Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

Downregulation of lncRNA ZNF582-AS1 due to DNA hypermethylation promotes clear cell renal cell carcinoma growth and metastasis by regulating the N(6)-methyladenosine modification of MT-RNR1

Abstract

Background: Emerging evidence confirms that lncRNAs (long non-coding RNAs) are potential biomarkers that play vital roles in tumors. ZNF582-AS1 is a novel lncRNA that serves as a potential prognostic marker of cancers. However, the specific clinical significance and molecular mechanism of ZNF582-AS1 in ccRCC (clear cell renal cell carcinoma) are unclear.

Methods: Expression level and clinical significance of ZNF582-AS1 were determined by TCGA-KIRC data and qRT-PCR results of 62 ccRCCs. DNA methylation status of ZNF582-AS1 promoter was examined by MSP, MassARRAY methylation and demethylation analysis. Gain-of-function experiments were conducted to investigate the biological roles of ZNF582-AS1 in the phenotype of ccRCC. The subcellular localization of ZNF582-AS1 was detected by RNA FISH. iTRAQ, RNA pull-down and RIP-qRT-PCR were used to identify the downstream targets of ZNF582-AS1. rRNA MeRIP-seq and MeRIP-qRT-PCR were utilized to examine the N(6)-methyladenosine modification status. Western blot and immunohistochemistry assays were used to determine the protein expression level.

Results: ZNF582-AS1 was downregulated in ccRCC, and decreased ZNF582-AS1 expression was significantly correlated with advanced tumor stage, higher pathological stage, distant metastasis and poor prognosis. Decreased ZNF582-AS1 expression was caused by DNA methylation at the CpG islands within its promoter. ZNF582-AS1 overexpression inhibited cell proliferative, migratory and invasive ability, and increased cell apoptotic rate in vitro and in vivo. Mechanistically, we found that ZNF582-AS1 overexpression suppressed the N(6)-methyladenosine modification of MT-RNR1 by reducing rRNA adenine N(6)-methyltransferase A8K0B9 protein level, resulting in the decrease of MT-RNR1 expression, followed by the inhibition of MT-CO2 protein expression. Furthermore, MT-RNR1 overexpression reversed the decreased MT-CO2 expression and phenotype inhibition of ccRCC induced by increased ZNF582-AS1 expression.

Conclusions: This study demonstrates for the first time that ZNF582-AS1 functions as a tumor suppressor gene in ccRCC and ZNF582-AS1 may serve as a potential biomarker and therapeutic target of ccRCC.

Keywords: DNA methylation; MT-RNR1; N(6)-methyladenosine modification; ZNF582-AS1; ccRCC.

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