丝裂霉素C通过调控miR-200b表达诱导人成纤维细胞凋亡的机制研究

We found that anti-tumor drugs could induce fibroblast apoptosis by several apoptosis pathway and prevent epidural adhesion,but mitomycin c had stronger effect than other drugs.We combined their specific inhibitors to block these apoptosis pathways,but found that they could not completely suppress fibroblast apoptosis induced by mitomycin c. The result of microarray showed that miR-200b expression in fibroblast stimulated by mitomycin c was upregulated compared with that in normal fibroblast. It was found and confirmed that P27 was a target gene of miR-200b by bioinformatics analysis and luciferase report gene system. P27 can inhibit the activity of the cyclin and its deopendent protein kinase and induce cell apoptosis.We speculate that mitomycin c can induce fibrobalst apoptosis by regulating miR-200b expression and P27 expression. In this study,we culture human epidural scar fibroblasts in vitro and investigate the mechanism of mitomycin c inducing fibrobalst apoptosis by regulating miR-200b expression and P27 expression. Moreover, we explore the effect of mitomycin c on regulating miR-200b expression by methylation modification of miR-200b promoter region using methylation sequencing assay. The study could illuminate the molecular mechanism of mitomycin c inducing fibrobalst apoptosis and preventing epidural scar adhesion by regulating miR-200b expression,which could provide a new way and a potential therapeutic target for preventing scar adhesion.

我们发现抗肿瘤药物能通过多种凋亡途径诱导成纤维细胞凋亡、防治硬膜外粘连，而丝裂霉素C（MMC）的防治效果更强，但应用特异性抑制剂联合阻断这些途径后，发现不能完全抑制MMC诱导的成纤维细胞凋亡。我们采用基因芯片技术检测miR-200b高表达于MMC刺激的人成纤维细胞。生物信息学分析及荧光素酶报告基因系统发现并证实P27是其靶基因。P27能抑制细胞周期蛋白及其激酶活性、诱导细胞凋亡。我们推测MMC可能通过调控miR-200b表达、调节P27来诱导成纤维细胞凋亡。本研究拟体外培养人硬膜外瘢痕成纤维细胞，探讨MMC通过调控miR-200b表达、调节P27诱导成纤维细胞凋亡的分子机制，并采用甲基化测序法明确MMC通过甲基化修饰miR-200b启动子区调控其表达的机制。本研究拟阐明MMC通过调控miR-200b表达诱导成纤维细胞凋亡、防治硬膜外粘连的分子机制，为瘢痕粘连的防治提供新思路和潜在治疗靶点。

研究背景和目的：成纤维细胞过度增生是形成硬膜外瘢痕粘连的主要原因，而MMC能够有效减少硬膜外粘连形成。本研究探讨MMC对人硬膜外瘢痕成纤维细胞凋亡的影响；MMC调控miR-200b表达、调节靶基因诱导人成纤维细胞凋亡的作用和机制；MMC对miR-200b启动子区甲基化的影响；MMC调控miR-200b表达减少大鼠硬膜外瘢痕粘连的研究。.实验方法：CCK-8检测MMC对人成纤维细胞活性的影响。Western blot、流式细胞技术、Hoechst 33342染色、TUNEL染色等方法检测不同处理因素诱导人成纤维细胞凋亡的情况。基因芯片和real time PCR检测miR-200b表达。荧光素酶报告基因检测验证miR-200b和P27、RhoE的靶向对应关系。亚硫酸氢盐测序法（BSP）检测MMC调控miR-200b启动子区发生甲基化的情况。组织学观察、TUNEL染色、羟脯氨酸含量测定评价MMC调控miR-200b减少大鼠硬膜外粘连的情况。.实验结果：MMC显著降低人成纤维细胞活性，呈现出用药浓度和时间依赖性。Western blot、Hoechst 33342染色、TUNEL染色和流式细胞技术结果显示MMC能够诱导人成纤维细胞凋亡。Real time PCR结果显示MMC能够下调miR-200b表达。荧光素酶报告基因结果显示P27和RhoE是miR-200b的靶基因。慢病毒转染敲除P27和RhoE能够减少靶蛋白表达，并显著降低MMC诱导的人成纤维细胞凋亡。下调miR-200b表达和MMC均能诱导成纤维细胞凋亡，上调miR-200b能够抑制MMC所诱导的人成纤维细胞凋亡。BSP测序结果显示MMC能够引起miR-200b启动子区部分发生甲基化。下调miR-200b表达（MMC诱导和慢病毒转染敲除）能够诱导成纤维细胞凋亡、减少胶原组织形成，有效减少大鼠硬膜外瘢痕粘连。.结论：MMC显著诱导人硬膜外瘢痕成纤维细胞凋亡，呈浓度和时间依赖性。MMC能够下调miR-200b表达、上调靶基因P27和RhoE表达，从而诱导人成纤维细胞凋亡。MMC能够通过下调miR-200b表达、诱导成纤维细胞凋亡，从而减少大鼠硬膜外粘连形成。本研究结果可为瘢痕粘连的防治提供新思路和潜在治疗靶点。