慢病毒介导STAT3-shRNA对胃癌细胞化疗敏感性的影响及机制

Lentiviral-shRNA-mediated STAT3 silencing enhances chemosensitivity to 5-fluorouracil in human gastric cancer cells

摘要:

目的：探讨慢病毒介导的shRNA靶向沉默信号传导及转录激活因子3（signal transducers and activators of transcription 3，STAT3）对人胃癌细胞SGC-7901化疗敏感性的影响及相关机制。方法：构建靶向STAT3基因的shRNA序列，以慢病毒为载体转染SGC-7901细胞。以real-time PCR及Western blot检测STAT3干扰效果；MTT法分析各组细胞对5-氟尿嘧啶（5-fluorouracil，5-Fu）化疗敏感性的变化；用流式细胞仪比较细胞凋亡率的差异；Western blot检测抗凋亡蛋白Ｂ细胞白血病－２（B-cell lymphoma-2，Bcl-2）、髓样细胞白血病－１（myeloid cell leukemia-1，Mcl-1）及survivin的变化。结果：成功构建靶向STAT3基因的慢病毒并转染SGC-7901细胞，有效抑制55％ STAT3 mRNA表达和40％STAT3蛋白表达（P＜0.05）。STAT3-shRNA转染后细胞的增殖抑制率及凋亡率均明显升高（P＜0.05），抗凋亡蛋白Bcl-2、Mcl-1及survivin蛋白水平明显下降（P＜0.05）。结论：慢病毒介导的STAT3-shRNA可抑制SGC-7901细胞增殖并诱导其凋亡，导致细胞对5-Fu的化疗敏感性增强，相关机制可能是通过抑制STAT3表达从而阻断STAT3信号通路，导致该通路下游的抗凋亡蛋白Bcl-2、Mcl-1、survivin的活性下调。

Abstract:

Objective：To investigate the effect and the mechanisms of lentiviral-shRNA-mediated signal transducers and activators of transcription 3（STAT3） silencing on the chemosensitivity to 5-fluorouracil（5-Fu）， of human gastric cancer cells SGC-7901. Meth－ods：The shRNA sequences targeting STAT3 was constructed and lentiviral vectors was used to transfect human gastric cancer cells SGC-7901. The interference effect of STAT3-shRNA was detected by real-time PCR and Western blot. The chemosensitivity to 5-Fu was analyzed by MTT assay and the flow cytometry was used to examine cell apoptotic rate. Western blot was used to compare the ex－pressions of anti-apoptotic proteins Bcl-2，Mcl-1，survivin. Results：Lentiviral vectors containing shRNA targeting STAT3 gene were successfully established and transfected into SGC-7901 cells．Real-time PCR and Western blot analysis showed that the STAT3-shRNA significantly inhibited the mRNA expression by 54% and protein expression by 40%（P<0.05）． The inhibitory rate of cell pro－liferation and the apoptotic rate were obviously higher in the cells treated by STAT3-shRNA（P<0.05）. STAT3-shRNA also induced downregulation of the anti-apoptotic proteins Bcl-2，Mcl-1 and survivin（P<0.05）. Conclusion：In summary，STAT3-shRNA could effectively inhibit the proliferation and accelerate the apoptosis of SGC-7901 cells，leading to the enhancement of their sensitivity to 5-Fu. The mechanism may be related with block of STAT3 signaling pathway though inhibiting STAT3 activation，thus decreasing ex－pressions of its downstream anti-apoptotic proteins Bcl-2，Mcl-1 and survivin.