Shanghai Jiaotong Daxue xuebao. Yixue ban (Jan 2023)
Role of methyltransferase like 3 regulating pri-miR-21 methylation in renal fibrosis of diabetes nephropathy
Abstract
Objective·To investigate the role of methyltransferase like 3 (METTL3) acting on N6-methyladenosine (m6A) and regulating pri-miR-21 methylation in the renal fibrosis of diabetic nephropathy (DN).Methods·Eight-week-old male db/db mice were used as DN models, and db/m mice were used as controls. The mice were randomly divided into 4 groups according to whether they received the treatment of 3-deazaadenosine (DAA) by tail vein injection or not (5 mice/group): db/m group, db/db group, db/m+DAA group and db/db+DAA group. From the age of 8 weeks, DAA was injected once per 5 d for a total of 8 times. After the DAA intervention, the mice were kept until they were 19 weeks old. The blood, the urine and the kidney tissue samples of the mice were collected, and blood glucose (BG), serum creatinine (Scr), and urinary albumin-to-creatinine ratio (ACR) were detected. The kidneys were stained with hematoxylin-eosin (H-E), Masson and sirius red to observe the pathological changes. The methylation level of m6A in total RNAs of the kidney was detected with the kit. The expression levels of METTL3 and fibrosis-related proteins in the kidney were detected by Western blotting. The overall pri-miR-21 and the mature miR-21 were detected by real-time quantitative PCR. After enrichment of the m6A-methylated RNAs in the kidney by immunomagnetic beads, the methylated pri-miR-21 at m6A was detected by PCR.Results·Compared with the db/m group, the levels of BG, Scr, and ACR, and METTL3, m6A methylation level, fibrosis-related proteins, overall pri-miR-21, m6A-methylated pri-miR-21 and mature miR-21 in the kidney in the db/db group significantly increased (P<0.05). Furthermore, the mesangial matrix in the kidney increased, glomerular basement membrane thickened, and the accumulation of collagen fibers increased significantly in the db/db group. Compared with the db/db group, the levels of BG, Scr, and ACR, and m6A methylation level, fibrosis-related proteins, m6A-methylated pri-miR-21 and mature miR-21 in the kidney in the db/db+DAA group decreased significantly (P<0.05) and the degree of renal injury and fibrosis was significantly reduced, but the expression level of overall pri-miR-21 significantly increased (P=0.000). The expression level of METTL3 protein did not change significantly.Conclusion·The m6A methylation modification of pri-miR-21 promotes the maturation of miR-21, thereby promoting the occurrence and development of renal fibrosis in DN mice; inhibition of METTL3 can inhibit renal fibrosis in DN mice by regulating m6A methylation of pri-miR-21.
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