Role of methyltransferase like 3 regulating pri-miR-21 methylation in renal fibrosis of diabetes nephropathy

doi:10.3969/j.issn.1674-8115.2023.01.001

Abstract

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.

Key words: diabetic nephropathy (DN), renal fibrosis, N⁶-methyladenosine (m⁶A), methyltransferase like 3 (METTL3), pri-miR-21, miR-21

CLC Number:

R587.1

WU Jiajin, ZHONG Chen, LI Dawei, CHEN Ruoyang, QU Junwen, ZHANG Ming. Role of methyltransferase like 3 regulating pri-miR-21 methylation in renal fibrosis of diabetes nephropathy[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(1): 1-7.

Figures/Tables 4

Tab 1 Primer sequences for real-time qPCR

Gene	Forward primer	Reverse primer
miR-21	5′-ACGTTGTGTAGCTTATCAGACTG-3′	5′-AATGGTTGTTCTCCACACTCTC-3′
pri-miR-21	5′-ACAGGCCAGAAATGCCTGGG-3′	5′-GATGGTCAGATGAAAGATAC-3′
U6	5′-CTCGCTTCGGCAGCACA-3′	5′-AACGCTTCACGAATTTGCGT-3′
Gapdh	5′-AATCCCATCACCATCTTCCAG-3′	5′-AAATGAGCCCCAGCCTTC-3′

Fig 1 Changes of blood glucose, Scr, ACR and kidney pathology in the mice of the 4 groups

Fig 2 Changes of levels of m6A methylation modification, METTL3 and fibrosis-related proteins in the 4 groups of mice kidneys

Fig 3 Changes of total pri-miR-21, methylated pri-miR-21, and mature miR-21 in the 4 groups of mice kidneys

TrendMD

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