微小RNA hsa-miR-93靶向STAT3提高鼻咽癌对放疗敏感性的机制研究

doi:10.11904/j.issn.1002-3070.2023.01.004

实用肿瘤学杂志 ›› 2023, Vol. 37 ›› Issue (1): 17-25.doi: 10.11904/j.issn.1002-3070.2023.01.004

微小RNA hsa-miR-93靶向STAT3提高鼻咽癌对放疗敏感性的机制研究

周绮纯¹, 王晰², 邓芷茵², 徐梦菲², 唐青¹, 吴万垠¹, 王苏美¹

1.广州中医药大学第二临床医学院中医药防治非小细胞肺癌临床与基础研究团队(广州 510120);
2.广州中医药大学第二临床医学院

收稿日期:2022-10-31 修回日期:2022-12-06 出版日期:2023-02-28 发布日期:2023-03-21
通讯作者: 王苏美,E-mail:wangsumei198708@163.com
作者简介:周绮纯,女,(1996-),硕士,研究实习员,从事肿瘤表观遗传学的研究。
基金资助:
广东省中医院科学技术专项(编号:YN2019QJ06);广东省中医院拔尖人才科研专项(编号:BJ2022KY13)

Research on the mechanism of hsa-miR-93 targeting STAT3 to improve the radiosensitivity of nasopharyngeal carcinoma

ZHOU Qichun¹, WANG Xi², DENG Zhiyin², XU Mengfei², TANG Qing¹, WU Wanyin¹, WANG Sumei¹

1. Clinical and Basic Research Team of TCM Prevention and Treatment of Non-Small Cell Lung Cancer,The Second Clinical College of Guangzhou University of Chinese Medicine,Guangzhou 510120,China;
2. Second Clinical College of Guangzhou University of Chinese Medicine

Received:2022-10-31 Revised:2022-12-06 Online:2023-02-28 Published:2023-03-21

摘要/Abstract

摘要： 目的研究hsa-miR-93在鼻咽癌放疗抗拒过程中的作用及分子机制。方法以人鼻咽癌细胞株CNE-1、CNE-2、CNE-2R、HONE1、C666.1以及鼻咽上皮永生细胞NP460为研究对象,采用实时荧光定量PCR(qRT-PCR)法检测各组细胞经放疗后hsa-miR-93的表达;采用Western blot法检测各组细胞经放疗后STAT3蛋白的表达;通过miRWalk软件和RNAHybrid软件预测hsa-miR-93和STAT3的结合作用及结合位点;通过双荧光素酶报告基因法检测hsa-miR-93对STAT3靶向结合情况;瞬时转染小分子模拟物或抑制剂调控hsa-miR-93的表达后,采用qRT-PCR和Western blot法检测STAT3的表达;转染siSTAT3沉默STAT3的表达后,通过细胞集落形成实验检测鼻咽癌细胞经放疗后的集落形成能力。结果与CNE-1、CNE-2细胞(相对放疗敏感)相比,hsa-miR-93在HONE1、CNE-2R细胞(相对放疗抗拒)中表达降低(P＜0.001),且各组鼻咽癌细胞经放疗后hsa-miR-93的表达呈剂量及时间依赖性降低(P＜0.05);与CNE-1、CNE-2细胞相比,STAT3在HONE1、CNE-2R细胞中表达升高,且各组鼻咽癌细胞经放疗后表达呈剂量及时间依赖性升高;经预测,hsa-miR-93可直接靶向结合STAT3。过表达hsa-miR-93可以在mRNA以及蛋白水平上显著抑制STAT3的表达(P＜0.05),而抑制hsa-miR-93的表达可以显著上调STAT3的表达(P＜0.05);过表达hsa-miR-93或沉默STAT3均使鼻咽癌细胞的集落形成显著减少(P＜0.001);抑制hsa-miR-93减弱了鼻咽癌对放疗的敏感性,而同时抑制hsa-miR-93和STAT3后,可提高鼻咽癌对放疗的敏感性。结论 hsa-miR-93可能通过靶向STAT3提高鼻咽癌对放疗的敏感性。

关键词: 鼻咽癌, 放疗, 微小RNA, hsa-miR-93, STAT3

Abstract: Objective The Objective of this study was to study the role and molecular mechanism of hsa-miR-93 in radiotherapy resistance of nasopharyngeal carcinoma(NPC). Methods Human NPC cell lines CNE-1,CNE-2,CNE-2R,HONE1,C666.1 and nasopharyngeal epithelial immortalized NP460 cell line were used as the research objects.The expression of hsa-miR-93 after radiotherapy in each group of cells was detected by qRT-PCR;The expression of STAT3 protein after radiotherapy in each group of cells was detected by Western blot;MiRwalk software and RNAHybrid software were used to predict the function and binding site of the combination of hsa-miR-93 and STAT3;The binding sites of hsa-miR-93 to STAT3 was detected by dual luciferase reporter gene assay;The expression of STAT3 was detected by qRT-PCR and Western blot;After the expression of STAT3 was silenced by transfection with siSTAT3,the clonogenic ability of NPC cells after radiotherapy was detected by cell colony formation assay. Results Compared with CNE-1 cells and CNE-2 cells(relative radiotherapy sensitivity),the expression of hsa-miR-93 in HONE1 cells and CNE-2R cells(relative radiotherapy resistance)decreased(P＜0.001),and the expression of hsa-miR-93 in each group was decreased in a dose- and time- dependent manner after radiotherapy(P＜0.05).Compared with CNE-1 cells and CNE-2 cells,the expression of STAT3 increased in HONE1 cells and CNE-2R cells,and the expression of STAT3 in NPC cells of each group after radiotherapy increased in a dose- and time-dependent manner.It was predicted that hsa-miR-93 could directly target STAT3.Overexpression of hsa-miR-93 could significantly inhibit the expression of STAT3 at mRNA and protein levels(P＜0.05),while inhibition of hsa-miR-93 could significantly upregulate the expression of STAT3(P＜0.05).Overexpression of hsa-miR-93 or silencing of STAT3 significantly reduced the number of NPC cell colony(P＜0.001).Inhibition of hsa-miR-93 attenuated the sensitivity of NPC to radiotherapy,while simultaneous inhibition of hsa-miR-93 and STAT3 could increase the sensitivity of NPC to radiotherapy. Conclusion hsa-miR-93 increases the sensitivity of NPC to radiotherapy by targeting STAT3.

Key words: Nasopharyngeal carcinoma, Radiotherapy, microRNA, hsa-miR-93, STAT3

中图分类号:

R739.6

周绮纯, 王晰, 邓芷茵, 徐梦菲, 唐青, 吴万垠, 王苏美. 微小RNA hsa-miR-93靶向STAT3提高鼻咽癌对放疗敏感性的机制研究[J]. 实用肿瘤学杂志, 2023, 37(1): 17-25.

ZHOU Qichun, WANG Xi, DENG Zhiyin, XU Mengfei, TANG Qing, WU Wanyin, WANG Sumei. Research on the mechanism of hsa-miR-93 targeting STAT3 to improve the radiosensitivity of nasopharyngeal carcinoma[J]. Journal of Practical Oncology, 2023, 37(1): 17-25.

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微小RNA hsa-miR-93靶向STAT3提高鼻咽癌对放疗敏感性的机制研究

Research on the mechanism of hsa-miR-93 targeting STAT3 to improve the radiosensitivity of nasopharyngeal carcinoma

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