摘要
目的研究miR-145-3p能否调控人牙周膜成纤维细胞(HPDLFs)的自噬及其可能存在的作用机制。方法收集12~18岁因正畸需要减数而拔除的健康前磨牙,采集和培养HPDLFs,并进行鉴定。将miR-145-3p类似物(miR-145-3p-mimics组)、miR-145-3p抑制物(miR-145-3p-inhibitor组)、阴性对照(miR-145-3p normal control,miR-145-3p-NC组)以及带有GFP-LC3的质粒转染至HPDLFs,24 h后荧光显微镜观察荧光发生情况。双荧光素酶报告实验验证miR-145-3p与HDAC4的靶向关系,Western Blot检测各转染组细胞内HDAC4、Beclin-1、P62和LC3的蛋白表达。结果免疫组化结果表明,波形蛋白为阳性染色而角蛋白呈阴性染色,证明其为无混杂细胞的HPDLFs。转染结果显示,miR-145-3p-mimics组的细胞内荧光强度最高,有大量自噬体形成,而miR-145-3p-inhibitor组荧光最弱。双荧光素酶报告实验证实miR-145-3p靶向抑制HPDLFs细胞中HDAC4的表达。4组细胞中miR-145-3p-inhibitor组P62蛋白表达最高(P<0.05),而其他3组差异无统计学意义(P>0.05);miR-145-3p-mimics组细胞内Beclin-1和LC3蛋白表达最高(P<0.05),其他3组差异无统计学意义(P>0.05)。结论miR-145-3p在HPDLFs中可调控自噬,这种作用可能是通过靶向抑制HDAC4表达实现的。
Objective To investigate the effects and action mechanism of miR-145-3p targeting HDAC4 to regulate autophagy of human periodontal ligament fibroblasts(HPDLFs).Methods The healthy premolars of chidren with 12-18 years of age were collected,and HPDLFs were collected,cultured and identified.The miR-145-3p-mimics,miR-145-3p-inhibitor,and miR-145-3p normal control(miR-145-3p-NC)and the plasmid with GFP-LC3 were transfected into HPDLFs,and the fluorescence occurrence was observed with a fluorescence microscope After 24 hours.Moreover the double luciferase report experiment was used to verify the targeting relationship between miR-145-3p and HDAC4.Western Blot was used to detect the expression levels of HDAC4,Beclin-1,P62 and LC3 in every transfection group.Results Immunohistochemical results showed that vimentin was positively stained,however,the keratin was negatively stained,proving that it was HPDLFs without promiscuous cells.Transfection results showed that the intracellular fluorescence intensity in miR-145-3p-mimics group was the highest,and a large number of autophagosomes were formed,however,which in miR-145-3p-inhibitor group was the weakest.Double luciferase reporting experiments confirmed that miR-145-3p targetedly inhibited the HDAC4 expression.The expression levels of P62 protein in miR-145-3p-inhibitor group were the highest(P<0.05),while the there were no significant differences among the other three groups.In addition the expression levels of Beclin-1 and LC3 protein were the highest in miR-145-3p-mimics group(P<0.05),but there were no significant differences among the other three groups(P>0.05).Conclusion The miR-145-3p can regulate the autophagy in HPDLFs,and its action mechanism may be related with targetedly inhibiting the HDAC4 expression.
作者
杨正祥
谢陆莉
邓琳
王瑜
YANG Zhengxiang;XIE Luli;DENG Lin(Department of Stomatology,The First People’s Hospital of Longquanyi District,Sichuan,Chengdu 610100,China)
出处
《河北医药》
CAS
2021年第9期1312-1315,1320,共5页
Hebei Medical Journal
