Exploring Long Noncoding HIF-1α Coactivating RNA Regulation through Short Hairpin RNA Knockdown of N-Methyladenosine Modification

Abstract

Previous research has shown that the long noncoding HIF-1α co-activating RNA (lncHIFCAR) is implicated in several disease progressions, such as oral cancer and osteoarthritis development. However, the regulatory mechanism of this RNA has largely remained unexplored. N6-methyladenosine (m6A) modifications of RNA are among the most prevalent in regulating long noncoding RNAs. Hence, determining these modification sites and the proteins responsible could provide the groundwork for further research on the role of m6A modification in lncHIFCAR. This study aims to identify the m6A-associated proteins interacting with lncHIFCAR and investigate their effects on its stability or degradation. To achieve this, bioinformatic analysis was performed utilizing tools such as cBioportal and GEPIA to explore coexpression within the writer/reader axis. Additionally, modification sites were predicted using algorithms like SRAMP and m6AMPred. The m6A-associated writers METTL16 and METTL4, as well as the m6A reader ELAVL1, were identified and knocked down in human tongue squamous cell carcinoma by short hairpin RNA. Gene expression levels of the m6A proteins and lncHIFCAR were measured via RT-qPCR to confirm successful knockdown and assess its impact on lncHIFCAR expression. Experimental results demonstrated a decrease in lncHIFCAR expression following the knockdown. However, these findings did not align with bioinformatics predictions, potentially due to limitations in the study where the methylation level is not directly measured. Hence, it remains unclear whether the observed decrease in lncHIFCAR is directly attributable to m6A modification. Future studies should incorporate direct measurement of methylation levels and additional experimental approaches, such as dose-dependent knockdown, to better understand the role of m6A in regulating lncHIFCAR.