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[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 observe the effect of silencing four times transmembrane superfamily protein 3 (Tspan8) on Rap1 and Rap1 GAP proteins in human colon cancer HT-29 cell xenografts and metastases. [Methods] By using bioinformatics method and combining with a gene chip data set in R2 platform, we screened the gene whose expression was significantly correlated with Tspan8 expression by Dukes staging and analyzed the KEGG pathway of Tspan8 related gene by KEGG tool. The expression of Tspan8 in human colon cancer cell line HT-29 was silenced by lentivirus silencing technique. The silencing effect of Tspan8 after HT-29 cells were detected by RT-PCR and immunohistochemistry was used to establish the animal model of HT-29 colon cancer. The lentivirus-silenced Tspan8 gene (shRNA + Tspan8 + HT-29 cells) in HT-29 cells was used as experimental group, empty vector group was used as control group, and the growth of two groups was observed. Western blot was used to detect the expression of Rap1 and Rap1 GAP in the liver metastases of nude mice. [Results] In the Tspan8-associated KEGG pathway, there was a correlation between Rap1 pathway and Tspan8 expression in DukeC and DukeD stages (P <0.05). RT-PCR results showed that Tspan8 mRNA expression level in the experimental group was significantly lower than that in the control group (P <0.05). Silencing Tspan8 reduced the number of liver metastases in nude mice and decreased the volume and weight. Immunohistochemistry showed that the expression of Tspan8 in liver metastases was significantly decreased in experimental group (P <0.05), while Western Blot showed that Rap1 The expression of Rap1GAP in the experimental group was significantly higher than that in the control group (P <0.05). [Conclusion] Silencing Tspan8 inhibits the hepatic metastasis of HT-29 cells by affecting Rap1 protein activity through Rap1GAP.