miR-125b表达对肝癌细胞增殖和PI3K/Akt信号通路的影响及其调控靶点分析

The effects of miR-125b on cell proliferation and the PI3K/Akt signaling pathway in hepatocellular carcinoma and target analysis

于歌 ¹穆瀚 ¹刘东明 ¹李慧锴 ¹崔云龙 ¹李强¹

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作者信息

1. 天津医科大学肿瘤医院肝胆肿瘤科国家恶性肿瘤临床医学研究中心天津市恶性肿瘤临床医学研究中心天津市肿瘤防治重点实验室,天津 300060
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摘要

目的分析微小RNA(miR)-125b对肝癌细胞增殖、磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路的影响以及是否通过靶向调控Polo样激酶4(PLK4)介导的.方法收集2022年3月至2023年3月在天津医科大学肿瘤医院手术切除的65例肝细胞癌患者的癌组织及癌旁组织,其中男性33例,女性32例,年龄(60.1±5.6)岁.实时荧光定量聚合酶链式反应检测肝癌、癌旁组织及肝癌细胞中miR-125a、miR-125b的表达,选择低表达肝癌细胞转染阴性对照(NC)miR、miR-125a、miR-125b序列,随后共转染miR-NC与NC质粒、miR-125b序列与NC质粒、miR-125b序列与PLK4质粒.细胞计数实验检测细胞增殖,Western印迹检测PLK4、磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)的表达,生物信息学分析和双荧光素酶报告基因检测miR-125b靶向PLK4.结果肝癌患者癌组织中miR-125a、miR-125b的相对表达分别为(0.62±0.08)、(0.58±0.07),低于癌旁组织的(1.00±0.12)和(1.00±0.13),差异均有统计学意义(t=21.24、22.93,P=0.005、P＜0.001).选择低表达miR-125a、miR-125b的肝癌HepG2细胞和抑制PI3K/Akt的miR-125b用于转染.生物信息学分析和双荧光素酶报告基因检测显示miR-125b与PLK4结合.miR-125b序列抑制HepG2细胞增殖及PI3K、Akt磷酸化,共转染miR-125b序列与PLK4质粒时,PLK4部分逆转miR-125b抑制HepG2细胞增殖及 PI3 K/Akt 的磷酸化(0.91±0.07)比(0.41±0.04).、(0.97±0.08)比(0.32±0.03),差异有统计学意义(t=13.87、17.01,均P＜0.001).结论 miR-125b抑制肝癌HepG2细胞增殖及PI3K/Akt磷酸化激活是由靶向抑制PLK4介导的.

Abstract

Objective To investigate the effects of microRNA(miR)-125b on the proliferation and phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway of hepatocellular carcinoma(HCC)cell by targeting Polo like kinases(PLK4).Methods The tumor tissues and adjacent tissues of 65 patients with HCC were collected from March 2022 to March 2023 in Tianjin Medical University Cancer Hospital,including 33 males and 32 females,aged(60.1±5.6)years.The expressions of miR-125a and miR-125b in liver cancer,adjacent tissues and liver cancer cells were detected by fluorescence quantitative polymerase chain reaction.Low expression liver cancer cell was selected to transfect negative control(NC)sequences of miR,miR-125a and miR-125b.Subsequently,miR-NC and NC plasmid,miR-125b sequence and NC plasmid,and miR-125b sequence and PLK4 plasmid were co-transfected.Cell proliferation was detected by cell counting assay,the expression of PLK4,phosphorylated PI3K(p-PI3K)and phosphorylated Akt(p-Akt)was detected by Western blot,and miR-125b-targeting PLK4 were detected by bioinformatics analysis and dual luciferase reporter gene.Results The relative expressions of miR-125a and miR-125b in HCC patients were(0.62±0.08)and(0.58±0.07),respectively,lower than those in adjacent tissues(1.00±0.12)and(1.00±0.13),and the differences were statistically significant(t=21.24,22.93,P=0.005,P＜0.001).HepG2 cells with low expression of miR-125a and miR-125b and miR-125b targe-ting PI3K/Akt were selected for transfection.Bioinformatic analysis and dual luciferase reporter gene assay confirmed that miR-125b binds to PLK4.Overexpression of miR-125b could inhibit the proliferation of HepG2 cells and the expression of p-PI3K and p-Akt,while overexpression of PLK4 could partially reverse the proliferation inhibition caused by miR-125b and the expression of p-PI3K and p-Akt,(0.91±0.07)vs(0.41±0.04),(0.97±0.08)vs(0.32±0.03)(t=13.87,17.01,both P＜0.001).Conclusion The inhibitory effect of miR-125b on HepG2 cell proliferation and PI3K/Akt signaling pathway is partly mediated by targeted inhibition of PLK4.

关键词

癌,肝细胞/微小RNA-125b/Polo样激酶4/增殖/磷脂酰肌醇-3激酶/蛋白激酶B

Key words

Carcinoma,hepatocellular/MicroRNA 125b/Polo like kinases 4/Proliferation/Phosphoinositide 3-kinase/protein kinase B

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出版年

2024

中华肝胆外科杂志

中华医学会

中华肝胆外科杂志

CSTPCDCSCD北大核心

影响因子：1.846

ISSN：1007-8118

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