AUTHOR=Li Yang , Luo Qingyang , Li Zun , Wang Yun , Zhu Chaoyang , Li Tieqiang , Li Xiaodong 

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

JOURNAL=Frontiers in Oncology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2020.01082

DOI=10.3389/fonc.2020.01082

ISSN=2234-943X

ABSTRACT=<p><bold>Aims:</bold> Long non-coding RNA <italic>IRAIN</italic> (lncRNA <italic>IRAIN</italic>) plays a critical role in numerous malignancies. However, the function of lncRNA <italic>IRAIN</italic> in renal carcinoma (RC) remains enigmatic. The purpose of this study is to characterize the effects of lncRNA <italic>IRAIN</italic> on RC progression.</p><p><bold>Methods:</bold> The expression pattern of lncRNA <italic>IRAIN</italic> 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 <italic>IRAIN</italic> 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 <italic>VEGFA</italic> promoter region. MSP was applied to detect the level of DNA methylation in RC cells. The interaction between lncRNA <italic>IRAIN</italic> and VEGFA was identified by RNA immunoprecipitation and RNA-protein pull down assays. Recruitment of DNA methyltransferases (Dnmt) to the <italic>VEGFA</italic> promoter region was achieved by chromatin immunoprecipitation. The subcellular localization of lncRNA <italic>IRAIN</italic> 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 <italic>IRAIN</italic>/VEGFA axis was confirmed in an <italic>in vivo</italic> tumor xenograft model.</p><p><bold>Results:</bold> LncRNA <italic>IRAIN</italic> was poorly expressed in RC tissues and cells with a primary localization in the nucleus, while VEGFA was highly expressed. Overexpression of lncRNA <italic>IRAIN</italic> or knockdown of <italic>VEGFA</italic> inhibited cell proliferation and migration and induced the apoptosis of RC cells. Bioinformatics analysis indicated the presence of CpG islands in the <italic>VEGFA</italic> promoter region. Lack of methylation at specific sites in the <italic>VEGFA</italic> promoter region was detected through MSP assay. We found that lncRNA <italic>IRAIN</italic> was able to inhibit VEGFA expression through recruitment of Dnmt1, Dnmt3a, and Dnmt3b to the <italic>VEGFA</italic> promoter region. LncRNA <italic>IRAIN</italic> was also able to suppress RC tumor growth <italic>via</italic> repression of VEGFA in an <italic>in vivo</italic> mouse xenograft model.</p><p><bold>Conclusion:</bold> Our data shows that by downregulating <italic>VEGFA</italic> expression in RC, the lncRNA <italic>IRAIN</italic> has tumor-suppressive potential.</p>