MAL expression downregulation through suppressive H3K27me3 marks at the promoter in HPV16-related cervical cancers is prognostically relevant and manifested by the interplay of novel MAL antisense long noncoding RNA AC103563.8, E7 oncoprotein and EZH2.

IF 5.4 3区材料科学 Q2 CHEMISTRY, PHYSICAL

ACS Applied Energy Materials Pub Date : 2024-03-10 DOI:10.1186/s13148-024-01651-9

Abarna Sinha, Abhisikta Ghosh, Arnab Ghosh, Sonia Mathai, Jaydip Bhaumik, Asima Mukhopadhyay, Arindam Maitra, Nidhan K Biswas, Sharmila Sengupta

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引用次数: 0

Abstract

Background: MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2.

Results: Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients.

Conclusion: AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.

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新型 MAL 反义长非编码 RNA AC103563.8、E7 肿瘤蛋白和 EZH2 相互作用，通过抑制启动子上的 H3K27me3 标记下调 HPV16 相关宫颈癌中的 MAL 表达，这与预后相关。

背景：MAL（T淋巴细胞成熟相关蛋白）在包括宫颈癌（CaCx）在内的大多数癌症中因启动子超甲基化而高度下调。长非编码 RNA 基因（lncGs）通过与人类乳头瘤病毒（HPV）编码的肿瘤蛋白相互作用，并通过表观遗传调控编码基因的表达，在 CaCx 发病机制中发挥着关键作用。因此，我们试图通过研究MAL反义lncRNA AC103563.8、E7肿瘤蛋白和PRC2复合蛋白EZH2的交互作用，破译MAL下调在HPV16相关CaCx发病机制中的影响和潜在机制：通过链特异性RNA测序，我们证实了MAL的表达下调与携带HPV16的CaCx患者总生存率低有关，同时也与MAL的反义长非编码RNA（lncRNA）AC103563.8有关。与正常人相比，MAL 与 AC103563.8 的正相关性在患者中明显较高。MAL的表达下调与携带HPV16的CaCx患者的总生存率较低有显著相关性，而AC103563.8则没有显示出这种相关性。我们利用 ChIP-qPCR 在 HPV16 阳性的 SiHa 细胞中证实了染色质抑制标记 H3K27me3 在 MAL 启动子处的富集。随后在此类细胞中敲除 E7 会显著增加 MAL 的表达，同时减少 EZH2 的表达和 MAL 启动子上的 H3K27me3 标记。硅学分析表明，E7和EZH2都有可能与AC103563.8在相同的结合域发生相互作用。分别用抗 EZH2 和抗 E7 抗体进行 RNA 免疫沉淀，随后在 E7 沉默和未受干扰的 SiHa 细胞中进行定量 PCR 分析，证实了 AC103563.8 与 EZH2 和 E7 的相互作用。很明显，AC103563.8 似乎排除了 EZH2 并与 E7 结合，未能阻断患者体内 EZH2 的功能。因此，在E7存在的情况下，EZH2表达的增强可能会通过H3K27me3标记使MAL启动子失活，这也证实了我们之前在患者体内发现的MAL表达下调的结果：因此，AC103563.8-E7-EZH2轴似乎在HPV16-CaCx发病机制中通过染色质失活对MAL的表达起着至关重要的调控作用，值得开发治疗策略。

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来源期刊

ACS Applied Energy Materials Materials Science-Materials Chemistry

CiteScore

10.30

自引率

6.20%

发文量

1368

期刊介绍： ACS Applied Energy Materials is an interdisciplinary journal publishing original research covering all aspects of materials, engineering, chemistry, physics and biology relevant to energy conversion and storage. The journal is devoted to reports of new and original experimental and theoretical research of an applied nature that integrate knowledge in the areas of materials, engineering, physics, bioscience, and chemistry into important energy applications.