Abstract
Methylation of the adenine base at the nitrogen 6 position (m6A) is the most common post-transcriptional epigenetic modification of RNA, and it plays a very important role in regulating gene expression. To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T–A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.
Highlights
The long noncoding RNA H19 plays a crucial role in the development of cancer [1]. miR675, derived from exon 1 of H19, has been shown to have an oncogenic role in liver cancers [2,3]
The cell apoptosis rate did not increase after point mutations of the m6A modification sites (Figure 2D,E)
We further explored the expression patterns of m6A-related genes after introducing point mutations in L02 and MHCC97L cells. Quantitative Real-time PCR (qPCR) results showed increased expression of ALKBH5 and decreased expression of METTL3, METTL14, WTAP, FTO, and YTHDF2 in MHCC97L cells compared with L02 cells (Figure 7A)
Summary
The long noncoding RNA (lncRNA) H19 plays a crucial role in the development of cancer [1]. miR675, derived from exon 1 of H19, has been shown to have an oncogenic role in liver cancers [2,3]. Previous studies have shown that the H19/miR675 axis can regulate cell apoptosis [5]. These results demonstrate that altered expression of H19 or miR675 can influence tumor cell behavior. Recent reports have suggested that m6A (methylation of the adenine base at the nitrogen 6 position) methylation plays an important role in the post-transcriptional modification of RNA [6], and it is known that this modification is regulated by adenosine methyltransferases and demethylases [7,8]. There is evidence that m6A modification in microRNA and lncNA affects cell development and fate [16] These data indicate that m6A modification might have a role in noncoding
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