rac 1 and Ras GTPases.Cell Biochem Biophys,2005,42:263-276.
32 Plaza-Menacho I,van der Sluis T,Hollema H,et al.Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c—fos promoter activation, cell mitogenicity, and transformation.J Biol Chem,2007,282:6415-6424.
33 Jayachandran QSazaki J,Nishizaki M,et al.Fragile histidine triad-mediated tumor suppression of lung cancer by targeting multiple components of the Ras/Rho GTPase molecular switch.Cancer Res,2007,67:10379-10388.
34 Wang Yong-xiang, Gao Liang, Ji Zongzheng. For the K-ras gene point mutations in antisense oligonucleotide on human pancreatic cancer cell line PC-2 rats. Surgery,2005,43 (21):1387-1390.
35 Zhang YA,Nemunaitis J,Scanlon KJ,et al. Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice. Gene Ther. 2000 Dec;7(23):2041-50.
36 Feldkamp MM,Lau N,Roncari L,et al.Isotype-specific Ras-GTP-levels predict the efficacy of famesyl transferase inhibitors against human astrocytomas regardless of Ras mutmional status. Cancer Res,2001,61:4425-4431.
37 Shuai Xiaoming. Mechanism of antisense research. Foreign Medical Sciences. MOLECULAR BIOLOGY,2001,23 (4):237-240.
38 Hou Y.Chromosomal localization of transforming oncogene BGC-Ha-ras in gastric cancer cell line.Zhonghua Zhong Liu Za Zhi,1990,12:95-97.
39 Brummelkamp TR,Bemards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell,2002:243-247.
40 Rubin LL,Philpott KL,Brooks SF.Apoptosis:the cell cycle and cell death.Curr Biol,1993,3:391-394.
41 Maciej Wiznerowicz,Didier Trono.Conditional suppression of cellular genes:lentivirus vector-mediated drug—inducible RNA interference.Journal of virology 2003,77(16):8957-8961.
42 Richard J Fish,Egbert KO Kruithof. Short—term cytotoxic effects and long—term instability of RNAi delivered using lentiviral vectors.BMC Molecular Biology 2004,5:9.
43 Li L,Liu X,Staver M,et al, Evaluating hypoxia-inducible factor-1 alpha as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res,2005; 65(16):7249-7258.
44 Lee TK, Man K, Ling MT, Wang XH, Wong YC, Lo CM, et al. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway. Carcinogenesis 2003; 24(11):1729-36.
45 Lyden D, Young AZ, Zagzaf D, et al. Id-1 and Id-3 are required for neurogenesis, angiofenesis and vascularization of tumour xenografts. Nature,1999, 401:670-677.
46 Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia:role of the HIF system. Nat Med,2003; 9:677-684.
47 Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism. Cancer Res,1996; 56(23):5309-5318.
48 Virials F, Reiriz J, Ambrosio S, Bartrons R, Rosa JL, Ventura F. BMP-2 decreases Mashl stability by increasing Idl expression. EMBO J 2004; 23(17):3527-37.
2Garzino-demoP, Dell'Acqua A, Dalmasso P, et al. Clinicopathologicalparameters and outcome of 245 patients operated for oral squamous cell carcinoma [J]. J Craniomaxillofac Surg,2006,34:344-350.
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7 Fong S, Itahana Y, Sumida T, et al. Id1 as a molecular target in therapy for breast cancer cell invasion and metastasis [J]. ProcNatl Acad Sci U S A,2003,100 (23):13543-13548.
8 Lyden D, Young AZ, Zagzag D, et al. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts [J]. Nature,1999,401 (6754): 670-677.
9 Ruzinova MB, Benezra R.Id proteins in development, cell cycle and cancer.Trends Cell Biol.2003；13(8)：410-8.
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11 Yokota Y, Mori S. Role of Id family proteins in growth control. J Cell Physiol, 2002,190:21-28.
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13 刘豫瑞,庄则豪,李友炳,等。Id-1,Ki-67及Bel-2在食管鳞状细胞癌中的表达及意义,中华消化内镜杂志,2005,10：327-330.
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1 Benezra R, Davis RL, Lockshon D, et al. The protein Id:a negative regulator of helix-loop-helix DNA binding proteins.Cell.1990 Apr 6; 61(1):49-59.
2 Massari ME, Murre C. Helix-loop-helix proteins:regulators of transcription in eukaryotic organisms. Mol Cell Biol.2000 Jan; 20(2):429-40.
3 Sun XH, Copeland NqJenkins NA, et al. Id proteins Id I and Id2 selectively inhibit DNA binding by one class ofhelix-loop-helix proteins. Mot Cell Biol.1991 Nov;I1(11):5603-11.
4 Christy BA, Sanders LK, Lau LF,et al.An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene. Proc Natl Acad Sci U S A.1991 Mar 1; 88(5):1815-9.
5 RiechmannMvan Cruchten I, Sablitzky F.The expression patern of IM, a novel dominantnegative helix-loop-helix protein, is distinct from Idl, Id2 and Id3.Nucleic Acids Res.1994 Mar 11; 22(5):749-55.
6 Uo T, Iavarone A. ID proteins at the cross-road of development and cancer. Oncogene,2001,20(58):8326-8333.
7 Coppe JP, Itahana Y, Moore DH, et al. Id-1 and Id-2 proteins as molecular markers for human prostate cancer progression [J].Clin Cancer Res,2004,10 (6): 2044-2051.
8 Desprez PY, Lin CQ, Thomasset N, et al.A novel pathway for mammary epithelial cell invasion induced by the helix-loop-helix protein Id-1[J]. Mol Cell Biol, 1998,18(8):4577-4588.
9 Fong S, Itahana Y, Sumida T, et al. Id1 as a molecular target in therapy for breast cancer cell invasion and metastasis[J]. ProcNatl Acad Sci U S A,2003, 100(23):13543-13548.
10 Lyden D, Young AZ, Zagzag D, et al. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts[J]. Nature,1999, 401(6754):670-677.
11 Zhao ZR, Zhang ZY, Zhang H, Jiang L, Wang MW, Sun XF. Overexpression of Id-1 protein is a marker in colorectal cancer progression. Oncol Rep.2008; 19 (2):419-24.
12 Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, and etc. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway. Int J Cancer.2009; 125(11):2576-85.
13 Xu B, Sun Y, Tang G, Xu C, Wang L, Zhang Y, and etc. Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor.Cancer Lett.2009; 278(2):220-9.
14 McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY.Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells. Mol Cancer Ther.2007; 6(11):2921-7.
15 Li J, Jia H, Xie L, Wang X, He H, Lin Y, Hu L. Correlation of inhibitor of differentiation 1 expression to tumor progression, poor differentiation and aggressive behaviors in cervical carcinoma. Gynecol Oncol.2009; 114(1):89-93.
16 NickoloffBJ,ChaturvediV,Bacon P,et a l.Id-1 delays senescence but does not immortalize keratinocytes[J] J Biol Chem,2000,275(36):27501-27504.
17 Ruzinova MB, Benezra R.Id proteins in development, cell cycle and cancer. Trends Cell Biol.2003 Aug; 13(8):410-8.
18 Chaudhary J,Schmidt M,Sadler-Riggleman I.Negative ACTINg HLH proteins Idl,ld2,ld3,and Id4 are expressed in prostate epithelial cell,Prostate,2005,64(3):253-264.
19 Noaon JD.ID helix-loop-helix proteins in cell growth,differentiation and tumorigenesis.J Cell Sci,2000,113(22):3897-3905.
20 Sikder HA,Devlin MK,Dunlap S,et al.Id proteins in cell growth and tumorigenesis.Cancer Cell,2003,3(6):525-530.
21 Fong S,Debs RJ,Desprez PY.1d genes and proteins as promising targets in cancer therapy.Trends Mol Med,2004,10(8):387-392.
22 Wong YC, Wang X, Ling MT.Id-1 expression and cell survival.Apoptosis,2004,9(3):279-289.
23 FireA, Xu S, Montgomery MK, et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditise legans. Nature,1998; 391(6669): 806-811.
24 Li L, Liu X,Staver M,et al, Evaluating hypoxia-inducible factor-lalpha as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res,2005; 65(16):7249-7258.
25 Lee TK, Man K, Ling MT, Wang XH, Wong YC, Lo CM, et al. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway. Carcinogenesis 2003; 24(11):1729-36.
26 Lyden D, Young AZ, Zagzaf D, et al. Id-1 and Id-3 are required for neurogenesis, angiofenesis and vascularization of tumour xenografts. Nature,1999, 401:670-677.
27 Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia:role of the HIF system. Nat Med,2003; 9:677-684.
28 Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism. Cancer Res,1996; 56(23):5309-5318.
29 Virials F, Reiriz J, Ambrosio S, Bartrons R, Rosa JL, Ventura F. BMP-2 decreases Mashl stability by increasing Idl expression. EMBO J 2004; 23(17):3527-37.
30 hlani R M, Young A Z,and Shifflett C B.Id-1 regulation of cellular senescence through transcriptional repression of P16ink4a Proc.Natl.Acad.Sci.USA 2001.98:7812-7816.
31 Ling M T,Wang X,Ouyang X S,et al.Id-I expression promotes cell survival through activation of NF-kB signalling pathway inprostate cancer cel 1 s.Oncogene 2003.22(29):4498-4508.
1 Lister J,Forrester WC. Inhibition ofanerythroid differentiation switch by the helix-loop-helix protein Idl. J Biol Chem,1995,270; 17939-17946.
2 Katagiri T,Imada M,Yanai T.,et al,Identification of a BMP-responsive element in Id-l.the gene for inhibition of myogenesis. Genes CellS,2002,7:949-960.
3 Ruzinova M B, Benezra R.Id proteins in development, cell cycle and cancer. Trends Cell Biol 2003,13:410-418.
4 Norton J. D.,Deed R. W.,Craggs G. and Sablitzky F. (1998) Id helix-loop-helix proteins in cell growth and differentiation.Trends Cell Biol.,8,58-65.
5 Ishigum A, Spifin K, Shiohara M, et al. Expression of Id2 and Id3 mRNA Inhuman lymphocytes [J]. Leuk Res 1995:19 (12):989-996
6 Zhao ZR, Zhang ZY, Zhang H, Jiang L, Wang MW, Sun XF. Overexpression of Id-1 protein is a marker in colorectal cancer progression. Oncol Rep.2008; 19 (2):419-24.
7 Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, and etc. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway. Int J Cancer.2009; 125(11):2576-85.
8 Xu B, Sun Y, Tang G, Xu C, Wang L, Zhang Y, and etc. Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor. Cancer Lett.2009; 278(2):220-9.
9 McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells. Mol Cancer Ther.2007; 6(11):2921-7.
10 Li J, Jia H, Xie L, Wang X, He H, Lin Y, Hu L. Correlation of inhibitor of differentiation 1 expression to tumor progression, poor differentiation and aggressive behaviors in cervical carcinoma. Gynecol Oncol.2009; 114(1):89-93.
11 Alani R M, Young A Z, and Shifflett C B. Idl regulation of cellular senescence through transcriptional repression of P16ink4a Proc.Natl. Acad. Sci. USA,2001,98: 7812-7816
12 Ling M T, Wang X, Ouyang X S, et al.Id-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells. Oncogene 2003,22 (29):4498-4508
13 Barns L, Everson JW, Reichart P, et al. Pathology and genetics of head and neck tumors[M]. Lyon:IARC Press,2005:168-175.
14 Garzino-demo P, Dell'Acqua A, Dalmasso P, et al. Clinicopathological parameters and outcome of 245 patients operated for oral squamous cell carcinoma [J]. J Craniomaxillofac Surg,2006,34:344-350.
15 Nishimine M, Nakamura M, Mishima K, Kishi M, Kirita T, Sugimura M, Konishi N. Id proteins are overexpressed in human oral squamous cell carcinomas.J Oral Pathol Med.2003 Jul; 32(6):350-7.
16 Kyats P A, Geleff S,Batistatou A,et al.Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma[J].J Pathol,2005,206(2):170-177.
17 Fire A, Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998,39 1(6669):806-81 1.
18 Tao Peng,Zhang Jun,Tang Ni,et al. Potent specific inhibition of SARS-CoV antigen expression by RNA interference.Chinese Medical Journal 2005,11 8(9):714-719.
19 Yin-cheng Zhang, Xiao-Ming Jin. Oncogenes and tumor suppressor genes in salivary gland tumors. Of Oral Medicine,2000,14:60-62.
20 DAI Yu-cheng, Chen Mao Hang.Apoptosis in the tumor and treatment significance. Cancer,1995,15:279-282.
21 Leicht DT, Balan V,Kaplun A,et al.Raf kinases:function,regulation and role in human cancer.Biochim Biophys Acta,2007,1773:1196-1212.
22 Meister G, Tuschl T. Mechanisms of genes silencing by double-stranded RNA. Nature,2004; 431(7006):343-349.
23 Hannon GJ. RNA interference. Nature,2002; 418 (6894):244-251.
24 Davenport RJ. Gene silencing:a faster way to shut down genes. Science,2001; 292(5521):1469-1471.
25 Lan Chun-Hui, Zhang Xiao Yu, Xiao-Ling. Gastric cancer and precancerous lesions of epidermal growth factor receptor and p21ras protein expression. Chinese Medical Journal,2000,10 (7):17.
26 Ming Xue-Zhi, Hao-Ran Yin, Zheng-gang, et al. Gastric H. ras, and VEGF expression and angiogenesis and its clinical significance. Chinese Journal of Cancer, 2006,13 (5):357-360.
27 Miura S,Matsuo Y,Saku K.Simvastatin suppresses coronary artery endothelial tube formation by disrupting Ras/Raf/ERK signaling.Atherosclerosis,2004,175(2):23 5-243.
28 Zhao L,Lobo S,Dong X,et al.Erf4p and Erf2p form an endoplasmic reticulum-associated complex involved in the plasma membrane localization ofyeast Ras proteins.J Biol Chem,2002,277:49352-49359.
29 Young T,Mei F,Liu J,et al.Proteomics analysis of H-RAS.Mediated oncogenic transformation in a genetically defined human ovarian cancer model. Oncogene,2005, 24:6174-6184.
30 Yoo J, Robinson RA.H-ras gene mutations in salivary gland mucoepidermoid carcinomas.Cancer,2000,88:518-523.
31 Simon AR,Severgnini M,Takahashi S,et al.5-HT induction of c-fos gene expression requires reactive oxygen species and Rac 1 and Ras GTPases. Cell Biochem Biophys,2005,42:263-276.
32 Plaza-Menacho I,van der Sluis T,Hollema H,et al.Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation.J Biol Chem,2007,282:6415-6424.
33 Jayachandran G,Sazaki J,Nishizaki M,et al.Fragile histidine triad-mediated tumor suppression of lung cancer by targeting multiple components of the Ras/Rho GTPase molecular switch.Cancer Res,2007,67:10379-10388.
34 Wang Yong-xiang, Gao Liang, Ji Zongzheng. For the K-ras gene point mutations in antisense oligonucleotide on human pancreatic cancer cell line PC-2 rats. Surgery,2005,43 (21):1387-1390.
35 Zhang YA,Nemunaitis J,Scanlon KJ,et al.Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice. Gene Ther.2000 Dec;7(23):2041-50.
36 Feldkamp MM,Lau N,Roncari L,et al.Isotype-specific Ras-GTP-levels predict the efficacy of famesyl transferase inhibitors against human astrocytomas regardless of Ras mutmional status. Cancer Res,2001,61:4425-4431.
37 Shuai Xiaoming. Mechanism of antisense research. Foreign Medical Sciences. MOLECULAR BIOLOGY,2001,23 (4):237-240.
38 Hou Y.Chromosomal localization of transforming oncogene BGC-Ha-ras in gastric cancer cell line.Zhonghua Zhong Liu Za Zhi,1990,12:95-97.
39 Brummelkamp TR,Bemards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell,2002:243-247.
40 Rubin LL, Philpott KL,Brooks SF.Apoptosis:the cell cycle and cell death.Curr Biol,1993,3:391-394.
41 Maciej Wiznerowicz, Didier Trono.Conditional suppression of cellular genes:lentivirus vector-mediated drug-inducible RNA interference.Journal of virology 2003,77(16):8957-8961.
42 Richard J Fish,Egbert KO Kruithof. Short—term cytotoxic effects and long—term instability of RNAi delivered using lentiviral vectors. BMC Molecular Biology 2004,5:9.
43 Li L, Liu X,Staver M,et al, Evaluating hypoxia-inducible factor-1 alpha as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res,2005; 65(16):7249-7258.
44 Lee TK, Man K, Ling MT, Wang XH, Wong YC, Lo CM, et al. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway. Carcinogenesis 2003; 24(11):1729-36.
45 Lyden D, Young AZ, Zagzaf D, et al. Id-1 and Id-3 are required for neurogenesis, angiofenesis and vascularization of tumour xenografts. Nature,1999, 401:670-677.
46 Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia:role of the HIF system. Nat Med,2003; 9:677-684.
47 Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism. Cancer Res,1996; 56(23):5309-5318.
48 Virials F, Reiriz J, Ambrosio S, Bartrons R, Rosa JL, Ventura F. BMP-2 decreases Mashl stability by increasing Idl expression. EMBO J 2004; 23(17):3527-37.
49 Ouyang XS,Wang X,Ling MT,el al.Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p I 6(INK4a)/Prb pathway.Carcinogenesis,2002,23(5):721-725.
50 Ling MT,Wang X,Ouyang XS,et al.Activation of MAPK signaling pathway is essential for Id-1 induced serum independent prostate cancer cell growth.Oncogene,2002,21(55):8498-8505.
51 Ling MT, Wang X, Ouyang XS, et al.ld-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells.Oncogene, 2003,22(29):4498-4508.
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[7]Desprez PY, Lin CQ, Thomasset N, et al.A novel pathway for mammary epithelial cell invasion induced by the helix-loop- helix protein Id- 1 [J]. Mol Cell Biol, 1998,18 (8):4577-4588.
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[9]Lyden D, Young AZ, Zagzag D, et al. Idl and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts[J]. Nature,1999, 401 (6754):670-677.
[10]Zhao ZR, Zhang ZY, Zhang H, Jiang L, Wang MW, Sun XF. Overexpression of Id-1 protein is a marker in colorectal cancer progression. Oncol Rep.2008; 19 (2):419-24.
[11]Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, and etc. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway. Int J Cancer.2009; 125 (11):2576-85.
[12]Xu B, Sun Y, Tang G, Xu C, Wang L, Zhang Y, and etc. Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor. Cancer Lett.2009; 278 (2):220-9.
[13]McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells. Mol Cancer Ther.2007; 6(11):2921-7.
[14]Li J, Jia H, Xie L, Wang X, He H, Lin Y, Hu L. Correlation of inhibitor of differentiation 1 expression to tumor progression, poor differentiation and aggressive behaviors in cervical carcinoma. Gynecol Oncol.2009;114(1):89-93.
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1 Lister J,Forrester WC. Inhibition ofanerythroid differentiation switch by the helix-loop-helix protein Idl. J Biol Chem,1995,270; 17939-17946.
2 Katagiri T,Imada M,Yanai T.et al,Identification of a BMP-responsive element in Id-1.the gene for inhibition of myogenesis. Genes CellS,2002,7:949-960.
3 Ruzinova M B, Benezra R.Id proteins in development, cell cycle and cancer. Trends Cell Biol 2003,13:410-418.
4 Norton J. D.,Deed R. W.,Craggs G and Sablitzky F. (1998) Id helix-loop-helix proteins in cell growth and differentiation.Trends Cell Biol.,8,58-65.
5 Ishigum A, Spifin K, Shiohara M, et al. Expression of Id2 and Id3 mRNA Inhuman lymphocytes [J]. Leuk Res 1995:19 (12):989-996
6 Zhao ZR, Zhang ZY, Zhang H, Jiang L, Wang MW, Sun XF. Overexpression of Id-1 protein is a marker in colorectal cancer progression. Oncol Rep.2008; 19 (2):419-24.
7 Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, and etc. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway. Int J Cancer.2009; 125(11):2576-85.
8 Xu B, Sun Y, Tang G, Xu C, Wang L, Zhang Y, and etc. Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor. Cancer Lett.2009; 278(2):220-9.
9 McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez PY. Cannabidiol as a novel inhibitor of Id-I gene expression in aggressive breast cancer cells. Mol Cancer Ther.2007; 6(11):2921-7.
10 Li J, Jia H, Xie L, Wang X, He H, Lin Y, Hu L. Correlation of inhibitor of differentiation 1 expression to tumor progression, poor differentiation and aggressive behaviors in cervical carcinoma. Gynecol Oncol.2009; 114(1):89-93.
11 Alani R M, Young A Z, and Shifflett C B. Idl regulation of cellular senescence through transcriptional repression of P16ink4a Proc.Natl. Acad. Sci. USA,2001,98: 7812-7816
12 Ling M T, Wang X, Ouyang X S,et al.Id-1 expression promotes cell survival through activation of NF-kB signalling pathway in prostate cancer cells. Oncogene 2003,22 (29):4498-4508
13 Barns L, Everson JW, Reichart P, et al. Pathology and genetics of head and neck tumors[M]. Lyon:IARC Press,2005:168-175.
14 Garzino-demo P, Dell'Acqua A, Dalmasso P, et al. Clinicopathological parameters and outcome of 245 patients operated for oral squamous cell carcinoma [J]. J Craniomaxillofac Surg,2006,34:344-350.
15 Nishimine M, Nakamura M, Mishima K, Kishi M, Kirita T, Sugimura M, Konishi N. Id proteins are overexpressed in human oral squamous cell carcinomas.J Oral Pathol Med.2003 Jul; 32(6):350-7.
16 KyatsPA.,GeleffS.,Batistatou A,et al. Evidence for lymphangiogenesis and its prognostic implications in head and neck squamous cell carcinoma[J].J Pathol, 2005,206(2):170-177.
17 Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998,39 1(6669):806-81 1.
18 Tao Peng,Zhang Jun,Tang Ni,et al.Potent specific inhibition of SARS-CoV antigen expression by RNA interference.Chinese Medical Journal 2005,118(9): 714-719.
19 Yin-cheng Zhang, Xiao-Ming Jin. Oncogenes and tumor suppressor genes in salivary gland tumors. Of Oral Medicine,2000,14:60-62.
20 DAI Yu-cheng, Chen Mao Hang.Apoptosis in the tumor and treatment significance. Cancer,1995,15:279-282.
21 Leicht DT,Balan V,Kaplun A,et al.Raf kinases:function,regulation and role in human cancer.Biochim Biophys Acta,2007,1773:1196-1212.
22 Meister G, Tuschl T. Mechanisms of genes silencing by double-stranded RNA. Nature,2004; 431(7006):343-349.
23 Hannon GJ. RNA interference. Nature,2002; 418 (6894):244-251.
24 Davenport RJ. Gene silencing:a faster way to shut down genes. Science,2001; 292(5521):1469-1471.
25 Lan Chun-Hui, Zhang Xiao Yu, Xiao-Ling. Gastric cancer and precancerous lesions of epidermal growth factor receptor and p21ras protein expression. Chinese Medical Journal,2000,10 (7):17.
26 Ming Xue-Zhi, Hao-Ran Yin, Zheng-gang, et al. Gastric H. ras, and VEGF expression and angiogenesis and its clinical significance. Chinese Journal of Cancer, 2006,13 (5):357-360.
27 Miura S,Matsuo Y,Saku K.Simvastatin suppresses coronary artery endothelial tube formation by disrupting Ras/Raf/ERK signaling.Atherosclerosis,2004,1 75(2):235-243.
28 Zhao L,Lobo S,Dong X,et al.Erf4p and Erf2p form an endoplasmic reticulum—associated complex involved in the plasma membrane localization ofyeast Ras proteins.J Biol Chem,2002,277:49352-49359.
29 Young T,Mei F,Liu J,et al.Proteomics analysis of H-RAS.Mediated oncogenic transformation in a genetically defined human ovarian cancer model. Oncogene,2005, 24:6174-6184.
30 Yoo J, Robinson RA.H-ras gene mutations in salivary gland mucoepidermoid carcinomas.Cancer,2000,88:518-523.
31 Simon AR,Severgnini M,Takahashi S,et al.5-HT induction of c-fos gene expression requires reactive oxygen species and Rac 1 and Ras GTPases.Cell Biochem Biophys,2005,42:263-276.
32 Plaza-Menacho I,van der Sluis T,Hollema H,et al.Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c—fos promoter activation, cell mitogenicity, and transformation.J Biol Chem,2007,282:6415-6424.
33 Jayachandran QSazaki J,Nishizaki M,et al.Fragile histidine triad-mediated tumor suppression of lung cancer by targeting multiple components of the Ras/Rho GTPase molecular switch.Cancer Res,2007,67:10379-10388.
34 Wang Yong-xiang, Gao Liang, Ji Zongzheng. For the K-ras gene point mutations in antisense oligonucleotide on human pancreatic cancer cell line PC-2 rats. Surgery,2005,43 (21):1387-1390.
35 Zhang YA,Nemunaitis J,Scanlon KJ,et al. Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cell line heterotransplants in nude mice. Gene Ther. 2000 Dec;7(23):2041-50.
36 Feldkamp MM,Lau N,Roncari L,et al.Isotype-specific Ras-GTP-levels predict the efficacy of famesyl transferase inhibitors against human astrocytomas regardless of Ras mutmional status. Cancer Res,2001,61:4425-4431.
37 Shuai Xiaoming. Mechanism of antisense research. Foreign Medical Sciences. MOLECULAR BIOLOGY,2001,23 (4):237-240.
38 Hou Y.Chromosomal localization of transforming oncogene BGC-Ha-ras in gastric cancer cell line.Zhonghua Zhong Liu Za Zhi,1990,12:95-97.
39 Brummelkamp TR,Bemards R,Agami R.Stable suppression of tumorigenicity by virus-mediated RNA interference.Cancer Cell,2002:243-247.
40 Rubin LL,Philpott KL,Brooks SF.Apoptosis:the cell cycle and cell death.Curr Biol,1993,3:391-394.
41 Maciej Wiznerowicz,Didier Trono.Conditional suppression of cellular genes:lentivirus vector-mediated drug—inducible RNA interference.Journal of virology 2003,77(16):8957-8961.
42 Richard J Fish,Egbert KO Kruithof. Short—term cytotoxic effects and long—term instability of RNAi delivered using lentiviral vectors.BMC Molecular Biology 2004,5:9.
43 Li L,Liu X,Staver M,et al, Evaluating hypoxia-inducible factor-1 alpha as a cancer therapeutic target via inducible RNA interference in vivo. Cancer Res,2005; 65(16):7249-7258.
44 Lee TK, Man K, Ling MT, Wang XH, Wong YC, Lo CM, et al. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway. Carcinogenesis 2003; 24(11):1729-36.
45 Lyden D, Young AZ, Zagzaf D, et al. Id-1 and Id-3 are required for neurogenesis, angiofenesis and vascularization of tumour xenografts. Nature,1999, 401:670-677.
46 Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia:role of the HIF system. Nat Med,2003; 9:677-684.
47 Hakomori, S. Tumor malignancy defined by aberrant glycosylation and sphingo (glyco) lipid metabolism. Cancer Res,1996; 56(23):5309-5318.
48 Virials F, Reiriz J, Ambrosio S, Bartrons R, Rosa JL, Ventura F. BMP-2 decreases Mashl stability by increasing Idl expression. EMBO J 2004; 23(17):3527-37.