TNF-α通过调控miR-650/LATS1表达对结肠癌细胞增殖和凋亡的影响及其机制研究

Effects of TNF-α on proliferation and apoptosis of colon cancer cells by regulating expression of miR-650/LATS1 and its mechanism

目的:探究miR-650是否通过介导LATS1表达参与TNF-α对结肠癌细胞增殖和凋亡的调控。方法:双荧光素酶报告基因分析法验证miR-650对LATS1的靶向作用。用不同浓度TNF-α处理SW480细胞,分别构建miR-650抑制表达、LATS1过表达和经TNF-α处理的miR-650过表达SW480细胞株,qRT-PCR检测miR-650表达水平,Western blot检测LATS1、CyclinD1、Bcl-2、p21和Bax蛋白表达水平,MTT实验检测细胞增殖抑制率,流式细胞术检测细胞凋亡率。结果:LATS1是miR-650的靶基因,miR-650可负性调控LATS1的表达。TNF-α处理可抑制SW480细胞的增殖,促进细胞凋亡,并可抑制miR-650、CyclinD1和Bcl-2的表达,促进LATS1、Bcl-2和p21的表达。抑制miR-650表达或LATS1过表达均可抑制SW480细胞的增殖,促进细胞凋亡。miR-650过表达可部分逆转TNF-α对SW480细胞的增殖抑制和凋亡促进作用。结论:TNF-α通过下调miR-650/LATS1抑制结肠癌细胞的增殖,促进细胞凋亡。

Objective:To investigate whether miR-650 is involved in the regulation of TNF-αon the proliferation and apoptosis of colon cancer cells by mediating LATS1.Methods:Dual luciferase reporter assay was used to verify the possible target genes of miR-650.The SW480 cells were treated with TNF-αat different concentrations,and the SW480 cell lines with inhibition of miR-650,the SW480 cell lines with over-expression of LATS1 and the TNF-αtreated SW480 cell lines with over-expression of miR-650 were constructed.The expression of miR-650 was detected by qRT-PCR,the expression levels of LATS1,CyclinD1,Bcl-2,p21 and Bax proteins were detected by Western blot,the cell proliferation inhibition rate was detected by MTT assay,and the cell apoptosis rate was detected by flow cytometry.Results:LATS1 was the target gene of miR-650,and miR-650 could negatively regulate the expression of LATS1.TNF-αtreatment inhibited the proliferation of SW480 cells,promoted apoptosis,inhibited the expression of miR-650,CyclinD1 and Bcl-2,and promoted the expression of LATS1,Bcl-2 and p21.The inhibition of miR-650 or the over-expression of LATS1 inhibited the proliferation and promoted apoptosis of SW480 cells.The effect of TNF-αtreated on inhibiting proliferation and promoting apoptosis of SW480 cells was partially reversed by the over-expression of miR-650.Conclusion:TNF-αinhibited the proliferation and promoted the apoptosis of colon cancer cells by down-regulating miR-650/LATS1.