SMAD1通过调控miR-32表达影响结直肠癌细胞增殖、凋亡和迁移

SMAD1 affects the proliferation,apoptosis and migration of colorectal cancer cells by regulating miR-32 expression

目的:研究SMAD家族成员1(SMAD family member 1,SMAD1)通过调控微RNA(microRNA,miRNA,miR)-32对结直肠癌(colorectal cancer,CRC)细胞增殖、凋亡和迁移等生物学行为的影响,并探讨可能的作用机制。方法:实时荧光定量PCR法检测SMAD1在CRC细胞HCT-8、HCT-116和HT-29以及正常结肠上皮细胞株NCM460中的表达水平。采用脂质体法分别将特异性针对SMAD1基因的si RNA(siSMAD1)和si RNA-阴性对照(negative control,NC)(siNC)转染至人CRC细胞HCT-116中,用实时荧光定量PCR和蛋白质印迹法检测沉默效果。分别采用CCK-8法、FCM法、划痕愈合实验和Transwell小室实验检测沉默SMAD1表达对细胞增殖、凋亡和迁移的影响;实时荧光定量PCR法和蛋白质印迹法检测沉默SMAD1表达对HCT-116细胞中miR-32及其宿主基因TMEM245及靶基因PTEN表达的影响;实时荧光定量PCR法检测上皮-间质转化相关因子波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)mRNA的表达水平。采用染色质免疫共沉淀法检测SMAD1是否能直接与TMEM245/miR-32的启动子区相结合。分别将miR-32-模拟物(miR-32-mimics)以及阴性对照-模拟物(NC-mimics)转入HCT-116细胞,采用实时荧光定量PCR法检测转染效率和SMAD1的表达水平。分别同时共转染siSMAD1+miR-32-mimics以及siSMAD1+NC-mimics至HCT-116细胞,采用CCK-8法、FCM法、划痕愈合实验和Transwell小室实验分别检测共转染后细胞增殖、凋亡和迁移能力的改变,实时荧光定量PCR法和蛋白质印迹法检测转染后对PTEN和上皮-间质转化相关因子表达的影响。结果:CRC细胞HCT-8、HCT-116和HT-29中SMAD1 mRNA的表达水平均高于正常结肠上皮细胞株NCM460细胞(P均<0.05)。siSMAD1转入HCT-116细胞后,HCT-116细胞中SMAD1 mRNA和蛋白的表达水平均明显下调(P均<0.05)。沉默SMAD1基因表达后,HCT-116细胞的增殖和迁移能力均被抑制,而凋亡率明显增加(P均<0.05),miR-32和TMEM245 mRNA的表达水平下调,PTENmRNA和蛋白表达水平上调(P均<0.05),Vimentin和N-cadherin mRNA的表达下调,E-cadherin mRNA表达上调(P均<0.05)。染色质免疫共沉淀结果显示,SMAD1能与TMEM245/miR-32核心启动子区相结合(P<0.05)。与siSMAD1+NC-mimics共转染组相比,siSMAD1+miR-32-mimics共转染组细胞增殖、迁移能力增强,凋亡率减少,PTEN蛋白表达水平下调,Vimentin和N-cadherin mRNA的表达上调,E-cadherin mRNA表达下调(P均<0.05)。结论:沉默SMAD1表达可通过下调miR-32表达来上调PTEN的表达水平,进而抑制CRC细胞的增殖和迁移能力,促进细胞凋亡。

Objective:To research the effect of SMAD family member 1(SMAD1)on the proliferation,apoptosis and migration of colorectal cancer(CRC)cells by regulating microRNA(miRNA,miR)-32,and investigate the underlying molecular mechanism.Methods:Real-time quantitative PCR was used to detect the mRNA expression of SMAD1 in CRC cell lines HCT-8,HCT-116 and HT-29 as well as normal colonic epithelial cell line NCM460.The SMAD1 gene specific siRNA(siSMAD1)and siRNA negative control(siNC)were transfected into HCT-116 cells by liposome method,and the silencing effect was assessed by real-time quantitative PCR and Western blotting.CCK-8 assay,FCM assay,and wound-healing assay and transwell chamber test were performed to evaluate the effect of SMAD1 knock-down on cell proliferation,apoptosis and migration,respectively.The expression of miR-32 as well as its host gene TMEM245 and target gene PTEN in HCT-116 cells after SMAD1 silencing was examined by real-time quantitative PCR and Western blotting.The mRNA levels of epithelial mesenchymal transition factors Vimentin,E-cadherin and N-cadherin after SMAD1 knock-down were examined by real-time quantitative PCR.Chromatin immunoprecipitation was used to verify the binding of SMAD1 to the promoter region of TMEM245/miR-32.The miR-32-mimics and negative control-mimics(NC-mimics)were transfected into HCT-116 cells,and the expression levels of miR-32 and SMAD1 mRNA were evaluated by real-time quantitative PCR.After co-transfection of siSMAD1+miR-32-mimics or siSMAD1+NC-mimics,the proliferation,apoptosis and migration of the HCT-116 cells were analyzed by CCK-8 assay,FCM assay,and wounding-healing assay and transwell chamber test,respectively,and the expression of PTEN and epithelial mesenchymal transition factors was examined by real-time quantitative PCR and Western blotting.Results:The expression level of SMAD1 in HCT-8,HCT-116 and HT-29 cells was higher than that in NCM460 cells(all P<0.05).The expression levels of SMAD1 mRNA and protein in HCT-116 cells were significantly down-regulated after siSMAD1 transfection(both P<0.05).The proliferation and migration of HCT-116 cells was inhibited while the apoptosis rate increased after SMAD1 knock-down(all P<0.05).The knock-down of SMAD1 significantly inhibited the expression of miR-32,TMEM245,Vimentin and N-cadherin(all P<0.05)and promoted the expression of PTEN and E-cadherin(all P<0.05).The chromatin immunoprecipitation result showed that SMAD1 could bind to the core promoter region of TMEM245/miR-32(P<0.05).Compared with the siSMAD1+NC-mimics co-transfection group,the proliferation and migration of HCT-116 cells was enhanced while the apoptosis rate was decreased,the protein expression level of PTEN was reduced,and the mRNA levels of Vimentin and N-cadherin were up-regulated while that of E-cadherin was reduced significantly in the siSMAD1+miR-32-mimics co-transfection group(all P<0.05).Conclusion:SMAD1 silencing could inhibit the proliferation and migration while promote the apoptosis of CRC cells by up-regulating the expression level of PTEN through miR-32 expression inhibition.