CAJ | 학술논문

目的 MicroRNA (miR)-19b是通过芯片筛选出与心脏发育高度相关的一种miRNA,本研究旨在研究miR-19b通过Wnt/β-catenin信号通路对P19细胞的影响.方法生物信息学软件预测miR-19b潜在靶基因;双荧光素酶报告基因验证miR-19b是否作用于Wnt的预测靶点;脂质体2000转染miR-19b过表达质粒与空载质粒进入P19细胞;CCK-8法检测细胞增殖活性;磷脂酰丝氨酸外翻分析检测细胞凋亡;Westem blot检测Wnt1表达水平;实时定量-聚合酶链反应(PCR)检测miRNA-19b表达水平.结果 TargetScan 5.1软件预测Wnt1为miR-19b在P19细胞中发挥作用的潜在靶点,双荧光素酶报告基因进行了验证,miR-19b过表达显著降低了靶基因Wnt的活性(P＜O.05);miR-19b过表达降低了Wnt信号通路中Wnt1和β-catenin的表达水平,两者水平表达一致,于第2天至第10天均有统计学差异(P均＜0.05).与空载对照组相比,miRNA-19b可促进P19细胞的增殖(各时间点P＜0.05),并且抑制其凋亡(P＜0.05).结论 miR-19b可能通过Wnt/β-catenin信号通路促进P19细胞的增殖,抑制凋亡,为其以后用于心脏发育的进一步研究奠定基础.
목적 MicroRNA (miR)-19b시통과심편사선출여심장발육고도상관적일충miRNA,본연구지재연구miR-19b통과Wnt/β-catenin신호통로대P19세포적영향.방법 생물신식학연건예측miR-19b잠재파기인;쌍형광소매보고기인험증miR-19b시부작용우Wnt적예측파점;지질체2000전염miR-19b과표체질립여공재질립진입P19세포;CCK-8법검측세포증식활성;린지선사안산외번분석검측세포조망;Westem blot검측Wnt1표체수평;실시정량-취합매련반응(PCR)검측miRNA-19b표체수평.결과 TargetScan 5.1연건예측Wnt1위miR-19b재P19세포중발휘작용적잠재파점,쌍형광소매보고기인진행료험증,miR-19b과표체현저강저료파기인Wnt적활성(P＜O.05);miR-19b과표체강저료Wnt신호통로중Wnt1화β-catenin적표체수평,량자수평표체일치,우제2천지제10천균유통계학차이(P균＜0.05).여공재대조조상비,miRNA-19b가촉진P19세포적증식(각시간점P＜0.05),병차억제기조망(P＜0.05).결론 miR-19b가능통과Wnt/β-catenin신호통로촉진P19세포적증식,억제조망,위기이후용우심장발육적진일보연구전정기출.
Objective Previously study indicated that the microRNA (miR)-19b is highly correlated with heart development by chip screening.This study aims to explore the function of miR-19b overexpression on P19 cells through Wnt/beta-catenin signaling pathway.Methods Potential target gene of miR-19b was predicted by bioinformatics software such as TargetScan on line; Dual luciferase reporter gene system was applied to test whether the the predict target could bind with miR-19b and transfect miR-19b overexpression plasmid or vector into P19 cells by lipo 2000and stable cell lines was selected by Blasticidin;CCK-8 assay was adopted to detect cell proliferation activityed and cell apoptosis was detected with Phosphatidylserine eversion analysis.Wnt1 protein expression level and the miR-19b RNA expression levels were detected by Western blot and real-time quantitative PCR.Results Wnt1 was the potential targets of miR-19b in P19 cells by TargetScan 5.1 software,which was verified by dual luciferase reporter gene; The results of luciferase reporter gene system demonstrats that miR-19b ihibited the activity of Wnt not the Wnt mutation(P ＜0.05) ; Overexpression of miR-19b reduced Wnt1 and beta-catenin expression level in the Wnt signaling pathway,respectively (at most point P ＜ 0.05).Moreover,miR-19b could promote the proliferation of P19 cell (at most point P ＜0.05),and inhibit its apoptosis(P ＜ 0.05).Conclusions MiR-19b promotes the P19 cell proliferation,inhibits its apoptosis.In addition,miR-19b is indirectly interacted with target gene Wnt1 and affects P19 cell lines through Wnt/beta-catenin signaling pathway.This study shows a new insight of heart development and more efforts are needed on exploring the deep function of heart diseses.