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. 2024 Mar 10;16(1):40.
doi: 10.1186/s13148-024-01651-9.

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

Abarna Sinha  1 , Abhisikta Ghosh  1 , Arnab Ghosh  1 , Sonia Mathai  2 , Jaydip Bhaumik  2 , Asima Mukhopadhyay  3 , Arindam Maitra  1 , Nidhan K Biswas  1 , Sharmila Sengupta  4
Affiliations

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

Abarna Sinha et al. Clin Epigenetics. .

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.

Keywords: Antisense long noncoding RNA AC103563.8; Cervical cancer; EZH2-mediated H3K27me3 marks; HPV16-E7; MAL; Patient survival.

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Conflict of interest statement

None. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Fig. 1
Fig. 1
Visualisation of the expression and correlative relationship of the DEG pair: MAL- AC103563.8. A Unsupervised hierarchical clustering demarcating the CaCx patients from the normal individuals based on MAL and AC103563.8 gene expression profiles. The heatmap represents normalised counts, which were log2 transformed after adding constant 1 to all values of genes. The rows represent the gene names, and the columns represent the samples (blue bar represents the CaCx patient samples and pink bar represents the samples from normal individuals). Pearson’s correlation coefficient of MAL and AC103563.8 among (B) CaCx cases and (C) normal healthy individuals, respectively. Real-time qPCR based relative expression of D MAL and E AC103563.8 (MAL-AS1) in an additional cohort of HPV16-positive CaCx patients compared to healthy individuals. Relative gene expression reflects expression of the gene minus expression of the house-keeping gene (GAPDH), and is inversely proportional to the fold change. Thus, higher relative gene expression designates lower fold change. The data are represented as mean ± SD
Fig. 2
Fig. 2
Kaplan–Meier survival analysis of MAL. KM plotter showed MAL to be significantly associated with poor patient overall-survival at low expression in (A) our cohort and in (B) TCGA-CESC cohort
Fig. 3
Fig. 3
Knockdown of E7 by siRNA against E7 in HPV16-positive CaCx cell line, SiHa. A TaqMan-based qRT-PCR revealed 69% knockdown in HPV16 E7 expression in transfected group (T-SiHa) compared to the untransfected (UT-SiHa). B Upper panel: Western blot showing knockdown of E7 protein (19KDa) in transfected group (T-SiHa) compared to the untransfected (UT-SiHa). Lower panel: Western blot performed for house-keeping protein β-actin (42 KDa) for UT-SiHa (untransfected SiHa) and T-SiHa (transfected SiHa). The data are represented as mean ± SD from two independent experiments. Chromatin immunoprecipitation (ChIP)-qPCR with H3K27me3 antibody (C, D). C Fold enrichment showing the depletion of H3K27me3 at the MAL promoter in the transfected (T SiHa) when compared to that of untransfected (UT SiHa). EVX1 promoter is shown as the positive control for H3K27me3 in UT and T SiHa. The data are represented as mean ± SD from three independent experiments. D % input showing the depletion of H3K27me3 at the MAL promoter in transfected (T SiHa) when compared to that of untransfected (UT SiHa). Effect of E7 knockdown in HPV16-positive SiHa cells (E–G). E Relative expression of MAL F EZH2 G AC103563.8, respectively, in transfected (T SiHa) when compared to that of untransfected (UT SiHa). These plots depict the relative gene expression, i.e. expression of the gene—expression of the house-keeping gene (GAPDH). Relative gene expression is inversely proportional to the fold change. Thus, higher relative gene expression designates lower fold change. The data are represented as mean ± SD from two independent experiments
Fig. 4
Fig. 4
RNA immunoprecipitation (RIP)-qPCR with E7 and EZH2 antibody. A Percent input showing the enrichment of AC103563.8 in SiHa compared to that of IgG control upon E7 Immunoprecipitation. B Fold enrichment showing the enrichment of AC103563.8 in SiHa compared to that of IgG control upon E7 Immunoprecipitation. The data are represented as mean ± SD from two independent experiments. C Percent input showing the enrichment of AC103563.8 in transfected SiHa (T SiHa) compared to that of untransfected SiHa (UT SiHa) upon EZH2 Immunoprecipitation. D Fold enrichment showing the enrichment of AC103563.8 in transfected SiHa (T SiHa) compared to that of untransfected SiHa (UT SiHa) upon EZH2 Immunoprecipitation. NEAT1 is shown as the positive control for EZH2 interaction in UT and T SiHa. The data are represented as mean ± SD from two independent experiments
Fig. 5
Fig. 5
Hypothetical functional model. Interplay of HPV16-E7/AC103563.8/EZH2/MAL axis in CaCx pathogenesis
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