miR-25-3p下调ADAM10表达阻断Notch信号通路抑制P19细胞向心肌细胞分化

miR-25-3p down-regulates the expression of ADAM10 to inhibit the differentiation of P19 cells into cardiomyocytes by blocking the Notch signaling pathway

目的探讨miR-25-3p靶向解整合素金属蛋白酶10(ADAM10)调控Notch信号通路对P19细胞向心肌细胞分化的影响。方法采用二甲基亚砜(DMSO)诱导P19细胞向心肌细胞分化,分别收集诱导分化第0、5、10天的细胞,实时荧光定量PCR检测诱导分化过程中心肌分化标志物GATA结合蛋白4(GATA4)、心肌肌钙蛋白T(cTnT)、心房利钠尿多肽(ANP)的mRNA以及miR-25-3p水平, Western blot法检测诱导分化过程中ADAM10蛋白水平。通过逆转录病毒感染P19细胞过表达miR-25-3p,采用实时荧光定量PCR检测感染后P19细胞中miR-25-3p水平, Western blot法检测感染后P19细胞ADAM10蛋白水平。生物信息学软件预测并通过荧光素酶报告实验验证miR-25-3p与ADAM10之间的靶向结合作用。感染后的P19细胞经DMSO诱导分化10d后,免疫荧光实验检测细胞中心肌肌钙蛋白I(cTnI)蛋白的表达情况,并计算心肌细胞分化率;Western blot法检测GATA4、cTnT、ANP以及Notch信号通路相关蛋白Notch1、Hes家族bHLH转录因子1(Hes1)、Hairy相关转录因子1(Hey1)和Hey2蛋白水平。结果P19细胞向心肌细胞诱导分化的第0、5、10天过程中, GATA4、cTnT、ANP的mRNA水平及ADAM10蛋白水平均逐渐增加,而miR-25-3p水平逐渐降低;逆转录病毒感染后, P19细胞中miR-25-3p水平显著增加,而ADAM10蛋白水平显著降低;生物信息学分析证实ADAM10为miR-25-3p的靶基因;诱导分化10 d后,过表达miR-25-3p可显著降低心肌细胞分化率,下调GATA4、cTnT、ANP及Notch信号通路相关分子Notch1、Hes1、Hey1和Hey2的蛋白水平。结论miR-25-3p可显著抑制P19细胞向心肌细胞分化,其机制可能与其靶向抑制ADAM10表达,进而抑制Notch信号通路的激活有关。

Objective To investigate the effect of miR-25-3p targeting a disintegrin and metalloproteinase 10(ADAM10) on the differentiation of P19 cells into cardiomyocytes by regulating Notch signaling pathway. Methods P19 cells were induced to differentiate into cardiomyocytes with dimethyl sulfoxide(DMSO) and collected at 0, 5, and 10 days. The mRNA levels of myocardial differentiation markers GATA4, cTnT, atrial natriuretic polypeptide(ANP) and the level of miR-25-3p were detected by real-time quantitative PCR(qRT-PCR). The protein level of ADAM10 was assessed by Western blot analysis. The miR-25-3p was over-expressed in P19 cells by infected with retrovirus, and the expression levels of miR-25-3p and ADAM10 in the infected P19 cells were detected by qRT-PCR and Western blotting. Bioinformatics were used to predict the targeted matching relationship between miR-25-3p and ADAM10 gene, which was then verified by the luciferase reporter gene system. After infection, P19 cells were induced to differentiate by DMSO for 10 days. Then the protein expression of cTnI was detected by immunofluorescence assay to calculate the differentiation rate of cardiomyocytes, and the proteins expression of myocardial differentiation markers GATA4, cTnT, ANP and Notch signaling pathway-related molecules Notch1, hes family bHLH transcription factor 1(Hes1), Hey1, and Hey2 were detected by Western blotting. Results During the 0, 5 and 10 days of the differentiation of P19 cells into myocardial cells, the mRNA expression levels of GATA4, cTnT, ANP and the protein expression level of ADAM10 gradually increased, while the expression level of miR-25-3p gradually decreased. After retrovirus infection, the expression level of miR-25-3p in the infected P19 cells went up significantly, while the protein expression level of ADAM10 went down significantly. Subsequently, ADAM10 was confirmed as a target gene of miR-25-3p. After the 10 days of differentiation, over-expression of miR-25-3p significantly decreased the differentiation rate of cardiomyocytes, and down-regulated the levels of the markers of myocardial differentiation-related proteins GATA4, cTnT, ANP, and the Notch signaling pathway related-proteins, including Notch1, Hes1, Hey1 and Hey2. Conclusion The miR-25-3p can significantly inhibit the differentiation of P19 cells into cardiomyocytes, and the mechanism may be related to inhibite the activation of Notch signaling pathway by depressing ADAM10 expression.