Long non-coding RNA X-inactive specific transcript suppresses the progression of hepatocellular carcinoma through microRNA-221-3p-targeted regulation of O6-methylguanine-DNA methyltransferase

MicroRNA-221-3p (miR-221-3p) is an important regulator involved in the progression and prognosis of various cancers. In this study, we aimed to investigate the diagnostic and prognostic value of miR-221-3p expression along with long non-coding RNA X–inactive specific transcript (XIST), which was identified as its upstream regulator in hepatocellular carcinoma (HCC) by bioinformatics analysis, and further validated by RNA immunoprecipitation (RIP) and dual-luciferase reporter assays. Their expression was measured in tumor tissues and corresponding non-tumor tissues by quantitative real-time PCR (qRT-PCR), which revealed that XIST was weakly expressed in HCC cells and tumors, while miR-221-3p was overexpressed. Complete knockdown of XIST enhanced HCC cell proliferation and migration and inhibited apoptosis, as observed by MTT, transwell, and flow cytometry experiments, respectively. Animal studies validated that XIST knockdown induces tumor growth <i>in vivo</i>. In contrast, upregulation of XIST in HCC cells suppressed their proliferation and migration, stimulated apoptosis, and retarded the growth rate of tumors <i>in vivo</i>. These effects were partially reversed by upregulating miR-221-3p expression. Furthermore, we demonstrated that O⁶-methylguanine-DNA methyltransferase (MGMT) is a downstream target of miR-221-3p. It was weakly expressed in HCC cells and tumors and showed a negative correlation with miR-221-3p. Forced MGMT expression repressed proliferation and migration and enhanced apoptosis in HCC cells. Nevertheless, these anti-tumor effects induced by MGMT overexpression could be abolished by miR-221-3p upregulation. Collectively, our findings reveal that XIST blocks the development of HCC through miR-221-3p-targeted regulation of MGMT. This reveals a new mechanism involved in the development of HCC.

微小RNA-221-3p（MicroRNA-221-3p，miR-221-3p）是参与多种癌症发生发展与预后调控的重要分子。本研究旨在探究miR-221-3p与长链非编码RNA X染色体失活特异性转录本（long non-coding RNA X–inactive specific transcript，XIST）的表达水平对肝细胞癌（hepatocellular carcinoma，HCC）的诊断及预后价值；其中XIST经生物信息学分析被证实为miR-221-3p的上游调控分子，并通过RNA免疫沉淀（RNA immunoprecipitation，RIP）与双荧光素酶报告基因实验（dual-luciferase reporter assays）进一步验证。本研究通过实时定量聚合酶链式反应（quantitative real-time PCR，qRT-PCR）检测了肿瘤组织及配对癌旁组织中的二者表达水平，结果显示XIST在肝癌细胞与肿瘤组织中呈低表达，而miR-221-3p则呈过表达。分别通过MTT实验（MTT）、Transwell实验（Transwell）与流式细胞术（flow cytometry）检测发现，完全敲低XIST可促进肝癌细胞的增殖与迁移，并抑制其凋亡。动物实验验证了敲低XIST可在体内促进肿瘤生长。与之相反，在肝癌细胞中过表达XIST则可抑制其增殖与迁移，促进细胞凋亡，并在体内延缓肿瘤生长速度。上述效应可通过上调miR-221-3p的表达得到部分逆转。此外，本研究证实O⁶-甲基鸟嘌呤-DNA甲基转移酶（O⁶-methylguanine-DNA methyltransferase，MGMT）是miR-221-3p的下游靶基因。MGMT在肝癌细胞与肿瘤组织中呈低表达，且与miR-221-3p的表达呈负相关。过表达MGMT可抑制肝癌细胞的增殖与迁移，并促进其凋亡。然而，过表达MGMT所产生的上述抗肿瘤效应可因miR-221-3p的上调而被抵消。综上，本研究结果表明XIST通过靶向调控miR-221-3p进而介导对MGMT的表达调控，从而阻滞肝癌的发生发展；该研究揭示了肝癌发生发展的全新分子机制。