Wnt5a对非小细胞肺癌细胞增殖及侵袭潜能的影响
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摘要
研究背景
肺癌是全球发病率最高的恶性肿瘤,己成为人类因癌症死亡的主要原因。非小细胞肺癌(NSCLC)占新发肺癌病例的75-80%,虽然各种治疗手段不断改善,但其预后依然很差。其主要原因在于肺癌的发病机制、特别是肺癌侵袭转移的机制尚不清楚,弄清肺癌的发病及侵袭转移机制对于预测肺癌患者的预后及提高治疗效果意义重大。
Wnt5a是Wnt家族中的重要成员之一,既能活化经典的Wnt/β-catenin通路又能活化非经典的Wnt/Ca2+通路,而且Wnt/Ca2+通路与Wnt/β-catenin通路相互作用。文献报道Wnt5a与多种恶性肿瘤关系密切,在肿瘤细胞的增殖、分化及侵袭转移过程中起着重要的作用。但也有研究发现Wnt5a在不同的恶性肿瘤中生物学作用并不一致,有的认识尚存在重大分歧。部分研究认为Wnt5a具有癌基因的特性,在多种恶性肿瘤如恶性黑色素瘤、胃癌中高表达,可促进其发生及侵袭转移,Wnt5a的高表达与这些肿瘤细胞的转移潜能和患者的不良预后密切相关;而有的研究则认为Wnt5a基因具有抑癌基因的特性,可以减少或减缓如白血病、恶性淋巴瘤及结肠癌等的发生及转移。迄今国内外关于肺癌与Wnt5a的关系研究甚少,而且大多停留在肺癌标本中Wnt5a表达水平的检测上,因此有必要对Wnt5a与NSCLC的关系进行深入研究。
目的
探讨Wnt5a在NSCLC的表达与病理意义以及对NSCLC细胞增殖及侵袭潜能的影响,为寻找肺癌有效的治疗途径及预后判定指标提供依据。
方法
1.应用Northern-blot检测43例NSCLC癌组织中Wnt5a mRNA的表达,同时采用免疫组织化学SP法和Western blot技术检测Wnt5a蛋白的表达,统计分析它们与临床病理特征之间的关系并进行生存分析。
2.采用基因重组技术,构建人Wnt5a基因正义及RNAi真核表达载体,转染人H157和A549细胞;半定量RT-PCR和Western blot检测细胞转染前后Wnt5a mRNA和蛋白表达的变化。
3.应用MTT检测细胞的增殖能力;流式细胞仪检测细胞周期;划痕试验检测细胞迁移力的改变;改良Transwell小室法检测细胞侵袭力的变化。
结果
1.Wnt5a mRNA在NSCLC癌组织及正常肺组织中的阳性表达率分别为79.1%和45.4%,二者的表达水平也存在差异,前者为0.96±0.65,后者为0.44±0.23,Wnt5a mRNA在NSCLC中表达明显增高,与正常相比具有显著统计学意义(P<0.05=。
2.Wnt5a mRNA与NSCLC病理分期、细胞分化程度、有无淋巴结转移及组织学类型、是否吸烟有一定关系:Ⅲ~Ⅳ期(1.24±0.47)较Ⅰ~Ⅱ期(0.58±0.71)增高;细胞分化程度低的(1.27±0.46)较分化程度好的(0.65±0.47)也明显增高;有淋巴结转移的(1.10±0.63)较没有淋巴结转移的(0.67±0.66)增高;鳞癌(1.31±0.65)比腺癌(0.58±0.46)增高,吸烟者(1.26±0.56)较非吸烟者(0.57±0.62)增高(P<0.01或P<0.05)。
3.Wnt5a蛋白水平变化趋势与其mRNA基本一致,二者阳性表达之间存在着显著的正相关(相关系数0.74,P<0.01),Wnt5a mRNA与蛋白的表达具有一致性。
4.在NSCLC中,Wnt5a阳性患者2年生存率(31.1%,9/29)明显低于Wnt5a阴性患者(71.4%,10/14),差异具有统计学意义(P<0.01)。
5.分别对重组质粒pcDNA3.1+-XE58和pAVU6-siWnt5a质粒进行酶切鉴定,酶切片段与插入的目的片段大小相一致;进行测序鉴定所得测序结果经分析比对与GeneBank文献报道一致,未见缺失和突变。
6.未经干预的H157和A549对照组细胞增殖指数分别为37.26%和29.01%;转染正义基因组细胞H157-XE58与A549-XE58增殖指数(62.94%,54.49%)增加;RNAi组细胞H157-siWnt5a与A549-siWnt5a增殖指数下降(20.81%,17.66%),正义基因组细胞增殖指数明显高于对照组及RNAi组细胞,RNAi组细胞增殖指数明显低于对照组。
7.细胞迁移力和侵袭力的检测:未经干预的H157和A549对照组细胞具有一定的迁移力和侵袭力;正义基因组H157-XE58与A549-XE58细胞转染24h后迁移速度明显加快,划痕区细胞数明显增多(94.1±3.8, 91.1±3.9),细胞迁移距离较远,穿过Matrigel胶的细胞数明显增加(71.3±3.2 , 61.7±3.5) ; RNAi组H157-siWnt5a与A549-siWnt5a细胞的迁移距离明显小于正义基因组及对照组细胞,划痕区细胞数稀少(21.9±2.2,19.6±4.1),细胞迁移不明显,穿过Matrigel胶的细胞数明显减少(22.3±3.2,20.7±3.5)。正义基因组细胞迁移力和侵袭力均明显高于对照组及RNAi组细胞。RNAi组细胞迁移力和侵袭力明显低于对照组。
结论
1.Wnt5a在NSCLC中不论是mRNA水平还是蛋白水平均明显增高,且与NSCLC的细胞分化程度、组织类型及TNM分期密切相关,提示Wnt5a可能参与NSCLC的发生发展过程。
2.体外NSCLC细胞转染人Wnt5a正义基因后细胞增殖能力明显增强,转染RNAi质粒后增殖能力受到显著抑制,初步证实Wnt5a在NSCLC细胞增殖中起着重要作用。
3.体外NSCLC细胞转染人Wnt5a正义基因后细胞侵袭能力明显增强,转染RNAi质粒后侵袭能力受到显著抑制,初步证实Wnt5a对NSCLC细胞侵袭潜能有显著影响。
4.Wnt5a可以明显促进NSCLC细胞增殖及侵袭潜能增强,此作用可为特异性siRNA所阻断,Wnt5a有可能作为肺癌基因治疗的靶标或药物治疗的靶点。
5.Wnt5a表达水平越高,患者预后越差,生存期越短,提示Wnt5a的检测可能作为NSCLC预后的分子指标之一。
6.成功构建人Wnt5a基因正义及RNAi真核表达载体并建立稳定的转染细胞株,为进一步研究Wnt5a与肺癌的关系提供了实验平台。
Background: Lung cancers, which are the highest occurrence ratio in the whole world, are the leading cause of death among all kinds of caners. It has been reported that among the newly occurrence lung caners, 75-80% of them are non-small cell lung cancer (NSCLC). The prognosis of NSCLC is still very poor even with the accumulating improvements therapies, mainly because of the underlying mechanisms, especially that of metastasis, of the occurrence of lung cancers are still unclear. Therefore, it is very important to answer these questions in order to make better prognosis and improve the treatment of NSCLC. Wnt5a is one of the most important members of Wnt family, it activates the classic Wnt/β-catenin pathway as well as the non-classic Wnt/Ca2+ pathway, and these two pathways interact. Studies have shown that Wnt5a is closely related many malignant cancers, plays very important roles in the proliferation, differentiation and metastasis of tumor cells. Studies have also shown that Wnt5a acts differentially among different metastasis tumors. Some of them show this gene processes the characteristics of oncogene, high expression of Wnt5a was detected in most malignant tumors such as melanoma and stomach cancer, and it can promote tumor occurrence and metastasis, thus its high expression is related to the metastasis potential and poor prognosis; however, some other researches argue Wnt5a share the characteristics of anti-oncogenes, it can decrease or delay the occurrence and metastasis of leucocythemia, malignant lymphanode tumors as well as colon tumors. By now most of the studies about the relationship between Wnt5a and lung cancers were focused on the expression of Wnt5a in clinic samples, but the underlying mechanisms are poorly understood.
Aims: To explore the expression of Wnt5a and role on the proliferation and invasion potentiality in NSCLC, aim at help effective treatment and prognosis of lung cancer.
Methods:
1. Tumor tissues of 43 cases of NSCLC were examined by Northern Blot, and the protein expression of these tissues were examined by Western Blot and immunohis- tochemistry. Survival analysis was carried out between the expression and clinical pathological characteristics by statistics.
2. By gene recombination technique, human Wnt5a gene sense plasmid and its RNAi eukaryotic expression plasmid were constructed, human cell lines of H157 and A549 were transfected, and semi-quantitative RT-PCR and Western Blot were employed to detect the change of Wnt5a mRNA and protein before- and after transfection, respectively.
3. MTT was used to test the change of cell proliferation; flow cytometry was used to test the changes of cell cycle; nick test was used to examine cell migration, and modified Transwell chamber test was used to examine cell invasion.
Results:
1. The positive ratio of Wnt5a mRNA in the tumor tissue of NSCLC and tissue from normal lung was 79.1% and 45.4%, respectively. Difference of expression level also existed: in the former it was 0.96±0.65 and the later was 0.44±0.23. This suggested higher expression of Wnt5a was detected in the NSCLC, statistically significant when compared with normal (P<0.05).
2. There is some degree of relationship between Wnt5a expression and pathological grade, cell differential stage, with- or without lymphonode metastasis and its tissue type, and smoking or not. Higher expression was detectable inⅢ-Ⅳphase(1.24±0.47) compared withⅠorⅡphase(0.58±0.71); in lower differentiation(1.27±0.46) compared with higher differentiation(0.65±0.47); in with lymphanode metastasis(1.10±0.63) compared with non-lymphanode metastasis(0.67±0.66); in squamous cell carcinoma(1.31±0.65) compared with adenocarcinoma(0.58±0.46), and in smokers(1.26±0.56) compared with non-smokers(0.57±0.62)(P<0.01 or 0.05).
3. The protein expression of Wnt5a in all the tissue was in coincidence with the mRNA expression, showing a significant positive correlation (correlation coefficient: 0.74, P<0.01), which highly suggested a consistency.
4. In NSCLC patients, two-year surviving ratio in Wnt5a positive patients(31.1%, 9/29) was lower than Wnt5a negative controls (71.4%, 10/14), showing statistically significant(P<0.01).
5. Restrictive enzyme analysis was carried to identify the recombined plasmid pcDNA3.1+—XE58 and pAVU6-si Wnt5a, the results showed the size of the cutting segment was identical to the inserting target segment; Sequencing analysis showed it was identical to the reported sequence in GeneBank, no missing or mutation was detected.
6. The proliferation index in untreated H157 cell line and A549 cell line was 37.26% and 29.01%, respectively; it increased in sense-transfected H157-XE58 and A549-XE58 (62.94% and 54.49%, respectively); decreased RNAi treated H157 and A549 cells(20.81% and 17.66%, respectively), suggesting a positive role of Wnt5a in the promoting potential of cell proliferation.
7. Untreated H157 and A549 cells had some degree of migration and invasion, respectively. Sense-transfection increased the migration after 24h,the number of cells in the nick area increased significantly, and the distance of migration was longer, too; the number of cross Matrigel cells increased (71.3±3.2 and 61.7±3.5 in H157-XE58 and A549-XE58 cells, respectively). However, all the above parameters were obviously inhibited by siRNA treatment.
Conclusion:
1. In vivo studies showed an increased expression of both Wnt5a mRNA and protein in NSCLC, furthermore the expression level of Wnt5a was closely related to the cell differentiation, histology and TNM stages of NSCLC. These suggested Wnt5a may play an important role in the occurrenence and progression of NSCLC.
2. In vitro studies showed the proliferation increased with the overexpression of Wnt5a and the ability can be inhibited by RNAi targeting Wnt5a efficiently in vitro and in vivo. confirmed this gene may play an important role in the proliferateion of NSCLC.
3. In vitro studies showed the invasion increased with the overexpression of Wnt5a and the ability can be inhibited by RNAi targeting Wnt5a efficiently in vitro and in vivo. confirmed this gene may play an important role in the invasion of NSCLC.
4. Wnt5a gene may be the target of gene therapy of lung cancer, this open a new field for the clinical application of RNA interference or designer drug to aim directly at the gene.
5. Higher expression of Wnt5a was related to the poor prognosis and shorter survival, suggesting Wnt5a may be used as one of the prognosis index.
6. The successful construction of sense plasmids and RNAi eukaryotic expression plasmid provided an experimental platform for further study of the role of this gene .
肺癌是全球发病率最高的恶性肿瘤,己成为人类因癌症死亡的主要原因。非小细胞肺癌(NSCLC)占新发肺癌病例的75-80%,虽然各种治疗手段不断改善,但其预后依然很差。其主要原因在于肺癌的发病机制、特别是肺癌侵袭转移的机制尚不清楚,弄清肺癌的发病及侵袭转移机制对于预测肺癌患者的预后及提高治疗效果意义重大。
Wnt5a是Wnt家族中的重要成员之一,既能活化经典的Wnt/β-catenin通路又能活化非经典的Wnt/Ca2+通路,而且Wnt/Ca2+通路与Wnt/β-catenin通路相互作用。文献报道Wnt5a与多种恶性肿瘤关系密切,在肿瘤细胞的增殖、分化及侵袭转移过程中起着重要的作用。但也有研究发现Wnt5a在不同的恶性肿瘤中生物学作用并不一致,有的认识尚存在重大分歧。部分研究认为Wnt5a具有癌基因的特性,在多种恶性肿瘤如恶性黑色素瘤、胃癌中高表达,可促进其发生及侵袭转移,Wnt5a的高表达与这些肿瘤细胞的转移潜能和患者的不良预后密切相关;而有的研究则认为Wnt5a基因具有抑癌基因的特性,可以减少或减缓如白血病、恶性淋巴瘤及结肠癌等的发生及转移。迄今国内外关于肺癌与Wnt5a的关系研究甚少,而且大多停留在肺癌标本中Wnt5a表达水平的检测上,因此有必要对Wnt5a与NSCLC的关系进行深入研究。
目的
探讨Wnt5a在NSCLC的表达与病理意义以及对NSCLC细胞增殖及侵袭潜能的影响,为寻找肺癌有效的治疗途径及预后判定指标提供依据。
方法
1.应用Northern-blot检测43例NSCLC癌组织中Wnt5a mRNA的表达,同时采用免疫组织化学SP法和Western blot技术检测Wnt5a蛋白的表达,统计分析它们与临床病理特征之间的关系并进行生存分析。
2.采用基因重组技术,构建人Wnt5a基因正义及RNAi真核表达载体,转染人H157和A549细胞;半定量RT-PCR和Western blot检测细胞转染前后Wnt5a mRNA和蛋白表达的变化。
3.应用MTT检测细胞的增殖能力;流式细胞仪检测细胞周期;划痕试验检测细胞迁移力的改变;改良Transwell小室法检测细胞侵袭力的变化。
结果
1.Wnt5a mRNA在NSCLC癌组织及正常肺组织中的阳性表达率分别为79.1%和45.4%,二者的表达水平也存在差异,前者为0.96±0.65,后者为0.44±0.23,Wnt5a mRNA在NSCLC中表达明显增高,与正常相比具有显著统计学意义(P<0.05=。
2.Wnt5a mRNA与NSCLC病理分期、细胞分化程度、有无淋巴结转移及组织学类型、是否吸烟有一定关系:Ⅲ~Ⅳ期(1.24±0.47)较Ⅰ~Ⅱ期(0.58±0.71)增高;细胞分化程度低的(1.27±0.46)较分化程度好的(0.65±0.47)也明显增高;有淋巴结转移的(1.10±0.63)较没有淋巴结转移的(0.67±0.66)增高;鳞癌(1.31±0.65)比腺癌(0.58±0.46)增高,吸烟者(1.26±0.56)较非吸烟者(0.57±0.62)增高(P<0.01或P<0.05)。
3.Wnt5a蛋白水平变化趋势与其mRNA基本一致,二者阳性表达之间存在着显著的正相关(相关系数0.74,P<0.01),Wnt5a mRNA与蛋白的表达具有一致性。
4.在NSCLC中,Wnt5a阳性患者2年生存率(31.1%,9/29)明显低于Wnt5a阴性患者(71.4%,10/14),差异具有统计学意义(P<0.01)。
5.分别对重组质粒pcDNA3.1+-XE58和pAVU6-siWnt5a质粒进行酶切鉴定,酶切片段与插入的目的片段大小相一致;进行测序鉴定所得测序结果经分析比对与GeneBank文献报道一致,未见缺失和突变。
6.未经干预的H157和A549对照组细胞增殖指数分别为37.26%和29.01%;转染正义基因组细胞H157-XE58与A549-XE58增殖指数(62.94%,54.49%)增加;RNAi组细胞H157-siWnt5a与A549-siWnt5a增殖指数下降(20.81%,17.66%),正义基因组细胞增殖指数明显高于对照组及RNAi组细胞,RNAi组细胞增殖指数明显低于对照组。
7.细胞迁移力和侵袭力的检测:未经干预的H157和A549对照组细胞具有一定的迁移力和侵袭力;正义基因组H157-XE58与A549-XE58细胞转染24h后迁移速度明显加快,划痕区细胞数明显增多(94.1±3.8, 91.1±3.9),细胞迁移距离较远,穿过Matrigel胶的细胞数明显增加(71.3±3.2 , 61.7±3.5) ; RNAi组H157-siWnt5a与A549-siWnt5a细胞的迁移距离明显小于正义基因组及对照组细胞,划痕区细胞数稀少(21.9±2.2,19.6±4.1),细胞迁移不明显,穿过Matrigel胶的细胞数明显减少(22.3±3.2,20.7±3.5)。正义基因组细胞迁移力和侵袭力均明显高于对照组及RNAi组细胞。RNAi组细胞迁移力和侵袭力明显低于对照组。
结论
1.Wnt5a在NSCLC中不论是mRNA水平还是蛋白水平均明显增高,且与NSCLC的细胞分化程度、组织类型及TNM分期密切相关,提示Wnt5a可能参与NSCLC的发生发展过程。
2.体外NSCLC细胞转染人Wnt5a正义基因后细胞增殖能力明显增强,转染RNAi质粒后增殖能力受到显著抑制,初步证实Wnt5a在NSCLC细胞增殖中起着重要作用。
3.体外NSCLC细胞转染人Wnt5a正义基因后细胞侵袭能力明显增强,转染RNAi质粒后侵袭能力受到显著抑制,初步证实Wnt5a对NSCLC细胞侵袭潜能有显著影响。
4.Wnt5a可以明显促进NSCLC细胞增殖及侵袭潜能增强,此作用可为特异性siRNA所阻断,Wnt5a有可能作为肺癌基因治疗的靶标或药物治疗的靶点。
5.Wnt5a表达水平越高,患者预后越差,生存期越短,提示Wnt5a的检测可能作为NSCLC预后的分子指标之一。
6.成功构建人Wnt5a基因正义及RNAi真核表达载体并建立稳定的转染细胞株,为进一步研究Wnt5a与肺癌的关系提供了实验平台。
Background: Lung cancers, which are the highest occurrence ratio in the whole world, are the leading cause of death among all kinds of caners. It has been reported that among the newly occurrence lung caners, 75-80% of them are non-small cell lung cancer (NSCLC). The prognosis of NSCLC is still very poor even with the accumulating improvements therapies, mainly because of the underlying mechanisms, especially that of metastasis, of the occurrence of lung cancers are still unclear. Therefore, it is very important to answer these questions in order to make better prognosis and improve the treatment of NSCLC. Wnt5a is one of the most important members of Wnt family, it activates the classic Wnt/β-catenin pathway as well as the non-classic Wnt/Ca2+ pathway, and these two pathways interact. Studies have shown that Wnt5a is closely related many malignant cancers, plays very important roles in the proliferation, differentiation and metastasis of tumor cells. Studies have also shown that Wnt5a acts differentially among different metastasis tumors. Some of them show this gene processes the characteristics of oncogene, high expression of Wnt5a was detected in most malignant tumors such as melanoma and stomach cancer, and it can promote tumor occurrence and metastasis, thus its high expression is related to the metastasis potential and poor prognosis; however, some other researches argue Wnt5a share the characteristics of anti-oncogenes, it can decrease or delay the occurrence and metastasis of leucocythemia, malignant lymphanode tumors as well as colon tumors. By now most of the studies about the relationship between Wnt5a and lung cancers were focused on the expression of Wnt5a in clinic samples, but the underlying mechanisms are poorly understood.
Aims: To explore the expression of Wnt5a and role on the proliferation and invasion potentiality in NSCLC, aim at help effective treatment and prognosis of lung cancer.
Methods:
1. Tumor tissues of 43 cases of NSCLC were examined by Northern Blot, and the protein expression of these tissues were examined by Western Blot and immunohis- tochemistry. Survival analysis was carried out between the expression and clinical pathological characteristics by statistics.
2. By gene recombination technique, human Wnt5a gene sense plasmid and its RNAi eukaryotic expression plasmid were constructed, human cell lines of H157 and A549 were transfected, and semi-quantitative RT-PCR and Western Blot were employed to detect the change of Wnt5a mRNA and protein before- and after transfection, respectively.
3. MTT was used to test the change of cell proliferation; flow cytometry was used to test the changes of cell cycle; nick test was used to examine cell migration, and modified Transwell chamber test was used to examine cell invasion.
Results:
1. The positive ratio of Wnt5a mRNA in the tumor tissue of NSCLC and tissue from normal lung was 79.1% and 45.4%, respectively. Difference of expression level also existed: in the former it was 0.96±0.65 and the later was 0.44±0.23. This suggested higher expression of Wnt5a was detected in the NSCLC, statistically significant when compared with normal (P<0.05).
2. There is some degree of relationship between Wnt5a expression and pathological grade, cell differential stage, with- or without lymphonode metastasis and its tissue type, and smoking or not. Higher expression was detectable inⅢ-Ⅳphase(1.24±0.47) compared withⅠorⅡphase(0.58±0.71); in lower differentiation(1.27±0.46) compared with higher differentiation(0.65±0.47); in with lymphanode metastasis(1.10±0.63) compared with non-lymphanode metastasis(0.67±0.66); in squamous cell carcinoma(1.31±0.65) compared with adenocarcinoma(0.58±0.46), and in smokers(1.26±0.56) compared with non-smokers(0.57±0.62)(P<0.01 or 0.05).
3. The protein expression of Wnt5a in all the tissue was in coincidence with the mRNA expression, showing a significant positive correlation (correlation coefficient: 0.74, P<0.01), which highly suggested a consistency.
4. In NSCLC patients, two-year surviving ratio in Wnt5a positive patients(31.1%, 9/29) was lower than Wnt5a negative controls (71.4%, 10/14), showing statistically significant(P<0.01).
5. Restrictive enzyme analysis was carried to identify the recombined plasmid pcDNA3.1+—XE58 and pAVU6-si Wnt5a, the results showed the size of the cutting segment was identical to the inserting target segment; Sequencing analysis showed it was identical to the reported sequence in GeneBank, no missing or mutation was detected.
6. The proliferation index in untreated H157 cell line and A549 cell line was 37.26% and 29.01%, respectively; it increased in sense-transfected H157-XE58 and A549-XE58 (62.94% and 54.49%, respectively); decreased RNAi treated H157 and A549 cells(20.81% and 17.66%, respectively), suggesting a positive role of Wnt5a in the promoting potential of cell proliferation.
7. Untreated H157 and A549 cells had some degree of migration and invasion, respectively. Sense-transfection increased the migration after 24h,the number of cells in the nick area increased significantly, and the distance of migration was longer, too; the number of cross Matrigel cells increased (71.3±3.2 and 61.7±3.5 in H157-XE58 and A549-XE58 cells, respectively). However, all the above parameters were obviously inhibited by siRNA treatment.
Conclusion:
1. In vivo studies showed an increased expression of both Wnt5a mRNA and protein in NSCLC, furthermore the expression level of Wnt5a was closely related to the cell differentiation, histology and TNM stages of NSCLC. These suggested Wnt5a may play an important role in the occurrenence and progression of NSCLC.
2. In vitro studies showed the proliferation increased with the overexpression of Wnt5a and the ability can be inhibited by RNAi targeting Wnt5a efficiently in vitro and in vivo. confirmed this gene may play an important role in the proliferateion of NSCLC.
3. In vitro studies showed the invasion increased with the overexpression of Wnt5a and the ability can be inhibited by RNAi targeting Wnt5a efficiently in vitro and in vivo. confirmed this gene may play an important role in the invasion of NSCLC.
4. Wnt5a gene may be the target of gene therapy of lung cancer, this open a new field for the clinical application of RNA interference or designer drug to aim directly at the gene.
5. Higher expression of Wnt5a was related to the poor prognosis and shorter survival, suggesting Wnt5a may be used as one of the prognosis index.
6. The successful construction of sense plasmids and RNAi eukaryotic expression plasmid provided an experimental platform for further study of the role of this gene .
引文
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