miR-4505靶向SOX-10在先天性巨结肠中的作用及机制

doi:10.3969/j.issn.1006-5725.2023.18.008

实用医学杂志 ›› 2023, Vol. 39 ›› Issue (18): 2330-2334.doi: 10.3969/j.issn.1006-5725.2023.18.008

miR-4505靶向SOX-10在先天性巨结肠中的作用及机制

吴凯,王健俊,何继贤,陈钦明,余岱岳,路羿,范凯斯,张梦真,杨六成()

南方医科大学珠江医院小儿外科（广州 510282 ）

收稿日期:2023-03-17 出版日期:2023-09-25 发布日期:2023-10-10
通讯作者: 杨六成 E-mail:sdylc@aliyun.com
基金资助:
广东省自然科学基金项目(2019A1515011086)

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

Kai WU,Jianjun WANG,Jixian HE,Qinming CHEN,Daiyue YU,Yi LU,Kaisi FAN,Mengzhen ZHANG,Liucheng. YANG()

Department of Pediatric Surgery，Zhujiang Hospital of Southern Medical University，Guangzhou 510282，China Corrsponding author： YANG Liucheng E?mail：sdylc@aliyun. com

Received:2023-03-17 Online:2023-09-25 Published:2023-10-10
Contact: Liucheng. YANG E-mail:sdylc@aliyun.com

摘要/Abstract

摘要：

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，qRT-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，qRT-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。

关键词: 先天性巨结肠, 神经母细胞, miR-4505, SOX-10

Abstract:

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 tissuesspecimens.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 downstream 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.

Key words: hirschsprung disease, neural stem cell, miR-4505, SOX-10

中图分类号:

R726

吴凯,王健俊,何继贤,陈钦明,余岱岳,路羿,范凯斯,张梦真,杨六成. miR-4505靶向SOX-10在先天性巨结肠中的作用及机制[J]. 实用医学杂志, 2023, 39(18): 2330-2334.

Kai WU,Jianjun WANG,Jixian HE,Qinming CHEN,Daiyue YU,Yi LU,Kaisi FAN,Mengzhen ZHANG,Liucheng. YANG. miR-4505 downregulates cell proliferation and migration in Hirschsprung′s disease by targeting SOX-10[J]. The Journal of Practical Medicine, 2023, 39(18): 2330-2334.

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参考文献 20

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引物	序列
U6-Forward	CTCGCTTCGGCAGCACA
U6-Reverse	AACGCTTCACGAATTTGCGT
miR-4505-Forward	CGGGCAGGCTGGGCTG
miR-4505-Reverse	CAGCCACAAAAGAGCACAAT

分组	0 h	24 h	48 h	72 h	96 h
miR?4505 mimics	0.282 ± 0.013	0.333 ± 0.029	0.385 ± 0.038	0.448 ± 0.042	0.490 ± 0.048
NC	0.285 ± 0.015	0.345 ± 0.030	0.452 ± 0.039	0.548 ± 0.046	0.641 ± 0.052
miR?4505 inhibitors	0.285 ± 0.013	0.358 ± 0.026	0.489 ± 0.042	0.587 ± 0.051	0.712 ± 0.058

分组	miR?4505 mimics	NC
野生型SOX?10	0.214 ± 0.065	1.081 ± 0.102
突变型SOX?10	1.020 ± 0.082	1.142 ± 0.121

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

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