[1]李亚宁,李成乾.FTO介导m6A修饰的PRKD2调节SIRT1/HIF-1α通路抑制糖尿病肾病足细胞损伤的机制研究[J].现代检验医学杂志,2024,39(01):5-9+22.[doi:10.3969/j.issn.1671-7414.2024.01.002]
　LI Yaning,LI Chengqian.Mechanism of FTO-mediated and m6A-modified PRKD2 Inhibiting Podocyte Injury in Diabetic Kidney Disease through the SIRT1/HIF-1 Pathway[J].Journal of Modern Laboratory Medicine,2024,39(01):5-9+22.[doi:10.3969/j.issn.1671-7414.2024.01.002]
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FTO介导m6A修饰的PRKD2调节SIRT1/HIF-1α通路抑制糖尿病肾病足细胞损伤的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第39卷
期数:
2024年01期
页码:
5-9+22
栏目:
论著
出版日期:
2024-01-15

文章信息/Info

Title:
Mechanism of FTO-mediated and m6A-modified PRKD2 Inhibiting Podocyte Injury in Diabetic Kidney Disease through the SIRT1/HIF-1 Pathway
文章编号:
1671-7414（2024）01-005-06
作者:
李亚宁;  李成乾
（青岛大学附属医院内分泌科，山东青岛 264200）
Author(s):
LI Yaning;  LI Chengqian
（Department of Endocrinology, Affiliated Hospital of Qingdao University, Shandong Qingdao 264200, China）
关键词:
糖尿病肾病; 足细胞; N6 甲基腺苷修饰; 脂肪和肥胖相关蛋白; 丝氨酸- 苏氨酸激酶蛋白激酶D2
分类号:
R587.2；R392.11
DOI:
10.3969/j.issn.1671-7414.2024.01.002
文献标志码:
A
摘要:
目的　探究脂肪含量和肥胖相关蛋白（fat mass and obesity-associated protein，FTO）和丝氨酸- 苏氨酸激酶蛋白激酶D2（serine-threonine kinase protein kinase D2，PRKD2）在糖尿病肾病（diabetic kidney disease，DKD）进展中的调控作用和调节机制。方法　采用35 mmol/L 葡萄糖对足细胞（MPC5 细胞）进行高糖刺激24h 构建DKD 体外模型。采用FTO 过表达载体（pcDNA-FTO）和PRKD2 过表达载体（pcDNA-PRKD2），或空载体（vector）转染高糖诱导的MPC5 细胞。通过RT-qPCR 检测FTO 和PRKD2 过表达效率；MeRIP 检测PRKD2 mRNA 的N6- 甲基腺苷（N6-methyladenosine，m6A） 修饰水平；ELISA 检测Caspase-3 活性、IL-6，TNF-α 和单核细胞趋化蛋白-1（monocytechemotactic protein-1，MCP-1）分泌量；流式细胞术分析细胞凋亡率；Western blot 评估FTO 和PRKD2 蛋白水平，以及SIRT1/HIF-1α 通路关键蛋白表达水平；Pearson 分析FTO 和PRKD2 水平的相关性。结果　与无高糖诱导对照组比较，高糖诱导的足细胞中FTO 蛋白（0.51 ± 0.04 vs 1.00 ± 0.03）和PRKD2 蛋白（0.45 ± 0.03 vs 1.01 ± 0.04）水平显著下调，差异具有统计学意义（t=13.17，16.76，均P<0.001）。高糖诱导的足细胞中FTO 蛋白水平和PRKD2 蛋白水平呈正相关（r2=0.705 1，P<0.001）。与vector 组相比，pcDNA-FTO 组PRKD2 mRNA 的m6A 水平（0.56± 0.09 vs1.01± 0.13）降低，PRKD2 mRNA 水平（3.16±0.14 vs 1.03±0.02）显著升高，差异具有统计学意义（t=51.37，11.82，均P<0.001）。与control 组（IL-6：512.76 ± 61.85 pg/ml，TNF-α：28.17 ± 2.83 pg/ml，MCP-1：157.31 ± 17.69 pg/ml）和vector 组（IL-6：498.41 ± 87.51 pg/ml，TNF-α：26.35 ± 5.47 pg/ml，MCP-1：165.52 ± 16.87 pg/ml）比较，pcDNA-PRKD2 组IL-6（301.86 ± 21.85 pg/ml），TNF-α（11.06 ± 4.12 pg/ml），MCP-1 分泌量（81.45 ± 9.03pg/ml）显著减少，差异具有统计学意义（F=7.51，10.47，61.97，均P<0.01）。与control 组（Caspase-3：689.65 ± 79.5U/L，细胞凋亡率：22.31% ± 2.69%）和vector 组（Caspase-3：715.91 ± 113.58 U/L，细胞凋亡率：21.07% ± 3.28%）比较，pcDNA-PRKD2 组Caspase-3 活性（437.64 ± 104.76 U/L）和细胞凋亡率（8.41% ± 3.15%）下降，差异具有统计学意义（F=2.35，79.13，均P<0.01）。与control 组（SIRT1：1.01 ± 0.05，HIF-1α：1.03 ± 0.07）和vector 组（SIRT1：0.97± 0.05，HIF-1α：1.02 ± 0.03）相比，pcDNA-PRKD2 组SIRT1 蛋白（3.51 ± 0.15）水平升高，HIF-1α 蛋白（0.37 ± 0.07）水平降低，差异具有统计学意义（F=31.54，8.31，均P<0.01）。结论　FTO 介导m6A 修饰的PRKD2 通过SIRT1/HIF-1α 通路抑制高糖诱导的足细胞炎症反应和细胞凋亡。
Abstract:
Objective　To explore the regulatory role of fat mass and obesity-associated protein (FTO) and serine-threonine kinase protein kinase D2 (PRKD2) in progression of diabetic kidney disease (DKD) and its regulatory mechanisms. Methods　 DKD model in vitro was constructed by podocytes (MPC5 cells) treated with high glucose (HG, 35 mmol/L glucose) for 24 h. HG-induced MPC5 cells were transfected with FTO overexpression vector (pcDNA-FTO) and PRKD2 overexpression vector (pcDNA-PRKD2), or empty vector. The overexpression efficiency of FTO and PRKD2 were detected with RT-qPCR. MeRIP was used to detect the N6-methyladenosine(m6A) modification level of PRKD2 mRNA. The activity of Caspase-3 and the secretion of IL-6, TNF-α and monocyte chemotactic protein-1(MCP-1) were detected by ELISA. Cell apoptosis rate was analyzed by flow cytometry. The protein levels of FTO and PRKD2, as well as the key proteins in SIRT1/HIF-1α pathway, were evaluated by Western blot. Pearson analysis was used to analyze the correlation between FTO levels and PRKD2 levels. Results　Compared with the control group without HG-induction, the protein expression of FTO (0.51 ± 0.04 vs 1.00 ± 0.03) and PRKD2 (0.45 ± 0.03 vs 1.01 ± 0.04) was significantly down-regulated in HG-induced podocytes, and the differences were statistically significant (t=13.17, 16.76, all P<0.001). FTO protein levels were positively correlated with PRKD2 protein levels in HG-induced podocytes (r2=0.705 1, P<0.001). Compared with the vector group, the m6A levels of PRKD2 mRNA (0.56 ± 0.09 vs 1.01 ± 0.13) in the pcDNA-FTO group were decreased, and the mRNA levels of PRKD2 (3.16 ± 0.14 vs 1.03 ± 0.02) were increased, with significant differences (t=51.37, 11.82, all P<0.001). Compared with the control group (IL-6: 512.76 ± 61.85 pg/ml, TNF-α: 28.17 ± 2.83 pg/ml, MCP-1: 157.31 ± 17.69 pg/ml) and the vector group (IL-6: 498.41 ± 87.51 pg/ml, TNF-α: 26.35 ± 5.47 pg/ml, MCP-1: 165.52 ± 16.87 pg/ml), the secretion of IL-6 (301.86 ± 21.85 pg/ml), TNF-α (11.06 ± 4.12 pg/ml) and MCP-1 (81.45 ± 9.03 pg/ml) were significantly decreased in the pcDNA-PRKD2 group, and the differences were statistically significant (F=7.51, 10.47, 61.97, all P<0.01). Compared with the control group (caspase-3 activity: 689.65 ± 79.5 U/L, cell apoptosis: 22.31% ± 2.69%) and the vector group (Caspase-3 activity: 715.91 ± 113.58 U/L, cell apoptosis: 21.07% ± 3.28%), Caspase-3 activity (437.64 ± 104.76 U/L) and the rate of apoptosis (8.41% ± 3.15%) were significantly decreased in the pcDNA-PRKD2 group, and the differences were statistically significant (F=2.35, 79.13, all P<0.01). Compared with the control group (SIRT1: 1.01 ± 0.05, HIF-1α: 1.03 ± 0.07) and the vector group (SIRT1: 0.97 ± 0.05, HIF-1α: 1.02 ± 0.03), SIRT1 protein levels (3.51 ± 0.15) were increased and HIF-1α protein levels (0.37 ± 0.07) were decreased in the pcDNA-PRKD2 group, and the differences were statistically significant (F=31.54, 8.31, all P<0.01). Conclusion　FTO-mediated and m6A-modified PRKD2 suppresses inflammation and apoptosis in HG-induced podocytes through the SIRT1/HIF-1 pathway.

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备注/Memo

备注/Memo:
作者简介：李亚宁（1985-），女，本科，硕士研究生在读，副主任医师，研究方向：糖尿病发病机制、糖尿病诊治，E-mail：he_554706@163.com。
通讯作者：李成乾（1974-），男，博士，副主任医师，E-mail：13754620843@163.com。
更新日期/Last Update: 2024-01-15