miR-381-3p靶向特异性蛋白1抑制mTOR/p70s6k信号通路调控食管癌细胞的增殖和凋亡

Study on the Regulatory Effect of miR-381-3p on the Proliferation and Apoptosis of Esophageal Carcinoma Cells by Targeting SP1 to Inhibit the mTOR/p70s6k Signaling Pathway

目的探讨miR-381-3p靶向特异性蛋白1(SP1)抑制mTOR/p70s6k信号通路对食管癌细胞的增殖和凋亡调控作用。方法RT-qPCR检测正常食管细胞和食管癌细胞中miR-381-3p与SP1mRNA的表达;利用Lipofectamine 2000将miR-381-3p mimic、miR-control、SP1质粒分别或联合转染进入EC9706细胞,基因预测软件预测miR-381-3p的靶基因,双荧光素酶报告检测靶向关系;MTT法检测细胞增殖情况;流式细胞术检测细胞凋亡;蛋白质印迹技术检测Ki67、增殖细胞核抗原(PCNA)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸蛋白酶3(caspase-3)、哺乳动物雷帕霉素靶蛋白(mTOR)、p-mTOR、p70s6激酶(p70s6k)、p-p70s6k和SP1蛋白表达水平。结果miR-381-3p在食管癌细胞EC9706中低表达(P<0.01),而SP1高表达(P<0.01);miR-381-3p与SP13′UTR区存在结合位点,且miR-381-3p直接靶向抑制SP1表达;过表达miR-381-3p后,食管癌细胞增殖明显降低(P<0.01),Ki67和PCNA蛋白表达明显下调(P<0.01),细胞凋亡率明显降升高(P<0.01),Bax/Bcl-2和cleaved caspase-3蛋白表达明显上调(P<0.01),mTOR和p70s6k磷酸化明显减少(P<0.01);而高表达SP1可以逆转miR-381-3p对食管癌细胞增殖抑制和凋亡诱导作用。抑制mTOR/p70s6k信号通路后,食管癌细胞增殖明显降低(P<0.01),细胞凋亡率明显增加(P<0.01),增殖标记蛋白Ki67表达明显下调(P<0.01),凋亡标记蛋白cleaved caspase-3表达明显上调(P<0.01),而SP1蛋白表达无明显变化。结论miR-381-3p可能通过靶向SP1抑制mTOR/p70s6k信号通路来抑制食管癌细胞的增殖、诱导细胞凋亡。

Objective To investigate the regulatory effect of miR-381-3 p on the proliferation and apoptosis of esophageal carcinoma cells by targeting specificity protein 1(SP1)to inhibit the mTOR/p70 s6 ksignaling pathway.Methods The expression of miR-381-3 p and SP1 mRNA in normal esophageal cells and esophageal carcinoma cells was detected by RT-qPCR.The miR-381-3 p mimic plasmid,miR-control plasmid and SP1 plasmid were respectively or jointly transfected into EC9706 cells by using Lipofectamine2000.The target gene of miR-381-3 p was predicted by the gene prediction software and the target relationship was detected by dual luciferase reporter gene assay.The cell proliferation was detected by MTT assay,the apoptosis was detected by flow cytometry,and the expression of Ki67,proliferating cell nuclear antigen(PCNA),B-celllymphoma(Bcl-2),Bcl-2 associated X protein(Bax),cysteine aspartic acid protease 3(Caspase-3),mammalian target of rapamycin(mTOR),p-mTOR,p70 s6 kinase(p70 s6 k),p-p70 s6 kand SP1 proteins was detected by Western blot.Results miR--381-3 p was lowly expressed in EC9706 cells(P<0.01),while SP1 was highly expressed(P<0.01).miR-381-3 p had a binding site with the SP13′UTR region,and miR-381-3 p directly targeted to inhibit the SP1 expression.After the overexpression of miR-381-3 p,the proliferation of esophageal carcinoma cells was significantly decreased(P<0.01),the expression of the Ki67 and PCNA proteins was significantly down-regulated(P<0.01),the apoptosis rate was significantly decreased(P<0.01),the expression of the Bax/Bcl-2 and cleaved caspase-3 proteins was significantly up-regulated(P<0.01),and the phosphorylation of mTOR and p70 s6 k was significantly reduced(P<0.01).The high expression of SP1 reversed the proliferation-inhibited and apoptosis-induced effect of miR-381-3 p on esophageal carcinoma cells.After inhibiting the mTOR/p70 s6 ksignaling pathway,the proliferation of esophageal carcinoma cells was significantly decreased(P<0.01),the apoptosis rate was significantly increased(P<0.01),the expression of the proliferation marker protein Ki67 was significantly down-regulated(P<0.01),and the expression of the apoptosis marker protein cleaved caspase-3 was significantly up-regulated(P<0.01),while the expression of SP1 protein was not significantly changed.Conclusion miR-381-3 p may inhibit the proliferation of esophageal carcinoma cells and induce their apoptosis by targeting SP1 to inhibit the mTOR/p70 s6 ksignaling pathway.