Long Non-coding RNA IRAIN Inhibits VEGFA Expression via Enhancing Its DNA Methylation Leading to Tumor Suppression in Renal Carcinoma

Abstract

<b>Aims:</b> Long non-coding RNA <i>IRAIN</i> (lncRNA <i>IRAIN</i>) plays a critical role in numerous malignancies. However, the function of lncRNA <i>IRAIN</i> in renal carcinoma (RC) remains enigmatic. The purpose of this study is to characterize the effects of lncRNA <i>IRAIN</i> on RC progression. <b>Methods:</b> The expression pattern of lncRNA <i>IRAIN</i> and the vascular endothelial growth factor A (VEGFA) in RC tissues and cells was characterized by RT-qPCR and Western blot analysis. The roles of lncRNA <i>IRAIN</i> and VEGFA in the progression of RC were studied by gain- or loss-of-function experiments. Bioinformatics data analysis was used to predict CpG islands in the <i>VEGFA</i> promoter region. MSP was applied to detect the level of DNA methylation in RC cells. The interaction between lncRNA <i>IRAIN</i> and VEGFA was identified by RNA immunoprecipitation and RNA-protein pull down assays. Recruitment of DNA methyltransferases (Dnmt) to the <i>VEGFA</i> promoter region was achieved by chromatin immunoprecipitation. The subcellular localization of lncRNA <i>IRAIN</i> was detected by fractionation of nuclear and cytoplasmic RNA. Cell viability was investigated by CCK-8 assay, cell migration was tested by transwell migration assay, and apoptosis was analyzed by flow cytometry. The expression of epithelial-mesenchymal transition-related and apoptotic factors was evaluated by Western blot analysis. Finally, the effect of the lncRNA <i>IRAIN</i>/VEGFA axis was confirmed in an <i>in vivo</i> tumor xenograft model. <b>Results:</b> LncRNA <i>IRAIN</i> was poorly expressed in RC tissues and cells with a primary localization in the nucleus, while VEGFA was highly expressed. Overexpression of lncRNA <i>IRAIN</i> or knockdown of <i>VEGFA</i> inhibited cell proliferation and migration and induced the apoptosis of RC cells. Bioinformatics analysis indicated the presence of CpG islands in the <i>VEGFA</i> promoter region. Lack of methylation at specific sites in the <i>VEGFA</i> promoter region was detected through MSP assay. We found that lncRNA <i>IRAIN</i> was able to inhibit VEGFA expression through recruitment of Dnmt1, Dnmt3a, and Dnmt3b to the <i>VEGFA</i> promoter region. LncRNA <i>IRAIN</i> was also able to suppress RC tumor growth <i>via</i> repression of VEGFA in an <i>in vivo</i> mouse xenograft model. <b>Conclusion:</b> Our data shows that by downregulating <i>VEGFA</i> expression in RC, the lncRNA <i>IRAIN</i> has tumor-suppressive potential.