摘要
目的:探究miR-130a-5p靶向Runt相关转录因子2(Runt-related transcription factor 2,Runx2)对牙槽骨成骨细胞(Alveolar osteoblast,AOB)增殖、凋亡、成骨分化的影响及其机制。方法:将AOB分为NC组、miR-NC组、miR-130a-5p组、miR-130a-5p+Runx2组和miR-130a-5p+SHH组。双荧光素酶报告验证miR-130a-5p对Runx2、Sonic hedgehog(SHH)的调控关系;CCK-8及EdU染色检测细胞增殖能力;AnnexinV-FITC/PI法检测细胞凋亡能力;ALP染色、茜素红染色检测细胞成骨分化能力;RT-qPCR检测miR-130a-5p、Runx2、SHH、神经胶质瘤关联癌基因同源物1(Gli1)mRNA水平;Western Blot检测增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)、Ki67、B细胞淋巴瘤/白血病-2(B-cell lymphoma/leukemia-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 related X protein,Bax)、碱性磷酸酶(Alkaline phosphatase,ALP)、骨钙素(Osteocalcin,OCN)、骨形态发生蛋白2(Bone morphogenetic protein 2,BMP2)、Runx2、SHH、Gil1水平。结果:miR-130a-5p靶向调控Runx2、SHH。过表达miR-130a-5p后,细胞24 h、48 h、72 h OD值及EdU阳性率降低,细胞凋亡率升高,成骨分化能力降低,miR-130a-5p、Bax蛋白水平升高,Runx2、PCNA、Ki67、Bcl-2、ALP、OCN、BMP2蛋白及SHH、Gil1 mRNA和蛋白水平均降低(P<0.05)。过表达miR-130a-5p和Runx2或过表达miR-130a-5p和SHH可减弱过表达miR-130a-5p对细胞增殖、凋亡、成骨分化的影响。结论:miR-130a-5p靶向Runx2抑制AOB增殖和成骨分化,并促进AOB凋亡,其可能通过抑制SHH信号通路发挥作用。
Objective To explore the effect of miR-130a-5p on proliferation,apoptosis and osteogenic differentiation of alveolar bone osteoblasts and its mechanism by targeting Runx2.Methods AOB was divided into NC group,miR-NC group,miR-130a-5p group,miR-130a-5p+Runx2 group and miR-130a-5p+SHH group.The double luciferase report was used to verify the regulatory relationship of miR-130a-5p on Runx2 and SHH;CCK-8 and EdU staining were used to detect proliferation ability;Annexin V-FITC/PI method was used to detect apoptosis ability.ALP staining and alizarin red staining were used to detect the osteogenic differentiation ability.RT-qPCR was used to detect miR-130a-5p,Runx2,SHH and Gil1 mRNA levels.Western blot was used to detect PCNA,Ki67,Bcl-2,Bax,ALP,OCN,BMP2,Runx2,SHH,Gli1 protein levels.Results MiR-130a-5p targeted Runx2 and SHH.After overexpression of miR-130a-5p,OD value at 24 h,48 h and 72 h and EdU positive rate of cells were decreased,and apoptosis rate was increased.The osteogenic differentiation ability was decreased,miR-130a-5p and Bax protein levels were increased,and Runx2,PCNA,Ki67,Bcl-2,ALP,OCN,BMP2 protein levels,SHH,Gil1 mRNA and protein levels were decreased(P<0.05).Overexpression of miR-130a-5p and Runx2 or overexpression of miR-130a-5p and SHH could attenuate the effect of overexpression of miR-130a-5p on proliferation,apoptosis and osteogenic differentiation ability.Conclusion MiR-130a-5p inhibits AOB proliferation and osteogenic differentiation,and promoted AOB apoptosis by targeting Runx2,which may play a role by inhibiting Sonic hedgehog signal pathway.
作者
张义林
侯旭
汪莉
朗么磋
李相宜
ZHANG Yilin;HOU Xu;WANG Li;LANG Mecuo;LI Xiangyi(Department of Stomatology,West China Airport Hospital,Sichuan University,Chengdu Shuangliu First People's Hospital,Chengdu 610200,Sichuan,China)
出处
《中国美容医学》
CAS
2024年第6期51-55,共5页
Chinese Journal of Aesthetic Medicine
