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

Abstract

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

LncRNA-mediated DNA methylation: an emerging mechanism in cancer and beyond

“…Currently, a growing number of studies suggest that lncRNA might recruit DNMTs directly to specific targets (Fig. 1 a), including both protein-coding genes [ 43 , 44 , 46 , 47 , 49 – 51 , 55 , 57 , 58 , 62 , 63 ] and non-coding genes such as miRNA [ 42 , 52 , 93 ]. For instance, in esophageal cancer (EC), lncRNA ADAMTS9-AS2 was reported to recruit DNMT1/3 to CDH3 promoter, inhibiting the cancer cell proliferation, invasion, and migration [ 50 ].…”

Long non‐coding RNA ELFN1‐AS1‐mediated ZBTB16 inhibition augments the progression of gastric cancer by activating the PI3K/AKT axis

“…LncRNAs can mediate DNA methylation at the promoter region of specific genes to affect gene expression 16 . Therefore, we surmised that ELFN1‐AS1 probably controls DNA methylation to repress the transcription of ZBTB16.…”

“…LncRNAs can mediate DNA methylation at the promoter region of specific genes to affect gene expression. 16 Therefore, we surmised that ELFN1-AS1 probably controls DNA methylation to repress the transcription of ZBTB16. To validate this, the promoter sequence of The sequence of Island 2 was too short to construct the qMSP primer, and Island 1 and Island 3 were selected for subsequent analysis.…”

SPATS2 is positively activated by long noncoding RNA SNHG5 via regulating DNMT3a expression to promote hepatocellular carcinoma progression

“…A recent study found that lncRNAs act as ceRNAs to affect the expression of DNMT3s through miRNAs [ 14 ]. Moreover, in addition to acting as molecular sponges for microRNAs [ 15 , 16 ], lncRNAs can also inhibit target gene expression through the recruitment of DNMTs to their promoters, such as lncRNA IRAIN/DNMT3s/VEGFA [ 17 ], lncRNA MCM3AP-AS1/DNMT1/DNMT3(A/B)/NPY1R [ 18 ], and lncRNA ROIT/DNMT3a/Nkx6.1 [ 19 ]. Here, to achieve the goal of elucidating the regulatory mechanism of SPATS2 in HCC, we raise the question of whether SNHG5 could regulate SPATS2 expression through DNA demethylation.…”