二甲双胍调控miR-340-5p/DDX17轴对高糖诱导成骨细胞增殖、凋亡的影响及分子机制  

Effects of metformin on the proliferation and apoptosis of osteoblasts induced by high glucose by regulating the miR-340-5p/DDX17 axis and its molecular mechanism

作　　者：黄璐捷 干开丰 张挺 周珂 胡珂琦 Huang Lujie;Gan Kaifeng;Zhang Ting;Zhou Ke;Hu Keqi(Department of Geriatrics,Ningbo Medical Center Li Huili Hospital,Ningbo 315000,China;Department of Orthopaedics,Ningbo Medical Center Li Huili Hospital,Ningbo 315000,China)

机构地区：[1]宁波市医疗中心李惠利医院老年医学科,宁波315000 [2]宁波市医疗中心李惠利医院骨科,宁波315000

出　　处：《中华内分泌外科杂志（中英文）》2024年第2期267-273,共7页Chinese Journal of Endocrine Surgery

基　　金：浙江省自然科学基金(LQ21H060002)。

摘　　要：目的探讨二甲双胍调控miR-340-5p/DEAD-box解旋酶17(DEAD-box helicase 17,DDX17)轴对高糖诱导成骨细胞增殖、凋亡的影响。方法MC3T3-E1细胞分为对照组、高糖组、二甲双胍组、inhibitor NC组、miR-340-5p inhibitor组、二甲双胍+mimic NC组、二甲双胍+miR-340-5p mimic组,qRT-PCR检测miR-340-5p表达;Western blot检测DDX17、细胞周期素D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、裂解的天冬氨酸特异性半胱氨酸蛋白酶-3(Cleaved aspartate-specific cysteine protease-3,Cleaved caspase-3)蛋白;CCK-8、5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,EdU)染色检测细胞增殖;流式细胞术检测细胞凋亡;荧光原位杂交实验、双荧光素酶分别验证miR-340-5p与DDX17的定位、靶向关系。结果与对照组比较,高糖组miR-340-5p表达、细胞凋亡率及Bax、Cleaved caspase-3蛋白表达升高(t=19.81,39.47,24.61,21.62,P均<0.001),OD_(450)值、EdU阳性细胞率及DDX17、Cyclin D1、PCNA蛋白表达降低(t=7.624,30.100,26.54,19.46,21.20,P均<0.001);与高糖组比较,二甲双胍组miR-340-5p表达、细胞凋亡率及Bax、Cleaved caspase-3蛋白表达降低(t=13.37,28.55,18.35,18.78,P均<0.001),OD_(450)值、EdU阳性细胞率及DDX17、Cyclin D1、PCNA蛋白表达升高(t=7.97,25.76,18.48,12.39,18.98,P均<0.001);与高糖组、inhibitor NC组比较,miR-340-5p inhibitor组miR-340-5p表达、细胞凋亡率及Bax、Cleaved caspase-3蛋白表达降低(F=85.92,301.11,142.64,170.35,P均<0.001),OD_(450)值、EdU阳性细胞率及DDX17、Cyclin D1、PCNA蛋白表达升高(F=26.54,447.72,249.31,113.46,393.27,P均<0.001);与二甲双胍组、二甲双胍+mimic NC组比较,二甲双胍+miR-340-5p mimic组miR-340-5p表达、细胞凋亡率及Bax、Cleaved caspase-3蛋白表达升高(F=36.27,395.56,94.90,18.59,P均<0.001),OD_(450)值、EdU阳性细胞率及DDX17、Cyclin D1、PCNA蛋白表达降低(F=21.44,128.47,24.28,25.89,18.06,P均<0.Objective To investigate the effects of metformin on the proliferation and apoptosis of osteoblasts induced by high glucose by regulating the miR-340-5p/DEAD-box helicase 17(DDX17)axis.Methods MC3T3-E1 cells were divided into control group,high glucose group,metformin group,inhibitor NC group,miR-340-5p inhibitor group,metformin+mimic NC group,and metformin+miR-340-5p mimic group.qRT-PCR was used to detect the expression of miR-340-5p;Western blot detect the protein expression of DDX17,cyclin D1,proliferating cell nuclear antigen(PCNA),Bcl-2 associated X protein(Bax),and cleaved aspartate-specific cysteine protease-3(Cleaved caspase-3);CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU)staining was used to detect the proliferation of cells;Flow cytometry was used to detect cell apoptosis;Fluorescence in situ hybridization experiment and dual luciferase assay was used to verify the localization and targeting relationship between miR-340-5p and DDX17,respectively.Results Compared with the control group,the expression of miR-340-5p,apoptosis rate,and the expression of Bax and Cleaved caspase-3 proteins in the high glucose group increased(t=19.81,39.47,24.61,21.62,all P<0.001),the OD_(450) value,EdU positive cell rate,and the expression of DDX17,Cyclin D1,and PCNA proteins decreased(t=7.62,30.10,26.54,19.462,21.20,all P<0.001);Compared with high glucose group,the expression of miR-340-5p,apoptosis rate,and the expression of Bax and Cleaved caspase-3 proteins in the Metformin group decreased(t=13.37,28.55,18.35,18.78,P all<0.001),the OD_(450) value,EdU positive cell rate,and the expression of DDX17,Cyclin D1,and PCNA proteins increased(t=7.97,25.76,18.48,12.39,18.98,P all<0.001);Compared with high glucose group and inhibitor NC group,the expression of miR-340-5p,apoptosis rate,and the expression of Bax and Cleaved caspase-3 proteins in the miR-340-5p inhibitor group decreased(F=85.92,301.11,142.64,170.35,all P<0.001),the OD_(450) value,EdU positive cell rate,and the expression of DDX17,Cyclin D1,and PCNA proteins increased(F=26.

关 键 词：二甲双胍 miR-340-5p DEAD-box解旋酶17 高糖 增殖 凋亡 糖尿病性骨质疏松症 

分 类 号：R587.2[医药卫生—内分泌] R580[医药卫生—内科学]