miR-191-5p通过靶向TJP1调控Cal-27细胞增殖、侵袭和迁移的体外研究

doi:10.13591/j.cnki.kqyx.2023.11.004

摘要/Abstract

摘要：

目的探讨miR-191-5p调控OSCC细胞的生物学功能及作用机制。方法利用生物信息学方法分析miR-191-5p在口腔鳞癌患者样本中的表达情况并通过相关数据库筛选出可能的靶基因TJP1,通过双荧光素酶实验验证二者的靶向关系。体外转染分为空白组、inhibitor NC(阴性对照组)和miR-191-5p inhibitor(敲低miR-191-5p组)。敲低miR-191-5p后通过qRT-PCR验证转染效率,CCK-8增殖实验、细胞克隆实验、Transwell、划痕实验分别检测miR-191-5p对Cal-27细胞的增殖能力、侵袭和迁移能力的影响;Western blotting检测TJP1蛋白及上皮间质转化相关蛋白的表达。将 siRNA-TJP1 转染到miR-191-5p低表达的细胞中,观察同时下调TJP1后对Cal-27细胞的增殖和迁移能力的影响。结果 miR-191-5p在OSCC组织和细胞中表达上调,而下调miR-191-5p可抑制Cal-27细胞的增殖、侵袭和迁移能力以及上皮间质转化趋势;miR-191-5p可靶向抑制TJP1的表达,同时TJP1的下调逆转了敲低miR-191-5p对Cal-27细胞增殖和迁移的抑制。结论 miR-191-5p通过靶向TJP1抑制OSCC细胞的增殖、侵袭和迁移,并可能通过调控EMT途径增加肿瘤的转移。

关键词: 口腔鳞癌, miR-191-5p, TJP1, 增殖, 侵袭迁移, 上皮间质转化

Abstract:

Objective To investigate the biological function and mechanism of miR-191-5p regulating OSCC cells. Methods The expression of miR-191-5p in oral squamous cell carcinoma patients was analyzed by bioinformatics method, and the possible target gene TJP1 was screened out through the relevant database. The targeting relationship between the two was verified by dual-luciferase experiment. In vitro transfection was divided into blank, inhibitor NC (negative control group) and miR-191-5p inhibitor (knockdown miR-191-5p group). After knocking down miR-191-5p, the transfection efficiency was verified by qRT-PCR, and the effects of miR-191-5p on the proliferation, invasion and migration ability of Cal-27 cells were detected by CCK-8 proliferation experiment, cell cloning experiment, Transwell and scratch experiment. Western blotting detected the expression of TJP1 protein and EMT-associated proteins. siRNA-TJP1 was transfected into miR-191-5p low-expressing cells and the effect of simultaneous down-regulation of TJP1 on the proliferation and migration capacity of Cal-27 cells was observed. Results miR-191-5p expression was up-regulated in OSCC tissues and cells, while down-regulation of miR-191-5p inhibited the proliferation, invasion and migration capacity of Cal-27 cells and EMT tendency. miR-191-5p targeted and inhibited the expression of TJP1, while down-regulation of TJP1 reversed the inhibition of Cal-27 cell proliferation and migration by knocking down miR-191-5p. Conclusion The proliferation, invasion and migration of OSCC cells is inhibited by targeting TJP1 under miR-191-5p, and miR-191-5p may increase tumor metastasis by regulating the EMT pathway.

Key words: oral squamous cell carcinoma, miR-191-5p, tight junction protein 1(TJP1), proliferation, invasion and migration, epithelial-mesenchymal transition

中图分类号:

R739.85

佟雪溪, 赵刚. miR-191-5p通过靶向TJP1调控Cal-27细胞增殖、侵袭和迁移的体外研究[J]. 口腔医学, 2023, 43(11): 981-988.

TONG Xuexi, ZHAO Gang. In vitro study on miR-191-5p targeting TJP1 to regulate the proliferation, invasion and migration of Cal-27 cells[J]. Stomatology, 2023, 43(11): 981-988.

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基因名称	上游引物(5'—3')	下游引物(5'—3')
U6	GGAACGATACAGAGAAGATTAGC	TGGAACGCTTCACGAATTTGCG
miR-191-5P	CCAACGGAATCCCAAAAGCAGC	TGGAACGCTTCACGAATTTGCG
GAPDH	TACTAGCGGTTTTACGGGCG	TCGAACAGGAGGAGCAGAGAGCGA
TJP1	CTGGTGAAATCCCGGAAAAATGA	TTGCTGCCAAACTATCTTGTGA