外泌体miR-150-5p通过靶向调控p53促进肝细胞癌的增殖、侵袭和迁移

Exosomal miR-150-5p suppresses the proliferation,invasion,and migration of Hepatocellular Carcinoma Cells via targeting p53

目的 探讨外泌体miR-150-5p通过靶向调控p53对肝癌细胞增殖、侵袭和迁移的影响及分子机制。方法 选取吉安市中心人民医院2019年1月至2022年12月收治的39例肝癌(HCC)患者癌组织及癌旁组织,采用差速离心法提取外泌体并鉴定,qRT-PCR检测肿瘤组织及外泌体中miR-150-5p和p53表达;采用双荧光素酶报告基因实验验证miR-150-5p与p53的互作;培养人肝癌细胞株(HCCLM3),给予癌组织及癌旁组织分离提取的外泌体处理(ExoNor和ExoHCC),同时给予GW 4869抑制细胞本身分泌外泌体,激光共聚焦显微镜对外泌体与受体细胞结合内化进行追踪;同时采用脂质体3000向HCCLM3细胞转染miR-150-5p mimics和miR-150-5p inhibitor;Transwell检测肝癌细胞迁移及侵袭能力,CCK-8检测细胞增殖,采用流式细胞术检测细胞凋亡率;qRT-PCR检测各组细胞miR-150-5p和p53表达,Western blot检测E-cadherin、N-cadherin和p53蛋白表达。结果 HCC组织中提取的外泌体miR-150-5p相对表达量明显高于邻近正常癌旁组织;与癌旁组织提取的外泌体相比,HCC细胞系HCCLM3能摄取肿瘤组织提取的外泌体miR-150-5p,从而增加细胞的增殖和迁移能力。外泌体能携带miR-150-5p并靶向负调控p53蛋白;共孵育ExoHCC或转染miR-150-5p mimics组HCCLM3细胞中N-cadherin表达较对照组明显降低,E-cadherin表达较对照组明显升高,而miR-150-5p inhibitor抑制了肿瘤的迁移和增殖能力,促进肿瘤细胞凋亡。结论 HCC细胞分泌的外泌体可被周围肿瘤细胞内吞;肝癌细胞分泌的外泌体及过表达肝癌细胞中的miR-150-5p可以靶向抑制周围细胞p53表达,增加细胞迁移及侵袭。

Objective To explore the effects and molecular mechanism of exosomal miR-150-5p on HCC cell proliferation,invasion and migration through targeted regulation of p53.Methods A total of 39 cases of Hepatocellular carcinoma(HCC)tissues and adjacent tissues of HCC patients in Ji′an Central People′s Hospital from January 2019 to December 2022 were selected.Exosomes were extracted and identified by differential centrifugation,and miR-150-5p and p53 expression in tumor tissues and exosomes were detected by qRT-PCR;Determination of dual luciferase reporter gene method was used to verify the interaction between miR-150-5p and p53;Human hepatcancer cell line(HCCLM3)was treated with exosomes isolated from tumor tissues and adjacent tissues,and GW4869 was given to inhibit the secretion of exosomes by cells themselves.L aser confocal microscopy to track binding and internalization of exosomes to recipient cells;Meanwhile,The miR-150-5p overexpression vector and miR-150-5p inhibitory expression vector were transfected into HCCLM3 cells use Lipofectamine 3000;The proliferation,the influence of HCC cell migration and invasion ability by Transwell and the detection of apoptosis rate by flow cytometry;The expression levels of miR-150-5p and p53 mRNA were detected by qRT-PCR.The expression levels of E-cadherin,N-cadherin and p53 protein were detected by Western blot.Results The relative expression of miR-150-5p from adjacent tissues was significantly lower than in HCC tissues;Compared with exosomes extracted from adjacent tissues,The HCCLM3 cells can uptake the exosomal miR-150-5p extracted from the tumor tissue,thereby increasing the cell proliferation and migration capacity.miR-150-5p could carried by exosomes and negatively regulated p53 protein;E-cadherin expression was increased in HCCLM3 cells incubated with Exo HCC or transfected with miR-150-5p mimics,compared with control cells,while miR-150-5p inhibitor inhibited tumor migration and proliferation capacity and promoted tumor cell apoptosis.Conclusion Exosomes secreted by HCC cells can be endocytosed by surrounding tumor cells;Exosomes secreted by HCC cells and overexpression of miR-150-5p in HCC cells can target and inhibit p53 expression in surrounding cells and increase cell migration and invasion.