miR-31-5p通过VPS53介导的自噬对结直肠癌细胞增殖的影响

Effect of miR-31-5p on colorectal cancer cell proliferation through VPS53-mediat⁃ed autophagy signaling pathway

目的探讨miR-31-5p通过VPS53介导的自噬对结直肠癌细胞增殖的影响。方法选择人正常结直肠黏膜细胞(FHC)及结直肠癌细胞株HT29、HCT116、SW480、LoVo进行研究。采用实时荧光定量PCR实验检测miR-31-5p在前述各种细胞中的表达,miR-31-5p对VPS53 RNA表达的影响,miR-31-5p、VPS53对Beclin1 RNA表达的影响。采用CCK-8法检测细胞活力。利用数据库Targetscan筛选与miR-31-5p靶向结合的靶基因,并通过双荧光素酶报告实验进行验证。利用数据库GEPIA分析VPS53和Beclin1表达的相关性。采用透射电子显微镜实验探究VPS53对细胞自噬的影响。采用蛋白免疫印迹实验(Western blot)分析VPS53对Beclin1蛋白表达的影响。结果与FHC细胞相比,HCT116、LoVo、HT29、SW480细胞的miR-31-5p表达水平均升高(均P<0.05)。过表达miR-31-5p可促进LoVo细胞增殖(P<0.05)。miR-31-5p与VPS53靶向结合。过表达miR-31-5p可下调VPS53的RNA表达水平(P<0.05)。过表达VPS53可抑制LoVo细胞增殖,可逆转过表达miR-31-5p对LoVo细胞增殖的促进作用(均P<0.05)。结直肠癌组织中的VPS53与Beclin1的表达呈正相关。过表达VPS53促进自噬小体的形成。过表达VPS53可上调Beclin1蛋白的表达水平(P<0.05)。自噬抑制剂可逆转过表达VPS53对LoVo细胞增殖的抑制作用(P<0.05)。过表达miR-31-5p可下调Beclin1的RNA表达水平,过表达VPS53可逆转该现象(均P<0.05)。结论miR-31-5p是结直肠癌发生发展中的重要调控因子,miR-31-5p在结直肠癌细胞中高表达。miR-31-5p可能通过下调VPS53来抑制Beclin1的表达,通过抑制自噬来促进结直肠癌细胞的增殖。

Objectives To investigate the effect of miR-31-5p on colorectal cancer(CRC)cell proliferation through VPS53-me⁃diated autophagy signaling pathway.Methods Human colorectal mucosal cell(FHC),CRC cell lines HT29,HCT116,SW480,and LoVo were selected.Expression of miR-31-5p in FHC and CRC cells,the effect of miR-31-5p on VPS53 RNA,and the effect of miR-31-5p and VPS53 on Beclin1 RNA were analyzed by qPCR.Cell vitality was measured by CCK-8.Genes that could target miR-31-5p were screened by Targetscan database and validated using dual-luciferase reporter assay.The correlation of VPS53 and Beclin1 expression was analyzed by GEPIA database.The effect of VPS53 on autophagy was assessed by electron microscopy.The effect of VPS53 on Beclin1 protein expression was assessed by Western blot analysis.Results The expression of miR-31-5p in HCT116,LoVo,HT29,SW480 cells were significantly higher than that in the FHC(P<0.05).Overexpression of miR-31-5p promoted LoVo cell proliferation(P<0.05).miR-31-5p could target VPS53,the overexpression of miR-31-5p can low⁃er the expression of VPS53 RNA(P<0.05).Overexpression of VPS53 could inhibit LoVo cell proliferation and reverse the effect of miR-31-5p on promoting LoVo cell proliferation(P<0.05).The expression of VPS53 was correlated positively with that of Be⁃clin1 in CRC tissue.Overexpression of VPS53 could promote autophagosome formation and increase Beclin1 protein(P<0.05).The autophagy inhibitor could reverse the inhibitory effect of VPS53 on LoVo cell proliferation(P<0.05).Overexpression of miR-31-5p down-regulated the expression of Beclin1 RNA,but overexpression of VPS53 could reverse this down-regulation(P<0.05).Conclusion miR-31-5p is one of the important modulating factors in the development of CRC.The expression of miR-31-5p is high in CRC cells.miR-31-5p may inhibit the expression of Beclin1 through down-regulation of VPS53 and promote CRC cell proliferation through inhibiting autophagy.