MicroRNA-145-3p 在先天性巨结肠中的 作用及机制研究

Role and mechanism of microRNA-145-3p in Hirschsprung disease

摘要:

目的 探讨microRNA-145-3p（miR-145-3p）在先天性巨结肠（HD）中的表达及其发挥作用 的可能分子机制。方法 选取2013 年2 月—2018 年6 月南方医科大学珠江医院40 例HD 病变段肠管（HD 病 变肠管组）及40 例正常肠管（正常肠管组）组织标本。采用qRT-PCR 检测组织标本miR-145-3p 相对表达 量。过表达细胞miR-145-3p 后，CCK-8 法检测细胞的增殖情况；Transwell 检测细胞迁移情况；双荧光素酶 报告基因实验、Western blotting 及免疫组织化学染色明确miR-145-3p 对靶基因GDNF 的影响。结果 HD 病 变肠管组miR-145-3p 相对表达量高于正常肠管组（P <0.05）。过表达细胞miR-145-3p 后，细胞增殖能力下 降（P <0.05），细胞迁移能力降低（P <0.05）；靶基因预测软件筛选出GDNF 是miR-145-3p 的潜在靶点；双 荧光素酶报告基因实验证实，miR-145-3p 可以与GDNF 的3'-UTR 结合（P <0.05）；Western blotting 检测证实， miR-145-3p 可使SH-SY5Y 细胞的GDNF 蛋白相对表达量降低（P <0.05）；而免疫组织化学染色证实，GDNF 在miR-145-3p 高表达的HD 组织标本中存在高频低表达。结论 miR-145-3p 可以抑制神经母细胞的增殖及 迁移，是一个重要的HD 分子标志物，其作用机制可能与抑制GDNF 蛋白表达有关。

Abstract:

Objective To investigate the relationship between miR-145-3p and Hirschsprung disease (HD), and to explore the possible molecular mechanism. Methods In this study, a total of 80 human colon tissues including 40 HD stenotic colon segments and 40 matched normal colon segments who undertook operative treatment were collected from Zhujiang Hospital of Southern medical University, and qRT-PCR was used to detect the relative expression of miR-145-3p in colon tissues. Cell Counting Kit-8 (CCK-8) assay and Transwell assay were employed to investigate the biological function of miR-145-3p in human SH-SY5Y cell lines. Bioinformatic analysis, dualluciferase reporter assay, Western blot and immunohistochemistry were performed to evaluate the target for miR- 145-3p. Results We found that ganglion cell numbers were reduced while miR-145-3p was upregulated in HD tissues compared to that in normal colon tissues (P < 0.05). MiR-145-3p overexpression inhibited cell proliferation (P < 0.05) and migration (P < 0.05). Bioinformatic software data revealed that miR-145-3p could directly target the 3′-UTR of GNDF. Dual-luciferase reporter assay confirmed that the 3′-UTR of GDNF was a direct target to miR-145-3p (P < 0.05). Moreover, an increased level of miR-145-3p was inversely correlated with decreased levels of GDNF protein in cells (P < 0.05) and tissues. Conclusions Our study demonstrates that aberrant expression of miR-145-3p might play a crucial role in the development of HD partly by regulating GDNF expression.