Id-1基因沉默抑制口腔鳞状细胞癌生长转移的研究
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摘要
研究目的：
1.研究分化抑制因子Id-1在口腔鳞状细胞癌中的表达及意义,初步探讨Id-1在口腔鳞状细胞癌增殖、侵袭、血管新生以及淋巴新生的作用。
2.探讨慢病毒介导的RNAi对舌鳞状细胞癌Tca8113细胞中Id-1基因沉默后对细胞增殖、侵袭以及VEGF-C表达的影响。
3.探讨裸鼠舌癌原位移植瘤瘤内注射Id-1-siRNA-慢病毒颗粒对移植瘤生长以及淋巴新生的影响,并对其机制进行初步探讨。
4.探讨以Id-1基因作为靶基因抑制口腔鳞状细胞癌生长及淋巴转移的可行性。
研究方法：
1.37℃、5%CO2孵箱中培养三种口腔鳞状细胞癌细胞Tca8113舌鳞癌细胞、SAS舌癌细胞以及Buccal885颊癌细胞,当细胞达80～90%融合时,提取三种细胞的总RNA和总蛋白。应用RT-PCR方法检测三种细胞内Id-1mRNA的表达,应用Western-blot方法检测三种细胞内Id-1蛋白的表达。将培养的癌细胞进行细胞爬片处理,通过细胞免疫组化方法检测Id-1蛋白在口腔鳞状细胞癌细胞中的表达及蛋白定位。
2.收集2005-2008年山东大学齐鲁医院口腔颌面外科128例口腔鳞状细胞癌住院患者的肿瘤标本。128例患者按照年龄、性别、是否吸烟、是否饮酒、肿瘤发病部位、肿瘤大小、肿瘤临床分期、肿瘤分化程度、肿瘤有无淋巴转移、肿瘤有无复发分类。所有患者术前均未行放疗、化疗以及其它干预治疗,并同期行颈淋巴清扫术。提取肿瘤标本的总RNA和总蛋白,应用RT-PCR方法检测肿瘤标本内Id-1mRNA的表达,应用Western-blot方法检测肿瘤标本内Id-1蛋白的表达。制备肿瘤标本蜡块,应用免疫组化方法检测Id-1蛋白在口腔鳞状细胞癌中的表达定位,并对免疫组化结果进行评分分析,评价Id-1蛋白的表达与临床资料之间的关系。
3.应用免疫组化方法检测(?)Id-1、VEGF-C蛋白在128例口腔鳞状细胞癌标本中的表达,并以抗体CD34、LYVE-1标记肿瘤内血管和淋巴管。对Id-1、VEGF-C免疫组化结果进行评分分析,测定肿瘤微血管密度及淋巴管密度,评价Id-1蛋白的表达与血管新生、淋巴新生的关系。
4.37℃、5%CO2孵箱中培养舌鳞状细胞癌Tca8113细胞,将生长状态良好的细胞以4×104数目接种于6孔培养板中,每孔加入2ml培养基。48小时后,细胞融合率可达到30～50%。将细胞分为慢病毒实验组、慢病毒阴性对照组、ENi.S.空白对照组,分别加入Id-1-RNAi-Lentivirus(复感染指数MOI值为50)、NC-RFP-Lentivirus(MOI为50)和ENi.S.,37℃、5% CO2孵箱中继续常规培养。
RNA干扰后第五天,收集细胞,提取细胞总RNA,应用RT-PCR方法检测不同实验组中Id-1、VEGF-C的基因表达。RNA干扰后第六天,收集细胞,提取细胞总蛋白,应用Western-blot方法检测不同实验组中Id-1、VEGF-C的蛋白表达。同时收集RNA干扰后第六天细胞上清液,对分泌至细胞外的VEGF-C蛋白进行ELLISA检测。分析三个不同实验组中Tca8113细胞Id-1和VEGF-C的基因、蛋白表达情况。观察Id-1基因沉默后对VEGF-C表达的影响。
应用MTT法检测Id-1基因沉默后1-7天Tca8113细胞的生长情况,并绘制细胞生长曲线,观察Id-1基因沉默对Tca8113细胞增殖活性的影响。
RNA干扰后第六天收集不同实验组中Tca8113细胞,重悬于培养液中,使终浓度为2×106／ml,吸取100ul细胞悬液加入Transwell小室中。应用Transwell小室方法观察Id-1基因沉默对Tca8113细胞侵袭性的影响。
5.裸鼠舌癌肿瘤模型的建立：37℃、5%CO2孵箱中常规培养Tca8113舌鳞癌细胞,在细胞生长状态良好时配制细胞悬液,使细胞浓度达到2×106个／ml,备用。4周龄的BALB/c nu/nu雄性裸鼠,乙醚吸入麻醉后,注射0.1ml Tca8113细胞悬液(细胞数量为2×105个)于裸鼠的舌侧缘粘膜下。接种肿瘤细胞后,将裸鼠置于SPF饲养条件下饲养,每天观察裸鼠舌部粘膜的成瘤情况。
6.当裸鼠舌侧缘形成的肿瘤直径约3～5mm大小时,选择生存状态良好的成瘤裸鼠21只,随机分成慢病毒干扰实验组、慢病毒阴性对照组、ENi.S.空白对照组3个组,每组7只。分别抽取0. lml Id-1-RNAi慢病毒颗粒溶液(MOI值为50)、RFP慢病毒颗粒溶液(MOI值为50)和ENi.S。在裸鼠舌体肿瘤内行多点注射,每周注射两次,连续注射两周。每天观察肿瘤生长情况,3天测量一次肿瘤直径,计算肿瘤体积,探讨肿瘤内注射Id-1-siRNA-慢病毒颗粒后对裸鼠移植瘤生长的影响。
三周后脱颈处死三组裸鼠,收集肿瘤标本。将收集的裸鼠肿瘤部分提取RNA,检测瘤内注射Id-1-siRNA-慢病毒颗粒对Id-1、VEGF-C基因表达的影响。部分标本制备蜡块,以Id-1、VEGF-C、Ki67、LYVE-1为抗体,应用免疫组化方法检测三组裸鼠肿瘤标本中Id-1、VEGF-C、Ki67的表达及淋巴管密度,研究Id-1基因沉默后对裸鼠移植瘤增殖及淋巴新生的影响。
结果：
1.三种口腔鳞状细胞癌细胞中Id-1mRNA以及蛋白均存在过表达,免疫组化结果显示Id-1蛋白既位于细胞浆也位于细胞核。
2.在口腔正常粘膜组织中,Id-1表达阴性或者呈弱表达,而在口腔鳞状细胞癌中,大部分存在过表达,128例肿瘤组织的免疫组化显示Id-1阳性表达率为64.8%。Id-1蛋白的表达与患者的性别、年龄、吸烟、饮酒、肿瘤生长部位、肿瘤分化程度均无关,而与患者肿瘤的大小、临床分期、淋巴转移、肿瘤有无复发有关。在直径大于31mm的肿瘤组织中Id-1蛋白的表达比小于31mm的肿瘤表达强(3.35±1.907vs4.06±2.017,p=0.013)；III、IV期的患者肿瘤组织中Id-1蛋白的表达比I、II期相对强(4.19±1.875vs 3.31±2.054,p=0.031)；在存在淋巴转移的患者组织标本中,Id-1蛋白的表达强度相对强(4.37±1.788vs3.26±2.048,p=0.001)；肿瘤复发的患者其表达强度亦高(4.57±2.03vs3.46±1.96,p=-0.003)。
3.对128例口腔鳞状细胞癌组织中Id-1的表达情况与微血管密度、VEGF-C的表达以及癌周淋巴管密度进行Spearman相关性分析,结果发现Id-1的表达与微血管密度、VEGF-C的表达以及癌周淋巴管密度呈正相关(r=0.223、0.569、0.240,p=-0.011、0.000、0.006)。不同Id-1表达强度之间MVD的表达存在差异,在Id-1中等和强表达的标本中,其MVD明显高于无Id-1表达和Id-1弱表达的标本(40.15±7.72 vs 37.14±6.37,p=0.027)；在Id-1弱表达的组别中,VEGF-C的表达相对较低(1.87±1.30vs3.08±0.76,p=0.000),癌周淋巴管密度也相对较低(13.21±5.53vs16.06±6.16,p=0.012)。
4.应用慢病毒介导的Id-1-siRNA对Tca8113细胞进行Id-1基因沉默,结果发现在实验后第五天RNA干扰组Id-1基因表达明显降低,与对照组相比有显著性差异(p＜0.01)；实验后第六天RNA干扰组Id-1蛋白表达明显降低,与对照组相比有显著性差异(p＜0.01)。Id-1基因沉默后Tca8113细胞的VEGF-C基因表达明显降低(p<0.01)；Western-blot和ELISA检测发现VEGF-C的蛋白表达也明显降低(p＜0.01)
应用MTT法检测Id-1基因沉默后Tca8113细胞增殖活性,结果发现在干扰实验后的前三天,三组Tca8113细胞的增殖活性未见明显差异,第四天开始,干扰组细胞的增殖活性与两对照组相比出现降低,统计学上有显著性差异(4、5、6、7天p=0.036,0.004,0.001,0.002)。
应用Transwell小室方法观察Id-1基因沉默对Tca8113细胞侵袭性的影响,结果发现干扰组穿过滤膜的Tca8113细胞数量明显减少(p＜0.01),而两对照组无明显差异。
5.肿瘤细胞注射后,开始是在舌粘膜下形成水肿,4天后即可在裸鼠舌粘膜下触及肿瘤硬结。肿瘤持续生长,开始呈圆形或者椭圆形,后来可呈结节状。大约在10天左右,裸鼠舌侧缘即可形成直径约3～5mm大小的肿块。与皮下注射成瘤相比,舌体形成的肿瘤体积相对小,生长较缓慢。
6.裸鼠移植瘤瘤内注射Id-1-RNAi-慢病毒后肿瘤继续生长,各组肿瘤体积无明显差异,但Id-1-RNAi-慢病毒组生长相对较慢,12天后各组裸鼠移植瘤的体积出现差异,Id-1-RNAi-慢病毒组肿瘤体积相对较小(p＜0.01),免疫组化显示肿瘤增殖指数(PI)也降低(p＜0.01)。
Id-1-RNAi-慢病毒组裸鼠移植瘤中Id-1的基因和蛋白表达均降低,与慢病毒阴性对照组、ENi.S.空白对照组相比有显著性差异(p＜0.01,p＜0.01)。Id-1-RNAi-慢病毒组裸鼠移植瘤中VEGF-C的表达也相应降低,与慢病毒阴性对照组、ENi.S.空白对照组相比有显著性差异(p＜0.01,p＜0.05)。
裸鼠移植瘤的淋巴管主要位于癌周,淋巴管管腔开放,扩张的淋巴管内有时可见瘤栓形成。Id-1-RNAi-慢病毒组裸鼠移植瘤中淋巴管密度减少,与空白慢病毒对照组、ENi.S.阴性对照组相比有显著性差异(p<0.05)。
结论：
1.Id-1在口腔鳞状细胞癌中存在异常表达,其表达水平与肿瘤的大小、临床分期、淋巴转移、复发有关,与口腔鳞状细胞癌中的血管新生、淋巴新生有关。Id-1是评价口腔鳞状细胞癌预后的一个重要因子。
2.Id-1-RNAi-慢病毒可以明显抑制Tca8113细胞Id-1的表达,Id-1基因沉默后Tca8113细胞增殖能力、侵袭性明显降低,此外Id-1基因沉默后VEGF-C的表达明显降低,VEGF—C促进肿瘤淋巴新生能力降低。Id-1可以作为口腔鳞状细胞癌基因治疗的有效靶基因。
3.研究口腔鳞状细胞癌的基因治疗,特别是研究基因治疗对肿瘤淋巴转移的影响时,构建裸鼠原位移植瘤是理想的肿瘤动物模型。
4.裸鼠原位移植瘤瘤内注射Id-1-RNAi-慢病毒能够抑制裸鼠原位移植瘤的生长,并且能够抑制肿瘤的淋巴新生,其作用机制是Id-1基因沉默后,促进淋巴新生的因子-VEGF-C的表达受到抑制。
5.Id-1是基因治疗口腔鳞状细胞癌特别是治疗口腔鳞状细胞癌淋巴转移的理想的靶基因。
研究意义：
本研究明确了Id-1在口腔鳞状细胞癌中存在异常表达,其表达与肿瘤的生长、转移及不良预后有关,并首次分析了Id-1与血管新生及淋巴新生的关系。此外,应用慢病毒介导的RNAi技术靶向沉默Id-1基因能抑制Tca8113细胞的生长、增殖和侵袭性,使Id-1成为口腔鳞状细胞癌基因治疗的理想的靶基因。而建立裸鼠舌癌原位移植瘤模型在研究口腔癌淋巴转移方面具有重要的意义。通过瘤内注射Id-1-siRNA,不仅验证了应用该方法进行RNAi的实用性和有效性,还证实了沉默Id-1基因对移植瘤生长和淋巴新生的抑制作用。本研究第一次提出了Id-1与淋巴新生的关系,为口腔鳞状细胞癌的基因治疗提供了新的思路,具有重要的意义。
Objective:
1. To study the expression of Id-1 in oral squamous cell carcinoma and its correlation with various clinicopathologic parameters, then to investigate the effect of Id-1 overexpression on tumor proliferation, invasion, angiogenesis and lymphangiogenesis.
2. To investigate the effect of lentivirus-mediated Id-1 RNAi on cell growth, invasion and VEGF-C expression in Tca8113 cells.
3. To investigate the effect of lentivirus-mediated Id-1 RNAi on growth and lymphangiogenesis of human oral squamous cell carcinoma in BALB/c nude mice model.
4. To investigate the significance of gene silencing targeting Id-1 in the treatment of lymphatic metastasis of oral squamous cell carcinoma.
Methods:
1. Three cell lines of oral squamous cell carcinoma, including Tca8113 cell, SAS cell and Bucca1885 cell, were cultivated in the condition of 37℃,5% CO2.The gross RNA and proteins were extracted from the cells when they got 80-90% convergence. The level of Id-1 mRNA was tested by RT-PCR and the concentration of Id-1 protein was determined by Western-blot. The localization of Id-1 protein was examined by immunohistochemistry.
2. The tumor samples were collected from 128 patients with OSCC who were treated at Qilu Hospital of Shandong University from 2004 through 2008. The specimens of nonneoplastic oral mucosa were collected as control samples. All of the patients received local tumor resection and synchronal neck dissection. Informed consent was obtained from all patients. Clinicopathological parameters including the patients'age, sex, tumor size, tumor location, clinical stage, histological grade, lymphatic metastasis and tumor recurrence were summarized. The level of Id-1 mRNA in the tumor samples was tested by RT-PCR and the concentration of Id-1 protein was determined by Western-blot. The score of Id-1 expression was assessed based on the immunohistochemical examination. The correlation between the expression of Id-1 and various clinicopathologic parameters was investigated.
3. The microvessel and lymphatic vessel were marked with antibodies of CD34 and LYVE-1, and the numbers were counted. Spearman'coefficient of correlation was used to assess the correlation between Id-1 expression and MVD、LVD. Meanwhile, VEGF-C protein was also tested by immunohistochemistry and the association between the expression of Id-1 and VEGF-C was analysized.
4. The cells were cultivated in the condition of 37℃,5% CO2. Inoculating Tca8113 cells with the number of 4×104 in cultivate plate. The test was divided into three groups:Id-1-RNAi-Lentivirus group, NC-RFP-Lentivirus group and ENi.S.-control group. Each group was added with Id-1-RNAi-Lentivirus (MOI was 50), NC-RFP-Lentivirus and ENi.S., respectively. The level of Id-1, VEGF-C mRNA in cells was tested by Real Time PCR in the fifth day, and the concentration of Id-1 VEGF-C protein was determined by Western-blot in the sixth day. The concentration of VEGF-C protein in the supernate of the cells was tested by ELLISA.
Cell growth of Tca8113 was tested by MTT after silencing gene of Id-1. The growth curve was draw to assess Tca8113 cell proliferation activity. The invasion of the Tca8113 cells was assessed by Boyden chamber invasion assays.
5. Establishment of nude mice tumor model. Male BALB/c nu/nu nude mice of four weeks old was injected submucously with 0.1ml Tca8113 cells (cell number:2×105) under the anesthesia with aether. The nude mice were breed under SPF condition. The neoplasma was observed everyday.
6. When the diameter of the neoplasma was about 3～5mm,21 nude mice were divided into three groups randomly. The Id-1-RNAi-Lentivirus、NC-RFP-Lentivirus and ENi.S. was injected intratumorally respectively. The diameter of the neoplasma was measured every three days and the volume was calculated. Three weeks later, the nude mice were executed and the tumors were collected. The mRNA level of Id-1 and VEGF-C was examined by Real Time PCR. Immunohistochemistry was performed using the antibodies of Id-1、VEGF-C、Ki67 and LYVE-1. Results: 1. Id-1 was overexpressed in three cells of oral squamous cell carcinoma. The localization of Id-1 protein was found in both cytoplasm and nucleus of tumor cells.
2. We found that in nonneoplastic oral tissues, Id-1 was either absent or weakly expressed. However, in OSCC, Id-1 expression was dysregulated. Among those 128 cases,21 (16.4%) were negative,24 (18.8%) were weakly expressed,64 (50.0%) were moderately expressed, and 19 (14.8%) were strongly expressed. The localization of Id-1 protein was found in both cytoplasm and nucleus of tumor cells.
No significant correlation between Id-1 expression and patients'age, sex, smoking, drinking, tumor location and histological grade was found. While there was significant correlation between Id-1 expression and tumor size (p=0.013), Id-1 expression was significantly higher in tumors with diameter larger than 31mm (4.06±2.02 vs.3.35±1.91). Tumors in stage 3 and 4 also presented higher Id-1 expression than tumors in stagel and 2(4.19±2.02 vs.3.31±2.05, p=0.031). Furthermore, there was significant correlation between Id-1 expression and lymph node involvement (p=0.001), Id-1 expression was significantly stronger in cases with lymph node metastasis compared with those without lymph node metastasis (4.37±1.79 vs.3.26±2.05). And there was significant difference between Id-1 expression and tumor recurrence (4.57±2.03 v.s.3.46±.96,p=0.003).
3.Tumors with higher Id-1 expression (moderate and strong) also showed significantly higher MVD、VEGF-C expression and LVD than tumors with lower Id-1 expression (negative and weak) (p=0.027,0.000,0.012). There was significant correlation between Id-1 expression and MVD、VEGF-C expression and LVD (r=0.223、0.569、0.240, p=0.011、0.000、0.006).
4.The gene expression of Id-1 decreased five days later after RNAi and the protein expression decreased six days later after RNAi (p<0.01). Meanwhile, the levels of VEGF-C mRNA and protein were both down-regulated (p＜0.01).
The cell proliferation of Tca8113 cells degraded four days later after RNAi (p<0.01), while there were no changes of the other two groups. The invasiveness of Tca8113 cells in the group transfected with Id-1 siRNA decreased dramatically in comparison to the other two groups (p<0.01).
5. The hydropsia formed first after injecting Tca8113 cells. A node could be touched four days later. The tumor grew continuously, round or ellipse shaped. Ten days later a neoplasma formed in a diameter about 3-5mm. Compared to the neoplasma formed subcutaneously, the neoplasma formed submucously grew slowly and the volume was smaller.
6. The neoplasma grew continuously after injecting intratumorally with Id-1-RNAi-Lentivirus, NC-RFP-Lentivirus and ENi.S.. Compared to the neoplasma of the other two groups, the neoplasma injected with Id-1-RNAi-Lentivirus grew slowly and its volume was smaller (p＜0.01), the PI also decreased(p<0.01).
The gene and protein expressions of Id-1 decreased in the tumors injectsd with Id-1-RNAi-Lentivirus (p＜0.01, p＜0.01). Meanwhile, the levels of VEGF-C mRNA and protein were both down-regulated (p＜0.01, p<0.05).
The lymph vessel mostly located in the pericancer, the lymphatic vessel was open and sometimes there was tumor embolus in it. The LVD of the tumor in the group injected with Id-1-RNAi-Lentivirus decreased dramatically in comparison to the other two groups (p<0.05).
Conclusion:
1. Id-1 was overexpressed in OSCC, its expression was associated with tumor size, lymphatic metastasis, tumor stage, tumor recurrence, tumor angiogenesis and lymphangiogenesis. Id-1 might be a novel prognostic factor for OSCC.
2. Id-1-RNAi-Lentivirus could inhibit the expression of Id-1 in Tca8113 cells. The proliferation activity and invasion decreased after RNAi. The expression of VEGF-C also decreased, which meant that Id-1 was associated with lymphangiogenesis. Inactivation of Id-1 gene might be a promising strategy for OSCC.
3. The orthotopic transplantation tumor model was an ideal animal tumor model for the study of gene therapy of OSCC, especially in the treatment of tumor lymphatic metastasis.
4. Intratumorally injecting Id-1-RNAi-Lentivirus could inhibit the growth and lymphangiogenesis of the orthotopic transplantation tumor, its mechanism might be the lower expression of VEGF-C.
5. Id-1 was an ideal target gene for gene therapy, especially in the treatment of tumor lymphatic metastasis of OSCC.
Significance:
We identified the significance of Id-1 overexpression in OSCC. Silencing Id-1 could inhibit cell growth, invasion and VEGF-C expression in Tca8113 cells. Furthermore, intratumorally injecting Id-1-RNAi-Lentivirus could inhibit the growth and lymphangiogenesis of orthotopic transplantation tumor. We first showed the association between Id-1 and lymphangiogenesis.We thought that Id-1 was an ideal target gene for gene therapy, especially in the treatment of tumor lymphatic metastasis of OSCC.
1.研究分化抑制因子Id-1在口腔鳞状细胞癌中的表达及意义,初步探讨Id-1在口腔鳞状细胞癌增殖、侵袭、血管新生以及淋巴新生的作用。
2.探讨慢病毒介导的RNAi对舌鳞状细胞癌Tca8113细胞中Id-1基因沉默后对细胞增殖、侵袭以及VEGF-C表达的影响。
3.探讨裸鼠舌癌原位移植瘤瘤内注射Id-1-siRNA-慢病毒颗粒对移植瘤生长以及淋巴新生的影响,并对其机制进行初步探讨。
4.探讨以Id-1基因作为靶基因抑制口腔鳞状细胞癌生长及淋巴转移的可行性。
研究方法：
1.37℃、5%CO2孵箱中培养三种口腔鳞状细胞癌细胞Tca8113舌鳞癌细胞、SAS舌癌细胞以及Buccal885颊癌细胞,当细胞达80～90%融合时,提取三种细胞的总RNA和总蛋白。应用RT-PCR方法检测三种细胞内Id-1mRNA的表达,应用Western-blot方法检测三种细胞内Id-1蛋白的表达。将培养的癌细胞进行细胞爬片处理,通过细胞免疫组化方法检测Id-1蛋白在口腔鳞状细胞癌细胞中的表达及蛋白定位。
2.收集2005-2008年山东大学齐鲁医院口腔颌面外科128例口腔鳞状细胞癌住院患者的肿瘤标本。128例患者按照年龄、性别、是否吸烟、是否饮酒、肿瘤发病部位、肿瘤大小、肿瘤临床分期、肿瘤分化程度、肿瘤有无淋巴转移、肿瘤有无复发分类。所有患者术前均未行放疗、化疗以及其它干预治疗,并同期行颈淋巴清扫术。提取肿瘤标本的总RNA和总蛋白,应用RT-PCR方法检测肿瘤标本内Id-1mRNA的表达,应用Western-blot方法检测肿瘤标本内Id-1蛋白的表达。制备肿瘤标本蜡块,应用免疫组化方法检测Id-1蛋白在口腔鳞状细胞癌中的表达定位,并对免疫组化结果进行评分分析,评价Id-1蛋白的表达与临床资料之间的关系。
3.应用免疫组化方法检测(?)Id-1、VEGF-C蛋白在128例口腔鳞状细胞癌标本中的表达,并以抗体CD34、LYVE-1标记肿瘤内血管和淋巴管。对Id-1、VEGF-C免疫组化结果进行评分分析,测定肿瘤微血管密度及淋巴管密度,评价Id-1蛋白的表达与血管新生、淋巴新生的关系。
4.37℃、5%CO2孵箱中培养舌鳞状细胞癌Tca8113细胞,将生长状态良好的细胞以4×104数目接种于6孔培养板中,每孔加入2ml培养基。48小时后,细胞融合率可达到30～50%。将细胞分为慢病毒实验组、慢病毒阴性对照组、ENi.S.空白对照组,分别加入Id-1-RNAi-Lentivirus(复感染指数MOI值为50)、NC-RFP-Lentivirus(MOI为50)和ENi.S.,37℃、5% CO2孵箱中继续常规培养。
RNA干扰后第五天,收集细胞,提取细胞总RNA,应用RT-PCR方法检测不同实验组中Id-1、VEGF-C的基因表达。RNA干扰后第六天,收集细胞,提取细胞总蛋白,应用Western-blot方法检测不同实验组中Id-1、VEGF-C的蛋白表达。同时收集RNA干扰后第六天细胞上清液,对分泌至细胞外的VEGF-C蛋白进行ELLISA检测。分析三个不同实验组中Tca8113细胞Id-1和VEGF-C的基因、蛋白表达情况。观察Id-1基因沉默后对VEGF-C表达的影响。
应用MTT法检测Id-1基因沉默后1-7天Tca8113细胞的生长情况,并绘制细胞生长曲线,观察Id-1基因沉默对Tca8113细胞增殖活性的影响。
RNA干扰后第六天收集不同实验组中Tca8113细胞,重悬于培养液中,使终浓度为2×106／ml,吸取100ul细胞悬液加入Transwell小室中。应用Transwell小室方法观察Id-1基因沉默对Tca8113细胞侵袭性的影响。
5.裸鼠舌癌肿瘤模型的建立：37℃、5%CO2孵箱中常规培养Tca8113舌鳞癌细胞,在细胞生长状态良好时配制细胞悬液,使细胞浓度达到2×106个／ml,备用。4周龄的BALB/c nu/nu雄性裸鼠,乙醚吸入麻醉后,注射0.1ml Tca8113细胞悬液(细胞数量为2×105个)于裸鼠的舌侧缘粘膜下。接种肿瘤细胞后,将裸鼠置于SPF饲养条件下饲养,每天观察裸鼠舌部粘膜的成瘤情况。
6.当裸鼠舌侧缘形成的肿瘤直径约3～5mm大小时,选择生存状态良好的成瘤裸鼠21只,随机分成慢病毒干扰实验组、慢病毒阴性对照组、ENi.S.空白对照组3个组,每组7只。分别抽取0. lml Id-1-RNAi慢病毒颗粒溶液(MOI值为50)、RFP慢病毒颗粒溶液(MOI值为50)和ENi.S。在裸鼠舌体肿瘤内行多点注射,每周注射两次,连续注射两周。每天观察肿瘤生长情况,3天测量一次肿瘤直径,计算肿瘤体积,探讨肿瘤内注射Id-1-siRNA-慢病毒颗粒后对裸鼠移植瘤生长的影响。
三周后脱颈处死三组裸鼠,收集肿瘤标本。将收集的裸鼠肿瘤部分提取RNA,检测瘤内注射Id-1-siRNA-慢病毒颗粒对Id-1、VEGF-C基因表达的影响。部分标本制备蜡块,以Id-1、VEGF-C、Ki67、LYVE-1为抗体,应用免疫组化方法检测三组裸鼠肿瘤标本中Id-1、VEGF-C、Ki67的表达及淋巴管密度,研究Id-1基因沉默后对裸鼠移植瘤增殖及淋巴新生的影响。
结果：
1.三种口腔鳞状细胞癌细胞中Id-1mRNA以及蛋白均存在过表达,免疫组化结果显示Id-1蛋白既位于细胞浆也位于细胞核。
2.在口腔正常粘膜组织中,Id-1表达阴性或者呈弱表达,而在口腔鳞状细胞癌中,大部分存在过表达,128例肿瘤组织的免疫组化显示Id-1阳性表达率为64.8%。Id-1蛋白的表达与患者的性别、年龄、吸烟、饮酒、肿瘤生长部位、肿瘤分化程度均无关,而与患者肿瘤的大小、临床分期、淋巴转移、肿瘤有无复发有关。在直径大于31mm的肿瘤组织中Id-1蛋白的表达比小于31mm的肿瘤表达强(3.35±1.907vs4.06±2.017,p=0.013)；III、IV期的患者肿瘤组织中Id-1蛋白的表达比I、II期相对强(4.19±1.875vs 3.31±2.054,p=0.031)；在存在淋巴转移的患者组织标本中,Id-1蛋白的表达强度相对强(4.37±1.788vs3.26±2.048,p=0.001)；肿瘤复发的患者其表达强度亦高(4.57±2.03vs3.46±1.96,p=-0.003)。
3.对128例口腔鳞状细胞癌组织中Id-1的表达情况与微血管密度、VEGF-C的表达以及癌周淋巴管密度进行Spearman相关性分析,结果发现Id-1的表达与微血管密度、VEGF-C的表达以及癌周淋巴管密度呈正相关(r=0.223、0.569、0.240,p=-0.011、0.000、0.006)。不同Id-1表达强度之间MVD的表达存在差异,在Id-1中等和强表达的标本中,其MVD明显高于无Id-1表达和Id-1弱表达的标本(40.15±7.72 vs 37.14±6.37,p=0.027)；在Id-1弱表达的组别中,VEGF-C的表达相对较低(1.87±1.30vs3.08±0.76,p=0.000),癌周淋巴管密度也相对较低(13.21±5.53vs16.06±6.16,p=0.012)。
4.应用慢病毒介导的Id-1-siRNA对Tca8113细胞进行Id-1基因沉默,结果发现在实验后第五天RNA干扰组Id-1基因表达明显降低,与对照组相比有显著性差异(p＜0.01)；实验后第六天RNA干扰组Id-1蛋白表达明显降低,与对照组相比有显著性差异(p＜0.01)。Id-1基因沉默后Tca8113细胞的VEGF-C基因表达明显降低(p<0.01)；Western-blot和ELISA检测发现VEGF-C的蛋白表达也明显降低(p＜0.01)
应用MTT法检测Id-1基因沉默后Tca8113细胞增殖活性,结果发现在干扰实验后的前三天,三组Tca8113细胞的增殖活性未见明显差异,第四天开始,干扰组细胞的增殖活性与两对照组相比出现降低,统计学上有显著性差异(4、5、6、7天p=0.036,0.004,0.001,0.002)。
应用Transwell小室方法观察Id-1基因沉默对Tca8113细胞侵袭性的影响,结果发现干扰组穿过滤膜的Tca8113细胞数量明显减少(p＜0.01),而两对照组无明显差异。
5.肿瘤细胞注射后,开始是在舌粘膜下形成水肿,4天后即可在裸鼠舌粘膜下触及肿瘤硬结。肿瘤持续生长,开始呈圆形或者椭圆形,后来可呈结节状。大约在10天左右,裸鼠舌侧缘即可形成直径约3～5mm大小的肿块。与皮下注射成瘤相比,舌体形成的肿瘤体积相对小,生长较缓慢。
6.裸鼠移植瘤瘤内注射Id-1-RNAi-慢病毒后肿瘤继续生长,各组肿瘤体积无明显差异,但Id-1-RNAi-慢病毒组生长相对较慢,12天后各组裸鼠移植瘤的体积出现差异,Id-1-RNAi-慢病毒组肿瘤体积相对较小(p＜0.01),免疫组化显示肿瘤增殖指数(PI)也降低(p＜0.01)。
Id-1-RNAi-慢病毒组裸鼠移植瘤中Id-1的基因和蛋白表达均降低,与慢病毒阴性对照组、ENi.S.空白对照组相比有显著性差异(p＜0.01,p＜0.01)。Id-1-RNAi-慢病毒组裸鼠移植瘤中VEGF-C的表达也相应降低,与慢病毒阴性对照组、ENi.S.空白对照组相比有显著性差异(p＜0.01,p＜0.05)。
裸鼠移植瘤的淋巴管主要位于癌周,淋巴管管腔开放,扩张的淋巴管内有时可见瘤栓形成。Id-1-RNAi-慢病毒组裸鼠移植瘤中淋巴管密度减少,与空白慢病毒对照组、ENi.S.阴性对照组相比有显著性差异(p<0.05)。
结论：
1.Id-1在口腔鳞状细胞癌中存在异常表达,其表达水平与肿瘤的大小、临床分期、淋巴转移、复发有关,与口腔鳞状细胞癌中的血管新生、淋巴新生有关。Id-1是评价口腔鳞状细胞癌预后的一个重要因子。
2.Id-1-RNAi-慢病毒可以明显抑制Tca8113细胞Id-1的表达,Id-1基因沉默后Tca8113细胞增殖能力、侵袭性明显降低,此外Id-1基因沉默后VEGF-C的表达明显降低,VEGF—C促进肿瘤淋巴新生能力降低。Id-1可以作为口腔鳞状细胞癌基因治疗的有效靶基因。
3.研究口腔鳞状细胞癌的基因治疗,特别是研究基因治疗对肿瘤淋巴转移的影响时,构建裸鼠原位移植瘤是理想的肿瘤动物模型。
4.裸鼠原位移植瘤瘤内注射Id-1-RNAi-慢病毒能够抑制裸鼠原位移植瘤的生长,并且能够抑制肿瘤的淋巴新生,其作用机制是Id-1基因沉默后,促进淋巴新生的因子-VEGF-C的表达受到抑制。
5.Id-1是基因治疗口腔鳞状细胞癌特别是治疗口腔鳞状细胞癌淋巴转移的理想的靶基因。
研究意义：
本研究明确了Id-1在口腔鳞状细胞癌中存在异常表达,其表达与肿瘤的生长、转移及不良预后有关,并首次分析了Id-1与血管新生及淋巴新生的关系。此外,应用慢病毒介导的RNAi技术靶向沉默Id-1基因能抑制Tca8113细胞的生长、增殖和侵袭性,使Id-1成为口腔鳞状细胞癌基因治疗的理想的靶基因。而建立裸鼠舌癌原位移植瘤模型在研究口腔癌淋巴转移方面具有重要的意义。通过瘤内注射Id-1-siRNA,不仅验证了应用该方法进行RNAi的实用性和有效性,还证实了沉默Id-1基因对移植瘤生长和淋巴新生的抑制作用。本研究第一次提出了Id-1与淋巴新生的关系,为口腔鳞状细胞癌的基因治疗提供了新的思路,具有重要的意义。
Objective:
1. To study the expression of Id-1 in oral squamous cell carcinoma and its correlation with various clinicopathologic parameters, then to investigate the effect of Id-1 overexpression on tumor proliferation, invasion, angiogenesis and lymphangiogenesis.
2. To investigate the effect of lentivirus-mediated Id-1 RNAi on cell growth, invasion and VEGF-C expression in Tca8113 cells.
3. To investigate the effect of lentivirus-mediated Id-1 RNAi on growth and lymphangiogenesis of human oral squamous cell carcinoma in BALB/c nude mice model.
4. To investigate the significance of gene silencing targeting Id-1 in the treatment of lymphatic metastasis of oral squamous cell carcinoma.
Methods:
1. Three cell lines of oral squamous cell carcinoma, including Tca8113 cell, SAS cell and Bucca1885 cell, were cultivated in the condition of 37℃,5% CO2.The gross RNA and proteins were extracted from the cells when they got 80-90% convergence. The level of Id-1 mRNA was tested by RT-PCR and the concentration of Id-1 protein was determined by Western-blot. The localization of Id-1 protein was examined by immunohistochemistry.
2. The tumor samples were collected from 128 patients with OSCC who were treated at Qilu Hospital of Shandong University from 2004 through 2008. The specimens of nonneoplastic oral mucosa were collected as control samples. All of the patients received local tumor resection and synchronal neck dissection. Informed consent was obtained from all patients. Clinicopathological parameters including the patients'age, sex, tumor size, tumor location, clinical stage, histological grade, lymphatic metastasis and tumor recurrence were summarized. The level of Id-1 mRNA in the tumor samples was tested by RT-PCR and the concentration of Id-1 protein was determined by Western-blot. The score of Id-1 expression was assessed based on the immunohistochemical examination. The correlation between the expression of Id-1 and various clinicopathologic parameters was investigated.
3. The microvessel and lymphatic vessel were marked with antibodies of CD34 and LYVE-1, and the numbers were counted. Spearman'coefficient of correlation was used to assess the correlation between Id-1 expression and MVD、LVD. Meanwhile, VEGF-C protein was also tested by immunohistochemistry and the association between the expression of Id-1 and VEGF-C was analysized.
4. The cells were cultivated in the condition of 37℃,5% CO2. Inoculating Tca8113 cells with the number of 4×104 in cultivate plate. The test was divided into three groups:Id-1-RNAi-Lentivirus group, NC-RFP-Lentivirus group and ENi.S.-control group. Each group was added with Id-1-RNAi-Lentivirus (MOI was 50), NC-RFP-Lentivirus and ENi.S., respectively. The level of Id-1, VEGF-C mRNA in cells was tested by Real Time PCR in the fifth day, and the concentration of Id-1 VEGF-C protein was determined by Western-blot in the sixth day. The concentration of VEGF-C protein in the supernate of the cells was tested by ELLISA.
Cell growth of Tca8113 was tested by MTT after silencing gene of Id-1. The growth curve was draw to assess Tca8113 cell proliferation activity. The invasion of the Tca8113 cells was assessed by Boyden chamber invasion assays.
5. Establishment of nude mice tumor model. Male BALB/c nu/nu nude mice of four weeks old was injected submucously with 0.1ml Tca8113 cells (cell number:2×105) under the anesthesia with aether. The nude mice were breed under SPF condition. The neoplasma was observed everyday.
6. When the diameter of the neoplasma was about 3～5mm,21 nude mice were divided into three groups randomly. The Id-1-RNAi-Lentivirus、NC-RFP-Lentivirus and ENi.S. was injected intratumorally respectively. The diameter of the neoplasma was measured every three days and the volume was calculated. Three weeks later, the nude mice were executed and the tumors were collected. The mRNA level of Id-1 and VEGF-C was examined by Real Time PCR. Immunohistochemistry was performed using the antibodies of Id-1、VEGF-C、Ki67 and LYVE-1. Results: 1. Id-1 was overexpressed in three cells of oral squamous cell carcinoma. The localization of Id-1 protein was found in both cytoplasm and nucleus of tumor cells.
2. We found that in nonneoplastic oral tissues, Id-1 was either absent or weakly expressed. However, in OSCC, Id-1 expression was dysregulated. Among those 128 cases,21 (16.4%) were negative,24 (18.8%) were weakly expressed,64 (50.0%) were moderately expressed, and 19 (14.8%) were strongly expressed. The localization of Id-1 protein was found in both cytoplasm and nucleus of tumor cells.
No significant correlation between Id-1 expression and patients'age, sex, smoking, drinking, tumor location and histological grade was found. While there was significant correlation between Id-1 expression and tumor size (p=0.013), Id-1 expression was significantly higher in tumors with diameter larger than 31mm (4.06±2.02 vs.3.35±1.91). Tumors in stage 3 and 4 also presented higher Id-1 expression than tumors in stagel and 2(4.19±2.02 vs.3.31±2.05, p=0.031). Furthermore, there was significant correlation between Id-1 expression and lymph node involvement (p=0.001), Id-1 expression was significantly stronger in cases with lymph node metastasis compared with those without lymph node metastasis (4.37±1.79 vs.3.26±2.05). And there was significant difference between Id-1 expression and tumor recurrence (4.57±2.03 v.s.3.46±.96,p=0.003).
3.Tumors with higher Id-1 expression (moderate and strong) also showed significantly higher MVD、VEGF-C expression and LVD than tumors with lower Id-1 expression (negative and weak) (p=0.027,0.000,0.012). There was significant correlation between Id-1 expression and MVD、VEGF-C expression and LVD (r=0.223、0.569、0.240, p=0.011、0.000、0.006).
4.The gene expression of Id-1 decreased five days later after RNAi and the protein expression decreased six days later after RNAi (p<0.01). Meanwhile, the levels of VEGF-C mRNA and protein were both down-regulated (p＜0.01).
The cell proliferation of Tca8113 cells degraded four days later after RNAi (p<0.01), while there were no changes of the other two groups. The invasiveness of Tca8113 cells in the group transfected with Id-1 siRNA decreased dramatically in comparison to the other two groups (p<0.01).
5. The hydropsia formed first after injecting Tca8113 cells. A node could be touched four days later. The tumor grew continuously, round or ellipse shaped. Ten days later a neoplasma formed in a diameter about 3-5mm. Compared to the neoplasma formed subcutaneously, the neoplasma formed submucously grew slowly and the volume was smaller.
6. The neoplasma grew continuously after injecting intratumorally with Id-1-RNAi-Lentivirus, NC-RFP-Lentivirus and ENi.S.. Compared to the neoplasma of the other two groups, the neoplasma injected with Id-1-RNAi-Lentivirus grew slowly and its volume was smaller (p＜0.01), the PI also decreased(p<0.01).
The gene and protein expressions of Id-1 decreased in the tumors injectsd with Id-1-RNAi-Lentivirus (p＜0.01, p＜0.01). Meanwhile, the levels of VEGF-C mRNA and protein were both down-regulated (p＜0.01, p<0.05).
The lymph vessel mostly located in the pericancer, the lymphatic vessel was open and sometimes there was tumor embolus in it. The LVD of the tumor in the group injected with Id-1-RNAi-Lentivirus decreased dramatically in comparison to the other two groups (p<0.05).
Conclusion:
1. Id-1 was overexpressed in OSCC, its expression was associated with tumor size, lymphatic metastasis, tumor stage, tumor recurrence, tumor angiogenesis and lymphangiogenesis. Id-1 might be a novel prognostic factor for OSCC.
2. Id-1-RNAi-Lentivirus could inhibit the expression of Id-1 in Tca8113 cells. The proliferation activity and invasion decreased after RNAi. The expression of VEGF-C also decreased, which meant that Id-1 was associated with lymphangiogenesis. Inactivation of Id-1 gene might be a promising strategy for OSCC.
3. The orthotopic transplantation tumor model was an ideal animal tumor model for the study of gene therapy of OSCC, especially in the treatment of tumor lymphatic metastasis.
4. Intratumorally injecting Id-1-RNAi-Lentivirus could inhibit the growth and lymphangiogenesis of the orthotopic transplantation tumor, its mechanism might be the lower expression of VEGF-C.
5. Id-1 was an ideal target gene for gene therapy, especially in the treatment of tumor lymphatic metastasis of OSCC.
Significance:
We identified the significance of Id-1 overexpression in OSCC. Silencing Id-1 could inhibit cell growth, invasion and VEGF-C expression in Tca8113 cells. Furthermore, intratumorally injecting Id-1-RNAi-Lentivirus could inhibit the growth and lymphangiogenesis of orthotopic transplantation tumor. We first showed the association between Id-1 and lymphangiogenesis.We thought that Id-1 was an ideal target gene for gene therapy, especially in the treatment of tumor lymphatic metastasis of OSCC.
引文
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