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.
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. Journal of Shanghai Jiao Tong University (Medical Science)[J], 2023, 43(1): 1-7 doi:10.3969/j.issn.1674-8115.2023.01.001
糖尿病肾病（diabetic nephropathy，DN）是糖尿病常见的慢性微血管并发症之一［1-3］，其病理特征为细胞外基质（extracellular matrix，ECM）蛋白的沉积［4］。多种因素参与了DN发病，其中微RNA（microRNA，miRNA）在DN中的调控作用近年来备受关注。研究［5］证实miR-21与DN肾脏纤维化密切相关，它能促进肾脏的纤维生成和上皮损伤，是抗肾脏纤维化治疗的候选靶点。已有研究［6-7］证实miR-21可通过上调Sma和Mad相关蛋白3（Sma- and Mad-related protein，SMAD3）、下调SMAD7，以及与靶基因磷酸酯酶与张力蛋白同源物（phosphatase and tensin homolog，PTEN）基因结合抑制其表达等多种途径促进肾脏纤维化，但miR-21的上游调节机制对DN的影响仍不清楚。
N6-甲基腺苷（N6-methyladenosine，m6A）是一种丰富的mRNA修饰。研究［8-9］表明，哺乳动物的m6A是动态调节的，参与各种生物学进展，并与代谢性疾病密切相关，包括糖尿病和肥胖。已有研究［10］表明，miR-21基因转录后生成的pri-miR-21向成熟miR-21转化受到甲基转移酶3（methyltransferase like 3，METTL3）催化的m6A甲基化修饰调控，但miR-21转录后的m6A甲基化修饰在DN中的病理作用和调控机制尚未阐明，揭示其在DN肾脏纤维化中的作用将有助于为DN患者开发新的治疗策略。本研究采用db/db糖尿病小鼠作为DN模型小鼠，探究pri-miR-21的m6A甲基化修饰在DN肾脏纤维化中的生物学功能和潜在的分子调控机制，旨在为DN肾脏纤维化的治疗提供新的靶点。
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
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
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
The study was designed by WU Jiajin, ZHANG Ming and LI Dawei. The manuscript was drafted and revised by WU Jiajin, ZHANG Ming and LI Dawei. The experiments were performed by WU Jiajin, ZHONG Chen, CHEN Ruoyang and QU Junwen. The data were analyzed by WU Jiajin, ZHONG Chen, CHEN Ruoyang and QU Junwen. All the authors have read the last version of paper and consented for submission.
利益冲突声明
所有作者声明不存在利益冲突。
All authors disclose no relevant conflict of interests.
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... 糖尿病肾病（diabetic nephropathy，DN）是糖尿病常见的慢性微血管并发症之一［1-3］，其病理特征为细胞外基质（extracellular matrix，ECM）蛋白的沉积［4］.多种因素参与了DN发病，其中微RNA（microRNA，miRNA）在DN中的调控作用近年来备受关注.研究［5］证实miR-21与DN肾脏纤维化密切相关，它能促进肾脏的纤维生成和上皮损伤，是抗肾脏纤维化治疗的候选靶点.已有研究［6-7］证实miR-21可通过上调Sma和Mad相关蛋白3（Sma- and Mad-related protein，SMAD3）、下调SMAD7，以及与靶基因磷酸酯酶与张力蛋白同源物（phosphatase and tensin homolog，PTEN）基因结合抑制其表达等多种途径促进肾脏纤维化，但miR-21的上游调节机制对DN的影响仍不清楚. ...
1
... 糖尿病肾病（diabetic nephropathy，DN）是糖尿病常见的慢性微血管并发症之一［1-3］，其病理特征为细胞外基质（extracellular matrix，ECM）蛋白的沉积［4］.多种因素参与了DN发病，其中微RNA（microRNA，miRNA）在DN中的调控作用近年来备受关注.研究［5］证实miR-21与DN肾脏纤维化密切相关，它能促进肾脏的纤维生成和上皮损伤，是抗肾脏纤维化治疗的候选靶点.已有研究［6-7］证实miR-21可通过上调Sma和Mad相关蛋白3（Sma- and Mad-related protein，SMAD3）、下调SMAD7，以及与靶基因磷酸酯酶与张力蛋白同源物（phosphatase and tensin homolog，PTEN）基因结合抑制其表达等多种途径促进肾脏纤维化，但miR-21的上游调节机制对DN的影响仍不清楚. ...
1
... N6-甲基腺苷（N6-methyladenosine，m6A）是一种丰富的mRNA修饰.研究［8-9］表明，哺乳动物的m6A是动态调节的，参与各种生物学进展，并与代谢性疾病密切相关，包括糖尿病和肥胖.已有研究［10］表明，miR-21基因转录后生成的pri-miR-21向成熟miR-21转化受到甲基转移酶3（methyltransferase like 3，METTL3）催化的m6A甲基化修饰调控，但miR-21转录后的m6A甲基化修饰在DN中的病理作用和调控机制尚未阐明，揭示其在DN肾脏纤维化中的作用将有助于为DN患者开发新的治疗策略.本研究采用db/db糖尿病小鼠作为DN模型小鼠，探究pri-miR-21的m6A甲基化修饰在DN肾脏纤维化中的生物学功能和潜在的分子调控机制，旨在为DN肾脏纤维化的治疗提供新的靶点. ...
1
... N6-甲基腺苷（N6-methyladenosine，m6A）是一种丰富的mRNA修饰.研究［8-9］表明，哺乳动物的m6A是动态调节的，参与各种生物学进展，并与代谢性疾病密切相关，包括糖尿病和肥胖.已有研究［10］表明，miR-21基因转录后生成的pri-miR-21向成熟miR-21转化受到甲基转移酶3（methyltransferase like 3，METTL3）催化的m6A甲基化修饰调控，但miR-21转录后的m6A甲基化修饰在DN中的病理作用和调控机制尚未阐明，揭示其在DN肾脏纤维化中的作用将有助于为DN患者开发新的治疗策略.本研究采用db/db糖尿病小鼠作为DN模型小鼠，探究pri-miR-21的m6A甲基化修饰在DN肾脏纤维化中的生物学功能和潜在的分子调控机制，旨在为DN肾脏纤维化的治疗提供新的靶点. ...
1
... N6-甲基腺苷（N6-methyladenosine，m6A）是一种丰富的mRNA修饰.研究［8-9］表明，哺乳动物的m6A是动态调节的，参与各种生物学进展，并与代谢性疾病密切相关，包括糖尿病和肥胖.已有研究［10］表明，miR-21基因转录后生成的pri-miR-21向成熟miR-21转化受到甲基转移酶3（methyltransferase like 3，METTL3）催化的m6A甲基化修饰调控，但miR-21转录后的m6A甲基化修饰在DN中的病理作用和调控机制尚未阐明，揭示其在DN肾脏纤维化中的作用将有助于为DN患者开发新的治疗策略.本研究采用db/db糖尿病小鼠作为DN模型小鼠，探究pri-miR-21的m6A甲基化修饰在DN肾脏纤维化中的生物学功能和潜在的分子调控机制，旨在为DN肾脏纤维化的治疗提供新的靶点. ...
