miR-143-3p靶向MCL1调节线粒体氧化磷酸化抑制宫颈癌细胞的增殖和迁移研究

MiR-143-3p targets MCL1 to regulate mitochondrial oxidative phosphorylation and inhibit the proliferation and migration of cervical cancer cells

摘要

目的 本研究旨在探讨miR-143在宫颈癌(CC)细胞增殖和迁移中的作用机制,特别是其通过靶向骨髓细胞白血病1(MCL1)基因调节线粒体氧化磷酸化过程的影响。方法 将宫颈癌细胞系HeLa分成6组:NC-mimic组(转染阴性对照NC-mimic)、miR-143-3p mimic(转染miR-143-3p mimic),oe-NC(转染阴性对照oe-NC)、oe-MCL1组(转染oe-MCL1)、miR-143-3p mimic+oe-NC(共转染miR-143-3p mimic和oe-NC)、miR-143-3p mimic+oe-MCL1(共转染miR-143-3p mimic和oe-MCL1)。采用实时荧光定量聚合酶链式反应(PCR)、蛋白免疫印迹、克隆形成实验、细胞划痕实验检测miR-143-3p与MCL1的表达水平、细胞增殖和迁移。分别检测其各组线粒体呼吸链复合体Ⅰ-Ⅴ酶活性、线粒体跨膜电位、细胞内三磷酸腺苷(ATP)含量以及线粒体活性氧(ROS)水平的变化。结果 在CC细胞系中,miR-143-3p表达显著下调(F=432.860,P<0.05),MCL1 mRNA和MCL1蛋白的表达显著上调(F=503.883、430.645,P<0.05)。与NC-mimic组相比,miR-143-3p mimic组的miR-143-3p的表达量与ROS水平显著增加(t=19.227、14.658,P<0.05);MCL1的mRNA和蛋白表达(t=19.941、19.078)、细胞克隆数和划痕修复率(t=12.709、9.333)、线粒体呼吸链复合体Ⅰ-Ⅴ的酶活性(t=9.177、6.123、7.607、5.777、96.12)、跨膜电位和ATP浓度(t=51.783、12.335)均显著降低(P<0.05)。与miR-143-3p mimic组相比,miR-143-3p mimic+oe-MCL1组的MCL1的mRNA和蛋白表达(t=7.703、8.908)、细胞克隆数和划痕修复率(t=5.282、4.713)、线粒体呼吸链复合体Ⅰ-Ⅴ的酶活性(t=5.918、4.772、5.637、4.189、5.717)、跨膜电位和ATP浓度(t=49.843、7.005)均显著增加(P<0.05);ROS水平显著降低(t=5.648,P<0.05)。结论 miR-143在宫颈癌细胞系中显著下调,上调miR-143-3p可显著抑制宫颈癌细胞的氧化磷酸化,而抑制其增殖与迁移,其机制可能是通过miR-143-3p靶向抑制下游基因MCL1实现的。

Abstract

Objective This study aimed to investigate the mechanism of miR-143-3p in the proliferation and migration of cervical cancer (CC) cells,particularly its effect via targeting the myeloid cell leukemia 1 (MCL1) gene to regulate the mitochondrial oxidative phosphorylation process. Methods The CC cell line HeLa was divided into six groups: NC-mimic group (transfected with negative control NC-mimic),miR-143-3p mimic group (transfected with miR-143-3p mimic),oe-NC group (transfected with negative control oe-NC),oe-MCL1 group (transfected with oe-MCL1),miR-143-3p mimic+oe-NC group (co-transfected with miR-143-3p mimic and oe-NC),and miR-143-3p mimic+oe-MCL1 group (co-transfected with miR-143-3p mimic and oe-MCL1). Quantitative real-time polymerase chain reaction (PCR),western blotting,colony formation assays,and wound healing assays were performed to evaluate the expression levels of miR-143-3p and MCL1,as well as cell proliferation and migration. The activities of mitochondrial respiratory chain complexes Ⅰ-Ⅴ,mitochondrial membrane potential,intracellular adenosine triphosphate (ATP) content,and mitochondrial reactive oxygen species (ROS) levels were measured in each group. Results In CC cell lines,miR-143-3p expression was significantly downregulated (F=432.860,P<0.05),whereas the mRNA and protein expression levels of MCL1 were significantly upregulated (F=503.883 and 430.645,respectively,P<0.05). Compared with the NC-mimic group,the miR-143-3p mimic group showed significantly increased miR-143-3p expression and ROS levels (t=19.227 and 14.658,respectively,P<0.05). The mRNA and protein expression levels of MCL1 (t=19.941 and 19.078),the number of cell colonies and wound healing rate (t=12.709 and 9.333),the enzymatic activities of mitochondrial respiratory chain complexes Ⅰ-Ⅴ (t=9.177,6.123,7.607,5.777,and 96.12),as well as mitochondrial membrane potential and ATP levels (t=51.783 and 12.335) were all significantly decreased (P<0.05). Compared with the miR-143-3p mimic group,the miR-143-3p mimic+oe-MCL1 group exhibited significantly increased MCL1 mRNA and protein expression (t=7.703 and 8.908),cell colony number and wound healing rate (t=5.282 and 4.713),enzymatic activities of mitochondrial respiratory chain complexes Ⅰ-Ⅴ (t=5.918,4.772,5.637,4.189,and 5.717),as well as mitochondrial membrane potential and ATP levels (t=49.843 and 7.005) (P<0.05). The ROS level was significantly decreased (t=5.648,P<0.05). Conclusion miR-143 is significantly downregulated in CC cell lines. Upregulation of miR-143-3p markedly inhibits oxidative phosphorylation in CC cells,thereby suppressing their proliferation and migration. This effect may be mediated through the targeted inhibition of the downstream gene MCL1 by miR-143-3p.

关键词

miR-143-3p / 骨髓细胞白血病1 / 氧化磷酸化 / 宫颈癌细胞 / 增殖 / 迁移

Key words

miR-143-3p / myeloid cell leukemia 1 / oxidative phosphorylation / cervical cancer cell / proliferation / migration

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基金资助

河北省保定市科技计划项目(2241ZF195)