miR-194-5p靶向下调CD44抑制胃癌肿瘤干细胞上皮间质转化的机制研究

Mechanism of MiR-194-5p Targeted Downregulation of CD44 Inhibiting Epithelial Mesenchymal Transformation of Gastric Cancer Stem Cells

目的:探讨miR-194-5p靶向下调CD44抑制胃癌肿瘤干细胞(CSCs)上皮间质转化(EMT)的相关分子机制。方法:采用qRT-PCR检测胃腺癌细胞系(SGC-7901、MGC-803、SPAG-9和MNK-45)和胃粘膜细胞(GES-1)中miR-194-5p、CD44、Snail和膜型基质金属蛋白酶-1(MT1-MMP)表达量,Western blot检测CD44、Snail和MT1-MMP蛋白表达量。体外构建miR-194-5p过表达和低表达质粒载体并进行慢病毒转染,实验分为过表达组、对照组和低表达组,qRT-PCR和Western blot检测miR-194-5p、CD44、Snail和MT1-MMP的变化;CCK8法检测细胞增殖率,流式细胞术检测细胞凋亡率,Transwell实验检测细胞侵袭力,Western blot检测EMT标志物E-cadherin和N-cadherin蛋白表达量。结果:胃腺癌细胞系中miR-194-5p表达显著低于胃粘膜细胞,而CD44、Snail和MT1-MMP表达量显著升高;SGC-7901和MGC-803与胃粘膜细胞的表达差异最明显(P<0.05)。与对照组相比,过表达组miR-194-5p表达量明显升高,CD44、Snail和MT1-MMP表达量显著下降,细胞增殖率和侵袭力下降,凋亡率升高,E-cadherin上调,N-cadherin下调(P<0.05)。与对照组相比,低表达组miR-194-5p表达量明显下降,CD44、Snail和MT1-MMP表达量显著增加,细胞增殖率和侵袭力升高,凋亡率下降,E-cadherin下调,N-cadherin上调(P<0.05)。结论:胃癌中miR-194-5p低表达可能发挥抑癌作用,通过下调CSCs中CD44表达进而抑制EMT的发生。

Objective:To explore the molecular mechanism of miR-194-5p targeting down regulation of CD44 and inhibiting epithelial mesenchymal transition(EMT)in gastric cancer stem cells(CSCs).Methods:qRT-PCR was used to detect the expression levels of miR-194-5p,CD44,Snail,and membrane matrix metalloproteinase-1(MT1-MMP)in gastric adenocarcinoma cell lines(SGC-7901,MGC-803,SPAG-9,and MNK-45)and gastric mucosal cells(GES-1).Western blot was used to detect the expression levels of CD44,Snail,and MT1-MMP proteins.miR-194-5p over-expression and low-expression plasmid vectors were constructed in vitro then to transfect with lentivirus.The experiment was divided into over-expression group,control group,and low-expression group.qRT-PCR and Western blot were used to detect changes of miR-194-5p,CD44,Snail,and MT1-MMP;CCK8 assay was used to detect cell proliferation rate,flow cytometry was used to detect cell apoptosis rate,Transwell assay was used to detect cell invasiveness,and Western blot was used to detect the expression levels of EMT markers E-cadherin and N-cadherin proteins.Results:The expression of miR-194-5p was significantly lower in gastric adenocarcinoma cell lines than gastric mucosal cells,while the expression levels of CD44,Snail,and MT1-MMP were significantly higher;what's more,the expression differences of SGC-7901 and MGC-803 compared to gastric mucosal cells were the most(P<0.05).Compared with the control group,miR-194-5p expression in the over-expression group was significantly more,but CD44,Snail,and MT1-MMP expressions were significantly less,cell proliferation and invasiveness were lower,apoptosis rate was higher,E-cadherin was upregulated,and N-cadherin was downregulated(P<0.05).Compared with the control group,the expression level of miR-194-5p was significantly lower in the low-expression group,while the expression levels of CD44,Snail,and MT1-MMP were significantly higher,the cell proliferation rate and invasiveness were more,while the apoptosis rate was less,E-cadherin was downregulated,and N-cadherin was upregulated(P<0.05).Conclusion:Low expression of miR-194-5p in gastric cancer may exert an anti-tumor effect by downregulating the expression of CD44 in CSCs,thereby inhibiting the occurrence of EMT.