The YTHDC1 reader protein recognizes and regulates the lncRNA MEG3 following its METTL3-mediated m6A methylation: a novel mechanism early during radiation-induced liver injury

While apoptotic cell death is known to be central to the pathogenesis of radiation-induced liver injury (RILI), the mechanistic basis for this apoptotic activity remains poorly understood.

N6-methyladenosine (m6A) modifications are the most common form of reversible methylation observed on lncRNAs in eukaryotic cells, with their presence leading to pronounced changes in the

activity of a range of biological processes. The degree to which m6A modification plays a role in the induction of apoptotic cell death in response to ionizing radiation (IR) in the context

of RILI remains to be established. Here, IR-induced apoptosis was found to significantly decrease the levels of m6A present, with a pronounced decrease in the expression of

methyltransferase-like 3 (METTL3) at 2 d post radiation in vitro. From a mechanistic perspective, a methylated RNA immunoprecipitation assay found that lncRNA MEG3 was a major METTL3 target.

The expression of MEG3 was upregulated via METTL3-mediated m6A in a process that was dependent on YTHDC1, ultimately reversing the miR-20b-mediated inhibition of BNIP2 expression. Together,

these findings demonstrate that the responsivity of METTL3 activity to IR plays a role in IR-induced apoptotic cell death, leading to the reverse of miR-20b-mediated BNIP2 inhibition

through the YTHDC1-dependent m6A modification of MEG3, suggesting that this process may play a central role in RILI incidence.

The radiotherapeutic treatment of many tumors in the upper body or other regions in patients with certain cancers can result in the exposure of healthy hepatic tissue to ionizing radiation

(IR), leading to the incidence of radiation-induced liver injury (RILI). In the liver, radiation can induce forms of disease such as non-icteric hepatomegaly and ascites that most often

manifest within 2 weeks to 3 months post irradiation [1]. Following hepatic radiation at the lowest effective dose (30 Gy), the odds of RILI have been estimated at just 5% [2], yet they rise

to >50% at a dose of 40 Gy, and 76% of liver cancer patients who receive a dose of 60 Gy will ultimately succumb to acute liver failure following treatment [3]. At present, the mechanistic

basis for RILI has yet to be fully clarified, limiting any efforts to design effective interventional strategies. There is thus a pressing need to clarify the molecular etiology of RILI to

guide its prevention and treatment.

RILI incidence is known to be closely associated with apoptotic cell death, oxidative stress, and fibrosis [4], with apoptosis occurring early during RILI [5]. The debris produced through

extensive apoptotic cell death, together with inflammatory mediators and oxidative stress, are central to the induction of hepatic fibrosis [6].

It was observed that MEG3 expression was dramatically downregulated in BNL CL2 cells within 24 h following radiation exposure (Fig. 1C). The binding proteins associated with MEG3 were

predicted using the RBPDB database (http://rbpdb.ccbr.utoronto.ca/). Analysis revealed multiple binding sites between YTHDC1 and MEG3, suggesting potential methylation modifications of MEG3.

Methylation sites of MEG3 were further predicted using the SRAMP tool (https://www.cuilab.cn/sramp). In vitro experiments demonstrated an increase in METTL3 levels post-cell radiation.

Additionally, RNAhybrid version 2.2 was employed to predict the sponge targeting sites of MEG3 on miR-20b.