苦木碱F通过miR-331-3p调控宫颈癌HeLa细胞增殖及凋亡的机制研究

目的:探讨苦木碱F调控miR-331-3p在宫颈癌HeLa细胞增殖及凋亡中的作用及机制。方法:MTT法检测苦木碱F对宫颈癌HeLa细胞增殖的影响; 流式细胞术检测苦木碱F对宫颈癌HeLa细胞凋亡的影响; RT-qPCR检测苦木碱F处理HeLa细胞后miR-331-3p的表达; miR-331-3p mimic或miR-331-3p inhibitor 转染HeLa细胞,经苦木碱F处理后检测HeLa细胞的增殖及凋亡情况; 使用生物信息学和双荧光素酶报告基因实验预测及验证miR-331-3p与蛋白酶体活化复合物3(PSME3)的靶向关系; Western blot检测HeLa细胞中凋亡相关蛋白表达情况。结果:苦木碱F能显著抑制HeLa细胞增殖,促进凋亡,且具有浓度依赖性(P<0.01); 苦木碱F显著上调HeLa细胞中miR-331-3p的表达(P<0.01); miR-331-3p mimic能显著抑制HeLa细胞增殖能力,促进细胞凋亡; miR-331-3p inhibitor能显著降低苦木碱F对HeLa细胞增殖的抑制以及凋亡的促进作用(P<0.05); 生物信息学预测PSME3是miR-331-3p的靶基因之一,双荧光素酶报告基因结果显示miR-331-3p与PSME3 3'UTR靶向结合; 与苦木碱F+NC inhibitor +NC-siRNA组比较,苦木碱F+miR-331-3p inhibitor +NC-siRNA组中细胞存活率升高,细胞凋亡率降低,β-catenin和Bcl-2蛋白水平升高,Bax蛋白水平降低,而敲低PSME3能逆转这一结果,差异有统计学意义(P<0.05)。结论:苦木碱F通过介导miR-331-3p/PSME3轴抑制宫颈癌HeLa细胞增殖,促进凋亡,发挥抗肿瘤作用。

The mechanism of Picrasidine F regulating the proliferation and apoptosis of cervical cancer HeLa cells through miR-331-3p

Objective:To investigate the effect of Picrasidine F-mediated miR-331-3p on the proliferation and apoptosis of cervical cancer HeLa cells and its underlying mechanism.Methods:MTT assay was used to detect the effect of Picrasidine F on the proliferation of cervical cancer HeLa cells.The effect of Picrasidine F on apoptosis of cervical carcinoma HeLa cells was detected by flow cytometry.The expression of miR-331-3p in Hela cells treated with Picrasidine F was detected by RT-qPCR.MiR-331-3p mimic or miR-331-3p inhibitor was transfected into HeLa cells,and the proliferation and apoptosis of HeLa cells were detected after treatment with Picrasidine F.Bioinformatics and dual luciferase reporter gene assay were used to predict and verify the targeting relationship between miR-331-3p and proteasome activator complex subunit 3(PSME3).The expression of apoptosis-related protein in HeLa cells was detected by Western blot.Results:Picrasidine F significantly inhibited the proliferation and promoted apoptosis of HeLa cells in a concentration-dependent manner(P<0.01).Picrasidine F significantly up-regulated the expression of miR-331-3p in HeLa cells(P<0.01).MiR-331-3p mimic could significantly inhibit the proliferation of HeLa cells and promote cell apoptosis.MiR-331-3p inhibitor significantly reduced the inhibitory effect of Picrasidine F on HeLa cell proliferation and the promotion of apoptosis(P<0.05).Bioinformatics predicted that PSME3 was one of the target genes of miR-331-3p,and dual luciferase reporter gene results showed that miR-331-3p was targeted to bind PSME33’UTR.Compared with the Picrasidine F+NC inhibitor+NC-siRNA group,the survival rate and apoptosis rate of cells in the Picrasidine F+miR-331-3p inhibitor+NC-siRNA group were increased,the protein levels ofβ-caten in and Bcl-2 were increased,and the protein levels of Bax were decreased,while knockdown of PSME3 could reverse these results,and the difference was statistically significant(P<0.05).Conclusion:Picrasidine F played an anti-tumor role by mediating miR-331-3p/PSME3 axis to inhibit the proliferation and promote apoptosis of cervical cancer HeLa cells.