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目的探讨可调控RNA干扰载体通过抑制STAT3信号通路对膀胱癌细胞BIU-87侵袭性的影响。方法采用受CRE重组酶调控的RNA干扰载体pSico构建针对STAT3的shRNA表达载体,以pLVX-CRE作为CRE蛋白的表达载体,将BIU-87细胞分为pSico-shNeg、pSico-shNeg/CRE、pSico-shSTAT3、pSico-shSTAT3/CRE 4组,分别转染相应质粒,RT-PCR和Western blot法检测其干扰效率,Transwell实验检测其侵袭能力。结果双酶切及测序证实载体构建正确;各组细胞在转染重组质粒后EGFP的表达受CRE调控;RT-PCR结果显示STAT3的表达在干扰之后有显著降低。Westernblot结果显示,在无CRE时,pSico-shSTAT3组与pSico-shNeg组STAT3的表达无显著差异(P>0.05),在有CRE时,pSico-shSTAT3组STAT3相对表达量下降为pSico-shNeg组的(43±4.2)%(P<0.05);体外侵袭实验显示pSico-shNeg组透膜细胞数为(203.33±12.42)/视野、pSico-shNeg/CRE组(196.33±11.85)/视野、pSico-shSTAT3组(201.00±16.64)/视野,而pSico-shSTAT3/CRE组(42.00±3.00)/视野,较其他3组显著降低(P<0.01)。结论由可调控RNA干扰载体介导的CRE依赖性STAT3表达载体能降低细胞内STAT3信号水平,并降低BIU-87细胞体外侵袭迁移能力。
OBJECTIVE: To investigate the effect of RNA interference vector on the invasiveness of bladder cancer cells BIU-87 by inhibiting STAT3 signaling pathway. METHODS: shRNA expression vector targeting STAT3 was constructed by RNA interference vector pSico regulated by CRE recombinase. BIU-87 cells were divided into pSico-shNeg, pSico-shNeg / CRE and pSico- shSTAT3 and pSico-shSTAT3 / CRE4 were transfected into the corresponding plasmids respectively. The interference efficiency was detected by RT-PCR and Western blot, and the invasion ability was detected by Transwell assay. Results Double digestion and sequencing confirmed that the vector was constructed correctly. The expression of EGFP was controlled by CRE after transfection of recombinant plasmids. RT-PCR results showed that the expression of STAT3 was significantly decreased after interference. In the absence of CRE, there was no significant difference in STAT3 expression between pSico-shSTAT3 group and pSico-shNeg group (P> 0.05). When CRE was present, the relative expression level of STAT3 in pSico-shSTAT3 group decreased to pSico-shNeg group (43 ± 4.2)% (P <0.05). In vitro invasion assay showed that the number of transmembrane cells in pSico-shNeg group was (203.33 ± 12.42) / field, pSico-shNeg / CRE group (196.33 ± 11.85) / field, pSico-shSTAT3 Group (201.00 ± 16.64) / field of vision, while pSico-shSTAT3 / CRE group (42.00 ± 3.00) / field of vision was significantly lower than the other three groups (P <0.01). CONCLUSION: The CRE-dependent STAT3 expression vector, which can be regulated by RNA interference vector, can decrease the level of STAT3 in cells and reduce the invasion and migration of BIU-87 cells in vitro.