甲基转移酶3调控pri-miR-21甲基化修饰在糖尿病肾病肾脏纤维化中的作用

doi:10.3969/j.issn.1674-8115.2023.01.001

摘要/Abstract

摘要：

目的·探讨甲基转移酶3（methyltransferase like 3，METTL3）调控pri-miR-21的N⁶-甲基腺苷（N⁶-methyladenosine，m⁶A）甲基化修饰在糖尿病肾病（diabetic nephropathy，DN）小鼠肾脏纤维化发病机制中的作用。方法·采用8周龄雄性db/db小鼠作为DN模型小鼠，db/m小鼠作为对照，同时按照是否经尾静脉注射S-腺苷高半胱氨酸水解酶抑制剂3-脱氮腺苷（3-deazaadenosine，DAA），共随机分为4组（5只/组），分别为db/m组、db/db组、db/m+DAA组和db/db+DAA组；8周龄开始注射DAA，注射1次/5 d，共注射8次。DAA干预结束后继续饲养小鼠至19周龄，收取各组小鼠血、尿、肾脏组织标本。检测血糖、血肌酐、尿白蛋白肌酐比（albumin-to-creatinine ratio，ACR），肾脏行苏木精-伊红（H-E）染色、Masson染色及天狼星红染色观察病理变化；试剂盒检测肾脏总RNA中m⁶A的甲基化水平；Western blotting检测肾脏METTL3及纤维化相关蛋白表达；实时定量PCR检测肾脏总pri-miR-21和成熟miR-21；使用免疫磁珠富集肾脏组织中m⁶A甲基化RNA，并通过PCR检测其中m⁶A甲基化的pri-miR-21。结果·相较于db/m组，db/db组小鼠血糖，血肌酐，ACR，肾脏METTL3、m⁶A甲基化修饰水平、纤维化相关蛋白、总pri-miR-21、m⁶A甲基化pri-miR-21和成熟miR-21表达水平均显著增加（均P<0.05），小鼠肾脏系膜基质增多、肾小球基底膜增厚、胶原纤维累积显著增加。相较于db/db组，db/db+DAA组血糖，血肌酐，ACR，肾脏m⁶A甲基化修饰水平、纤维化相关蛋白、m⁶A甲基化pri-miR-21和成熟miR-21表达水平均显著下降（均P<0.05），总pri-miR-21表达水平显著升高（P=0.000），METTL3蛋白表达水平未见显著变化，小鼠肾脏损伤及纤维化程度显著减轻。结论·pri-miR-21的m⁶A甲基化修饰促进miR-21成熟，进而促进DN小鼠肾脏纤维化的发生发展；抑制METTL3可通过调控pri-miR-21的m⁶A甲基化修饰抑制DN小鼠肾脏纤维化。

关键词: 糖尿病肾病, 肾脏纤维化, N⁶-甲基腺苷, 甲基转移酶3, pri-miR-21, miR-21

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

中图分类号:

R587.1

吴佳晋, 钟晨, 李大伟, 陈若洋, 瞿俊文, 张明. 甲基转移酶3调控pri-miR-21甲基化修饰在糖尿病肾病肾脏纤维化中的作用[J]. 上海交通大学学报（医学版）, 2023, 43(1): 1-7.

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.

图/表 4

表1 实时qPCR引物序列

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′

图1 各组小鼠血糖、Scr、ACR及肾脏病理变化Note： A. Analysis of blood glucose levels in the 4 groups. B. Analysis of Scr levels in the 4 groups. C. Analysis of ACR levels in the 4 groups. D. H-E staining, Masson staining and sirius red staining of kidneys in the 4 groups (×200). ①P=0.006, ②P=0.033, ③P=0.000, ④P=0.016, ⑤P=0.023.

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

图2 各组小鼠肾脏m6A甲基化修饰水平、METTL3水平及纤维化相关蛋白表达变化Note： A. The m6A modification levels in the 4 groups. B. The protein expression levels of METTL3, α-SMA, FN, COL-Ⅰ, and COL-Ⅳ in the 4 groups. ①P=0.000, ②P=0.019, ③P=0.022, ④P=0.038, ⑤P=0.007, ⑥P=0.009, ⑦P=0.021, ⑧P=0.039, ⑨P=0.017, ⑩P=0.045.

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

图3 各组小鼠肾脏总pri-miR-21、甲基化pri-miR-21及成熟miR-21的表达变化Note： A. The levels of pri-miR-21 in the renal tissues in the 4 groups detected by qPCR. B. The levels of m6A methylated pri-miR-21 in the renal tissues in the 4 groups detected by immunocoprecipitation. C. The levels of mature miR-21 in the renal tissues in 4 groups detected by qPCR. ①P=0.008, ②P=0.000, ③P=0.004, ④P=0.001, ⑤P=0.006, ⑥P=0.005.

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

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