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长链非编码RNA-ROR介导上皮-间质转化对鼻咽癌细胞放疗抵抗作用的体外研究
薛晓成1△,张雪2△,黄水仙1,张燚1,鲁丹1,陈晓平1*
0
(1. 海军军医大学(第二军医大学)附属公利医院耳鼻咽喉头颈外科, 上海 200135;
2. 海军军医大学(第二军医大学)附属公利医院、上海市卫生健康委员会炎症与慢病管理人工智能重点实验室, 上海 200135
共同第一作者
*通信作者)
摘要:
目的 探讨lncRNA-ROR介导上皮-间质转化在鼻咽癌细胞放疗抵抗中的作用。方法 将鼻咽癌细胞CNE2分为空白组、阴性对照组、lncRNA-ROR沉默组,进行相应的处理。将CNE2细胞分为空白组、放疗组、放疗+阴性对照组、放疗+lncRNA-ROR过表达组(放疗处理为6 Gy射线照射24 h),进行相应的处理。用CCK-8法检测CNE2增殖能力,通过细胞划痕实验和Transwell 细胞迁移实验检测细胞迁移能力,用流式细胞术检测细胞凋亡的情况,用蛋白印迹法检测凋亡相关蛋白和上皮-间质转化相关蛋白的表达。结果 与空白组、阴性对照组相比,抑制lncRNA-ROR表达48、72 h后鼻咽癌细胞CNE2的增殖能力均减弱(均P<0.05)。抑制lncRNA-ROR表达后鼻咽癌细胞CNE2的迁移率低于阴性对照组(P<0.05),而放疗+lncRNA-ROR过表达组CNE2细胞的迁移能力高于放疗组与放疗+阴性对照组(均P<0.05)。与放疗组、放疗+阴性对照组相比,放疗+lncRNA-ROR过表达组CNE2细胞的凋亡率均降低(均P<0.05)。抑制lncRNA-ROR后,活化的caspase 3、caspase 9蛋白表达均较空白组和阴性对照组升高(均P<0.05);而放疗+lncRNA-ROR过表达组活化的caspase 3、caspase 9蛋白表达均较放疗组和放疗+阴性对照组下降(均P<0.05)。抑制lncRNA-ROR可导致上皮标志蛋白(E-钙黏蛋白、β-联蛋白)表达升高,间质标志蛋白(N-钙黏蛋白、波形蛋白)表达下降(均P<0.05);而与放疗组和放疗+阴性对照组相比,放疗+lncRNA-ROR过表达组CNE2细胞的上皮标志蛋白表达下降、间质标志蛋白表达升高(均P<0.05)。结论 lncRNA-ROR可通过调控鼻咽癌细胞增殖、迁移、凋亡及上皮-间质转化影响其放疗抵抗,是逆转鼻咽癌细胞放疗抵抗的潜在靶点。
关键词:  鼻咽肿瘤  放疗抵抗  上皮-间质转化  长链非编码RNA-ROR
DOI:10.16781/j.CN31-2187/R.20230506
投稿时间:2023-09-05修订日期:2023-10-10
基金项目:上海市浦东新区卫生系统优秀青年医学人才培养计划(PWRq-2020-63),上海市卫生健康委员会青年项目(20204Y0144),上海市浦东新区重点亚专科项目(PWZy2020-06),上海市浦东新区临床特色专科项目(PWYts2021-15).
Effect of long non-coding RNA-ROR mediating epithelial-mesenchymal transformation on radiotherapy resistance of nasopharyngeal carcinoma cells in vitro
XUE Xiaocheng1△,ZHANG Xue2△,HUANG Shuixian1,ZHANG Yi1,LU Dan1,CHEN Xiaoping1*
(1. Department of Otorhinolaryngology Head and Neck Surgery, Gongli Hospital, Naval Medical University (Second Military Medical University), Shanghai 200135, China;
2. Key Laboratory of Artificial Intelligence for Inflammation and Chronic Disease Management, Shanghai Municipal Health Commission, Gongli Hospital, Naval Medical University (Second Military Medical University), Shanghai 200135, China
Co-first authors.
* Corresponding author)
Abstract:
Objective To investigate the role of long non-coding RNA (lncRNA)-ROR in mediating epithelial-mesenchymal transformation (EMT) and its impact on radiotherapy resistance in nasopharyngeal carcinoma cells. Methods Nasopharyngeal carcinoma cells CNE2 were divided into blank group, negative control (NC) group and lncRNA-ROR siliencing group; or were divided into blank group, radiotherapy group, radiotherapy+NC group, and radiotherapy+lncRNA-ROR overexpression group (radiotherapy treated with 6 Gy radiation for 24 h). The CNE2 proliferation was detected by cell counting kit 8 method. The cell migration was detected by cell scratch test and Transwell cell migration test. The apoptosis ratio was detected by flow cytometry, and the apoptosis-related proteins and epithelial-mesenchymal transition proteins were detected by Western blotting. Results Compared with the blank group and NC group, the proliferation ability of nasopharyngeal carcinoma cells CNE2 was decreased after inhibition of lncRNA-ROR expression for 48 and 72 h (all P<0.05). The mobility of CNE2 cells after lncRNA-ROR expression inhibition was lower than that in the NC group (P<0.05). The migration ability of CNE2 cells in the radiotherapy+lncRNA-ROR overexpression group was higher than that in the radiotherapy group and radiotherapy+NC group (both P<0.05). Compared with the radiotherapy group and radiotherapy+NC group, the apoptosis rates of CNE2 cells in the radiotherapy+lncRNA-ROR overexpression group was decreased (both P<0.05). After lncRNA-ROR inhibition, the expression of activated caspase 3 and caspase 9 proteins was increased (both P<0.05), while the expression of activated caspase 3 and caspase 9 proteins was decreased in the radiotherapy+overexpressed lncRNA-ROR group (both P<0.05). Inhibition of lncRNA-ROR increased the expression of epithelial marker proteins (E-cadherin, β-catenin), and decreased the expression of interstitial marker proteins (N-cadherin, vimentin). The epithelial marker protein expression was decreased and interstitial marker protein expression was increased in CNE2 cells in the radiotherapy+lncRNA-ROR overexpression group compared with the radiotherapy group and radiotherapy+NC group (all P<0.05). Conclusion lncRNA-ROR can affect the radiotherapy resistance of nasopharyngeal carcinoma cells by regulating their proliferation, migration, apoptosis and EMT, and it is a potential target for reversing the radiotherapy resistance of nasopharyngeal carcinoma cells.
Key words:  nasopharyngeal neoplasms  radiotherapy resistance  epithelial-mesenchymal transformation  long non-coding RNA-ROR