Yang Sijia, Li Ang, Lv Lihong, Zheng Zhihua, Liu Peiqing, Min Jun, Wei Jinxing
Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 510120 Guangzhou, Guangdong, China.
Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 510120 Guangzhou, Guangdong, China.
J Cancer. 2024 Jun 17;15(14):4448-4466. doi: 10.7150/jca.96500. eCollection 2024.
Hepatocellular carcinoma (HCC) is a major global health challenge. Chemotherapy can cause HCC cells to become senescent. Senescent HCC cells play an important role in inhibiting or promoting cancer by producing extracellular vesicles with a senescence-associated secretory phenotype (EV-SASP). miRNA can be strongly upregulated in EV-SASP during the aging process and can substantially alter the phenotypic characteristics of cells. MiRNA microarray analysis revealed that miRNA-146a-5p was highly expressed in oxaliplatin- and HO-induced senescent Huh7 cells, and RT‒PCR confirmed its significant upregulation in exosomes. The transcriptome sequencing results of Huh7 cells overexpressing miRNA-146a-5p suggested that miRNA-146a-5p could regulate HCC cell glycolysis. Subsequently, a dual luciferase assay was used to verify whether miRNA-146a-5p can interact with IRF7 to promote aging. The key functions of miRNA-146a-5p and IRF7 in aerobic glycolysis in liver cancer cells were determined through experiments analyzing glucose uptake, lactate production, the oxygen consumption rate (OCR) and the proton efflux rate (PER). Subsequently, the regulatory effect of IRF7 on the key glycolytic gene PFKL was confirmed through luciferase reporter assays. The western blot experiment results showed that miR-146a-5p can activate CHK2 and p53 phosphorylated proteins by targeting IRF7, and upregulate p21 protein. Overexpression of miRNA-146a-5p effectively inhibited the aerobic glycolytic function of HCC cells. Moreover, silencing IRF7 effectively inhibited aerobic glycolysis. MiR-146a-5p. MiR-146a-5p can activate the phosphorylation of CHK2 phosphorylation protein and its downstream protein p53 by targeting IRF7, and the activated p53 upregulates the expression of p21. Our study revealed that exosomal miRNA-146a-5p produced by aging HCC cells, can inhibit HCC cell proliferation through inhibiting aerobic glycolysis and promote HCC cell aging by activating CHK2/p53/p21 signaling way by targeting IRF7.
肝细胞癌（HCC）是一项重大的全球健康挑战。化疗可使HCC细胞衰老。衰老的HCC细胞通过产生具有衰老相关分泌表型的细胞外囊泡（EV-SASP）在抑制或促进癌症中发挥重要作用。在衰老过程中，miRNA在EV-SASP中可被强烈上调，并可显著改变细胞的表型特征。miRNA微阵列分析显示，miRNA-146a-5p在奥沙利铂和HO诱导的衰老Huh7细胞中高表达，RT-PCR证实其在外泌体中显著上调。过表达miRNA-146a-5p的Huh7细胞的转录组测序结果表明，miRNA-146a-5p可调节HCC细胞糖酵解。随后，采用双荧光素酶测定法验证miRNA-146a-5p是否能与IRF7相互作用以促进衰老。通过分析葡萄糖摄取、乳酸生成、耗氧率（OCR）和质子外排率（PER）的实验，确定了miRNA-146a-5p和IRF7在肝癌细胞有氧糖酵解中的关键作用。随后，通过荧光素酶报告基因测定法证实了IRF7对关键糖酵解基因PFKL的调节作用。蛋白质免疫印迹实验结果表明，miR-146a-5p可通过靶向IRF7激活CHK2和p53磷酸化蛋白，并上调p21蛋白。miRNA-146a-5p的过表达有效抑制了HCC细胞的有氧糖酵解功能。此外，沉默IRF7可有效抑制有氧糖酵解。MiR-146a-5p。MiR-146a-5p可通过靶向IRF7激活CHK2磷酸化蛋白及其下游蛋白p53的磷酸化，激活的p53上调p21的表达。我们的研究表明，衰老的HCC细胞产生的外泌体miRNA-146a-5p可通过抑制有氧糖酵解抑制HCC细胞增殖，并通过靶向IRF7激活CHK2/p53/p21信号通路促进HCC细胞衰老。
