miR-4505靶向SOX-10在先天性巨结肠中的作用及机制

miR-4505 downregulates cell proliferation and migration in Hirschsprung′s disease by targeting SOX-10

目的 探讨miR-4505在先天性巨结肠(HSCR)中的作用及分子机制。方法 选取HSCR病变段肠管及扩张段肠管组织标本,q RT-PCR检测组织标本miR-4505表达量;分别选用miR-4505模拟物及抑制剂转染人神经母细胞株SH-SY5Y,q RT-PCR检测转染后miR-4505表达,CCK-8及Transwell检测miR-4505对细胞增殖及细胞迁移的影响;生物信息学预测、双荧光素酶报告基因实验、Western blot以及免疫组织化学在细胞及组织层面证实SOX-10是miR-4505的作用靶点。结果 与扩张段肠管相比,HSCR病变段肠管的miR-4505呈高频高表达(t=11.25,P <0.001);过表达miR-4505可以抑制细胞增殖及迁移,敲低其表达后细胞的增殖及迁移能力增强;靶基因预测及双荧光素酶报告基因检测证实miR-4505可与SOX-10的3′-UTR结合,Western blot证实其可降低细胞SOX-10蛋白含量,而免疫组化证实SOX-10在HSCR病变段肠管中呈低表达。结论 miR-4505在HSCR病变段及扩张段肠管之间存在表达差异,miR-4505可以抑制细胞的增殖及迁移,其在HSCR中发挥作用可能与抑制SOX-10基因表达有关。

Objective To explore the role and molecular mechanism of miR-4505 in Hirschsprung disease(HSCR).Methods Stenotic and dilated colon segments of HSCR patients operated at Zhujiang Hospital of Southern Medical University were collected.qRT-PCR was performed to detected the expression of miR-4505 in colon tissues-specimens.The miR-NC,miR-4505 mimics and miR-4505 inhibitors were transfected into human neuroblastoma cell line SH-SY5Y,followed by qRT-PCR to examine the expression of miR-4505 after transfection,CCK-8 assay and Transwell assay to detect the effect of miR-4505 on the proliferation and migration of SH-SY5Y cells.The down-stream target genes of miR-4505 were predicted through bioinformatics and detected by dual luciferase reporter gene assay;the target genes were verified by Western blot and Immunohistochemistry.Results miR-4505 was highly expressed in the stenotic colon compared to the dilated segment(t=11.25,P<0.001).Overexpression of miR-4505 inhibited cell proliferation and migration,and conversely,downregulation of miR-4505 enhanced cell proliferation and migration.Target gene prediction and dual-luciferase reporter gene assay suggested that the 3′-UTR of SOX-10 was a direct target of miR-4505.Moreover,Western blot confirmed that miR-4505 decreased SOX-10 protein levels in cells,while immunohistochemistry proved that SOX-10 was lowly expressed in HSCR disease colontissues.Conclusion Our study demonstrated that miR-4505 was differentially expressed between the diseased and dilated segments of HSCR.miR-4505 could inhibited cell proliferation and migration,and its role in HSCR may be achieved by suppressing the downstream SOX-10 gene expression.