Rationale: Lung cancer is the most prevalent form of cancer and has a high mortality rate, making it a global public health concern. The N6-methyladenosine (m6A) modification is a highly dynamic and reversible process that is involved in a variety of essential biological processes. Using in vitro, in vivo, and multi-omics bioinformatics, the present study aims to determine the function and regulatory mechanisms of the long non-coding (lnc)RNA zinc ribbon domain-containing 1-antisense 1 (ZNRD1-AS1).
Methods: The RNAs that were bound to the m6A 'reader' were identified using YTH domain-containing 2 (YTHDC2) RNA immunoprecipitation (RIP)-sequencing. Utilizing methylated RIP PCR/quantitative PCR, pull-down, and RNA stability assays, m6A modification and ZNRD1-AS1 regulation were analyzed. Using bioinformatics, the expression levels and clinical significance of ZNRD1-AS1 in lung cancer were evaluated. Using fluorescent in situ hybridization and quantitative PCR assays, the subcellular location of ZNRD1-AS1 was determined. Using cell migration, proliferation, and angiogenesis assays, the biological function of ZNRD1-AS1 in lung cancer was determined. In addition, the tumor suppressor effect of ZNRD1-AS1 in vivo was validated using a xenograft animal model. Through bioinformatics analysis and in vitro assays, the downstream microRNAs (miRs) and competing endogenous RNAs were also predicted and validated.
Results: This study provided evidence that m6A modification mediates YTHDC2-mediated downregulation of ZNRD1-AS1 in lung cancer and cigarette smoke-exposed cells. Low levels of ZNRD1-AS1 expression were linked to adverse clinicopathological characteristics, immune infiltration, and prognosis. ZNRD1-AS1 overexpression was shown to suppress lung cancer cell proliferation, migration, and angiogenesis in vitro and in vivo, and to reduce tumor growth in nude mice. ZNRD1-AS1 expression was shown to be controlled by treatment of cells with either the methylation inhibitor 3-Deazaadenosine or the demethylation inhibitor Meclofenamic. Furthermore, the miR-942/tensin 1 (TNS1) axis was demonstrated to be the downstream regulatory signaling pathway of ZNRD1-AS1.
Conclusions: ZNRD1-AS1 serves an important function and has clinical relevance in lung cancer. In addition, the findings suggested that m6A modification could mediate the regulation of the ZNRD1-AS1/miR-942/TNS1 axis via the m6A reader YTHDC2.
Keywords: Cigarette smoke; Immune infiltration; Lung cancer; TNS1; lncRNA ZNRD1-AS1; m6A RNA methylation; miR-942.
© 2022. The Author(s).