摘要
目的观察miR-93对常用于心脏发育研究的睾丸畸胎瘤细胞P19增殖与心肌分化的影响,并分析其与活化T细胞核转录因子3(NFATC3)在此过程中的调控关系。方法将P19细胞分为三组,miR-93组、NC组分别转染miR-93 mimic及阴性对照空白质粒,对照组不转染;采用CCK-8法观察转染0、24、48 h后miR-93对P19细胞增殖的影响,Western blotting法检测miR-93对心肌分化标志基因心肌肌钙蛋白T(cTnT)与锌指转录因子CATA结合蛋白4(GATA4)的影响。通过TargetScanhuman7.2数据库筛选发现NFATC3为miR-93的下游靶基因,通过双荧光素酶基因报告验证两者的结合情况;采用Western blotting法检测miR-93对P19细胞中NFATC3表达的影响。再将P19细胞另分为三组,miR-93组、miR-93+NFATC3组及NC组分别转染miR-93 mimic、miR-93 mimic+NFATC3 mimic及阴性对照空白质粒,采用CCK-8法、Western blotting法观察NFATC3对miR-93过表达所致细胞增殖及心肌分化的影响。结果miR-93组除0 h外同时点细胞OD值及cTnT、GATA4蛋白表达均低于NC组及对照组(P均<0.05),NC组与对照组各指标比较差异无统计学意义。转染NFATC3 wt+NC质粒的P19细胞荧光素酶相对活性与转染NFATC3 wt+miR-93 mimic质粒的细胞比较有明显差异(P<0.01);转染NFATC3 mut+NC质粒的P19细胞荧光素酶相对活性与转染NFATC3 mut+miR-93 mimic质粒的细胞比较无统计学差异。miR-93组NFATC3蛋白表达低于对照组及NC组(P均<0.05),除0 h外同时点细胞OD值及cTnT、GATA4蛋白表达NC组>miR-93+NFATC3组>miR-93组(P均<0.05)。结论miR-93可抑制P19细胞增殖及心肌分化,该作用可能通过沉默NFATC3表达来实现,或可为先天性心脏病提供潜在的治疗靶点。
Objective To observe the effects of miR-93 which is commonly used in the study of cardiac development on the proliferation and cardiomyocyte differentiation of the testicular teratoma cell line P19,and to analyze its regulatory relationship with nuclear factor of activated T cells 3(NFATC3).Methods P19 cells were divided into three groups:miR-93 group(transfected with miR-93 mimic),negative control(NC)group(transfected with empty plasmid),and control group(not transfected).Cell Counting Kit-8(CCK-8)was used to observe the effects of miR-93 on the proliferation of P19 cells at 0,24,and 48 h after transfection.Western blotting was used to determine the effects of miR-93 on the cardiomyocyte differentiation marker genes cardiac troponin T(cTnT)and GATA4.TargetScanHuman 7.2 predicted that NFATC3 was the downstream target gene of miR-93,and their binding was verified by the dual-luciferase reporter assay.Western blotting was used to measure the effect of miR-93 on the expression of NFATC3 in P19 cells.Then P19 cells were divided into another three groups:miR-93 group,miR-93+NFATC3 group,and NC group.The three groups were trans⁃fected with miR-93 mimic,miR-93 mimic plus NFATC3 mimic,and empty plasmid,respectively.CCK-8 and Western blotting were used to determine the role of NFATC3 in miR-93 overexpression-induced cell proliferation and cardiomyocyte differentiation.Results The miR-93 group showed significantly lower cell optical density(OD)and expression of cTnT and GATA4 proteins than the NC group and control group at all time points except 0 h(all P<0.05).There were no significant differences in those indicators between the NC group and the control group.The relative luciferase activity showed a significant difference between P19 cells transfected with the NFATC3 wt+NC plasmid and those transfected with the NFATC3 wt+miR-93 mimic plasmid(P<0.01),and there was no significant difference between P19 cells transfected with the NFATC3 mut+NC plasmid and those transfected with the NFATC3 mut+miR-93 mimic plasmid(P>0.05).The expression of NFATC3 protein in the miR-93 group was significantly lower than those in the control group and NC group(both P<0.05).Cell OD value and cTnT and GATA4 protein expression were as follows:NC group>miR-93+NFATC3 group>miR-93 group(except 0 h)(all P<0.05).Conclusions MiR-93 can inhibit P19 cell proliferation and cardiomyocyte differentiation,which may be achieved by silencing the expression of NFATC3.This may provide a potential therapeutic target for congenital heart disease.
作者
许耿
仲逢钰
顾海涛
XU Geng;ZHONG Fengyu;GU Haitao(Department of Pediatric Cardiothoracic Surgery,The First Affiliated Hospital of Nanjing Medical University,Nanjing 210009,China;不详)
出处
《山东医药》
CAS
2021年第29期1-5,共5页
Shandong Medical Journal
基金
江苏省卫生厅面上项目(H2017026)。
