Targeted point mutations of the m6A modification in miR675 using RNA-guided base editing induce cell apoptosis

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.

m6A Methylation in Cardiovascular Diseases: From Mechanisms to Therapeutic Potential

“…METTL3 and FTO have opposing effects on cardiomyocyte apoptosis, likely because of differential regulation of m6A methylation in different target mRNAs. Furthermore, studies have confirmed that targeting point mutations in the m6A modification sites of target transcripts using gene editing technologies can regulate apoptosis ( Hao et al, 2020 ); thus, m6A methylation is a potentially effective means of regulating apoptosis. However, target transcripts that affect apoptosis first need to be identified.…”

The Basis and Promise of Programmable RNA Editing and Modification

“…In order to observe the effects of m6A modifications on miRNA and long non-coding RNA, an adenine base editor system (ABE7.10) was used to induce single site base change. After the targeted mutation of an m6A site (T-A conversion) upstream of miR-675 in the H19 locus in HEK293T cells, miR-675, and H19 expression were observed to be suppressed, resulting in an increase in apoptosis (Hao et al, 2020). It was hypothesized that the reduced expression resulted in an increased presence of p53 protein, thus inducing cell death which indicates the role of m6A in regulating miR675 and H19 expression (Hao et al, 2020), and by extension, cell survival.…”

“…After the targeted mutation of an m6A site (T-A conversion) upstream of miR-675 in the H19 locus in HEK293T cells, miR-675, and H19 expression were observed to be suppressed, resulting in an increase in apoptosis (Hao et al, 2020). It was hypothesized that the reduced expression resulted in an increased presence of p53 protein, thus inducing cell death which indicates the role of m6A in regulating miR675 and H19 expression (Hao et al, 2020), and by extension, cell survival. As stated previously, m6A is highly involved in cancer development.…”

The Emerging Clinical Application of m6A RNA Modification in Inflammatory Bowel Disease and Its Associated Colorectal Cancer

“…This shows that the methylation modification of miRNA plays an important role in cell. 52 Furthermore, manipulation of miRNA expression or sequence can alter the modification level of m6A by regulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targets. The increase of m6A content promotes the reprogramming of mouse embryonic fibroblasts (MEFs) into pluripotent stem cells.…”