MiR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制研究

doi:10.3969/j.issn.1004-616x.2021.02.001

癌变·畸变·突变 ›› 2021, Vol. 33 ›› Issue (2): 81-88,94.doi: 10.3969/j.issn.1004-616x.2021.02.001

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MiR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制研究

张碟, 刘祥贺, 霍苗苗, 刘梅, 徐宁志, 朱红霞

国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院, 分子肿瘤学国家重点实验室, 北京 100021

收稿日期:2020-10-10 修回日期:2021-02-27 出版日期:2021-03-30 发布日期:2021-04-12
通讯作者: 朱红霞,E-mail:drhxzhu@cicams.ac.cn E-mail:drhxzhu@cicams.ac.cn
作者简介:张碟,E-mail:2113126781@qq.com。
基金资助:
中国医学科学院医学与健康科技创新工程（2016-I2M-1-001）

Effects of miR-195-5p on radiosensitivity of esophageal squamous cell carcinoma cells

ZHANG Die, LIU Xianghe, HUO Miaomiao, LIU Mei, XU Ningzhi, ZHU Hongxia

State Key Laboratory of Molecular Oncology, Center for Cancer Precision Medicine, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

Received:2020-10-10 Revised:2021-02-27 Online:2021-03-30 Published:2021-04-12

摘要/Abstract

摘要： 目的：探讨miR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制。方法：采用4和8 Gy X射线照射KYSE510和KYSE150细胞，以未照射细胞为对照。采用CCK-8法检测细胞生长情况，qPCR检测细胞内miR-195-5p及survivin mRNA的表达水平，Western blot检测细胞内survivin蛋白的表达水平。KYSE510细胞分为转染miR-195-5p类似物组、转染miR-195-5p拮抗物组和相应的对照组，并采用X射线照射后，Incucyte实时动态活细胞监测和EdU掺入实验测定细胞增殖情况，克隆形成实验和流式细胞术分别测定细胞克隆形成率和凋亡率，双荧光素酶报告基因实验验证各组KYSE150细胞的荧光素酶活性。结果：经X射线照射后的KESE510和KYSE150细胞生长均受到明显抑制，与未照射组相比，照射组细胞miR-195-5p表达降低，survivin蛋白表达升高（P<0.05或0.01）。转染miR-195-5p类似物组的KESE510细胞在接受X射线照射后的存活率较阴性对照组降低，EdU阳性率和克隆存活率降低，凋亡率显著升高，细胞中survivin mRNA和蛋白表达较阴性对照组均降低（P<0.05或0.01）；转染miR-195-5p拮抗物组KESE510细胞的EdU阳性率和克隆存活率较阴性对照组升高，细胞中survivin mRNA和蛋白表达均升高（P<0.01）。在KYSE150细胞中过表达miR-195-5p能够抑制BIRC5 3' UTR报告基因的荧光素酶活性（P<0.05）。结论：MiR-195-5p可能通过靶向抑制survivin基因的表达增强食管鳞癌细胞的放射敏感性。

关键词: miR-195-5p, survivin, 放射敏感性, 食管鳞癌细胞

Abstract: OBJECTIVE: To investigate effects and mechanism of miR-195-5p on radiosensitivity of esophageal squamous cell carcinoma cells in vitro. METHODS: KYSE510 and KYSE150 cells were exposed to an 8 Gy of X-rays,and non-irradiated cells were used as control. mRNA expression levels of miR-195-5p and of survivin were detected by qPCR. Protein expression levels of survivin were detected by Western blot. KYSE510 cells were transfected with different oligos which gave rise to the miR-195-5p mimic,antagomir and negative control groups. After KYSE510 cells were irradiated with X-rays,cell proliferation was measured by the Incucyte real-time dynamic living cell monitoring and EdU incorporation assays. Cell colony survival and apoptosis rates were measured by colony formation assay and flow cytometry analysis,respectively. Dual-luciferase reporter assay was used to verify the luciferase activity of KYSE150 cells in each group. RESULTS: Growth of KYSE510 and KYSE150 cells were significantly inhibited after irradiation. Compared with the non-irradiation group,expression of miR-195-5p in the irradiation group was decreased,and expression of survivin protein was increased (P<0.05 or 0.01). After X-ray irradiation,the survival rate of the miR-195-5p mimic group was lower than that of the negative control group (P<0.01). The rates for EdU positive cells and clonal survival were lower than that of the negative control group (P<0.05 or 0.01),and the apoptosis rate was significantly higher than that of the negative control group (P<0.01). However,the rates for EdU positive cells and clonal survival of the antagomir group was higher than that of the negative control group (P<0.01). The expressions of survivin mRNA and protein in the miR-195-5p mimic group was lower than that of the negative control group. In contrast,the expressions of survivin mRNA and protein in the miR-195-5p antagomir group was higher than that of the negative control group. Over-expression of miR-195-5p in KYSE150 cells inhibited luciferase activity of the BIRC5 3' UTR reporter gene (P<0.05). CONCLUSION: MiR-195-5p could enhance radiosensitivity of esophageal squamous cell carcinoma cells in vitro by targeting survivin gene.

Key words: miR-195-5p, survivin, radiosensitivity, esophageal squamous cell carcinoma

中图分类号:

R730.5

张碟, 刘祥贺, 霍苗苗, 刘梅, 徐宁志, 朱红霞. MiR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制研究[J]. 癌变·畸变·突变, 2021, 33(2): 81-88,94.

ZHANG Die, LIU Xianghe, HUO Miaomiao, LIU Mei, XU Ningzhi, ZHU Hongxia. Effects of miR-195-5p on radiosensitivity of esophageal squamous cell carcinoma cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(2): 81-88,94.

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MiR-195-5p对食管鳞癌细胞放射敏感性的影响及作用机制研究

Effects of miR-195-5p on radiosensitivity of esophageal squamous cell carcinoma cells

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