可调控STAT3干扰载体抑制BIU-87细胞侵袭的体外研究

Effect of CRE-dependent RNA interference targeting STAT3 on invasion and migration in human bladder cancer BIU-87 cells

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 CRE-dependent RNA interference targeting STAT3 on the invasion and migration of human bladder cancer BIU-87 cells. Methods RNA interfering vectors pSico was used to construct CRE-dependent shRNA expression plasmids targeting STAT3, and pLVX-CRE was used as an expression vector of CRE. BIU-87 cells were divided into 4 groups and were transfected with pSico-shNeg, pSico-shNeg/CRE, pSico-shSTAT3 and pSico-shSTAT3/CRE, respectively. RT-PCR and Western blot analysis were carried out to assess the efficiency of RNA interference. The abilities of invasion and migration of BIU-87 cells after CRE-dependent RNA interference of STAT3 were detected by Transwell chamber assay and wound-healing assay. Results Restriction analysis and DNA sequencing proved that the recombinant plasmid pSico-shSTAT3 was constructed successfully. CRE-dependent green fluorescent cells were detected after the transfection. The shRNA against STAT3 significantly inhibited STAT3 mRNA expression, and CRE and shSTAT3 transfection down-regulated the expression levels of STAT3 in BIU87 cells significantly （P〈0.05）. The number of migrating cells were significantly less in the pSico-shSTAT3 /CRE group （42.00±3.00） than in the pSico-shNeg （203.33±12.42）, pSico-shNeg/CRE （196.33±11.85） and pSico-shSTAT3 （201.00±16.64） groups （P〈0.01）. Conclusion RNA interference of STAT3 mediated by CRE-dependent shRNA expression plasmid can down-regulate intracellular STAT3 signal level and reduce the abilities of invasion and migration of BIU-87 cells.