摘要
[目的]观察沉默四次跨膜超家族蛋白3(Tspan8)对人结肠癌HT-29细胞移植瘤和转移灶中Rap1和Rap1GAP蛋白的影响。[方法]通过生物信息学方法,结合R2平台中的1个基因芯片数据集,以Dukes分期筛选出自身表达与Tspan8表达显著相关的基因,利用KEGG工具对Tspan8相关的基因进行KEGG通路分析。利用慢病毒沉默技术沉默Tspan8在人结肠癌细胞株HT-29中的表达,RT-PCR及免疫组化技术检测HT-29细胞干扰后Tspan8沉默效果,构建结肠癌HT-29细胞转移动物模型。慢病毒沉默HT-29细胞中的Tspan8基因(shRNA+Tspan8+HT-29细胞)为实验组、空载体组为对照组,观察2组移植瘤生长情况。Western blot技术检测裸鼠肝脏转移灶中Rap1、Rap1 GAP的表达情况。[结果]与Tspan8相关的KEGG通路中,Rap1通路在DukeC期及DukeD期中与Tspan8表达存在相关性(P<0.05)。RT-PCR结果表明Tspan8mRNA表达水平在实验组中较对照组明显下调(P<0.05)。沉默Tspan8后,裸鼠肝转移瘤的数目减少、体积和重量变小,免疫组化结果提示实验组肝脏转移瘤组织Tspan8表达均较对照组小鼠明显减少(P<0.05),Western Blot示Rap1、Rap1GAP在实验组肝脏肿瘤组织表达较对照组改变具有统计学意义(P<0.05)。[结论]沉默Tspan8通过Rap1GAP影响Rap1蛋白活性抑制HT-29细胞肝脏转移。
[Objective]To investigate the effects of Tspan8 on Rap1 and RapGAP proteins in xenograft and metastatic tumors of human colon cancer HT-29 Cell.[Methods]By bioinformatics method,combined with the data set of 1 genes in R2 platform,Dukes stages were selected to screen out the genes which were significantly associated with the expression of Tspan8.Then we used KEGG tools to find which KEGG pathway was related to Tspan8.The Tspan8 expression was silenced in human colorectal cancer cell line HT-29 by lentivirus vector.Tspan8 silencing effect was detected by RT-PCR and Immunohistochemistry.The HT-29 cells in animal colon cancer model were constructed and the growth of transplanted tumor in each group were observed.Western blot were used to investigate the protein expressions of Rap1 and Rap1 GAP in liver metastatic tumor tissues.[Results]The Tspan8 related Rap1 signaling pathway had statistical significant change in duke stage C phase and D phase（P〈0.05）.RT-PCR results showed that the expression of mRNA Tspan8 decreased with statistical significance（P〈0.05）.After silencing Tspan8,the number of liver metastases in nude mice was reduced,the volume and weight of the tumor became smaller.The results of immunohistochemistry showed that the expression of Tspan8 in the liver metastasis of the experimental group was significantly lower than that in the control group（P〈0.05）.Western blot showed that Rap1 and Rap1 GAP in the experimental group were less than the control group in the expression of liver tumor tissue（P〈0.05）.[Conclusion]Silencing Tspan8 inhibits the liver metastasis of HT-29 cells through Rap1 GAP affecting Rapl protein activity.
出处
《临床消化病杂志》
2017年第2期85-90,共6页
Chinese Journal of Clinical Gastroenterology
