miR-150-5p 靶向 SIRT1 提高肝癌细胞系HepG2放疗敏感性

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (3): 321-327.

miR-150-5p 靶向 SIRT1 提高肝癌细胞系HepG2放疗敏感性

吴大平^1*, 徐璐², 吴焕良¹, 郑文宏¹, 郑小妹¹, 谢文蕊¹

1.儋州市人民医院肿瘤科,海南儋州 571700;
2.海南医学院第一附属医院血液肿瘤科,海南海口 570000

收稿日期:2019-05-13 修回日期:2019-11-04 出版日期:2020-03-05 发布日期:2020-03-02
通讯作者: ^*2696057624@qq.com
基金资助:
海南省卫生计生行业科研项目(18A200157)

miR-150-5p enhances radiotherapy sensitivity of hepatocellular carcinoma cells line HepG2 through targeting SIRT1

WU Da-ping^1*, XU Lu², WU Huan-liang¹, ZHENG Wen-hong¹, ZHENG Xiao-mei¹, XIE Wen-rui¹

1. Department of Oncology, People's Hospital of Danzhou City,Danzhou 571700;
2. Department of Hematology, the First Affiliated Hospital of Hainan Medical College, Haikou 570000, China

Received:2019-05-13 Revised:2019-11-04 Online:2020-03-05 Published:2020-03-02
Contact: ^*2696057624@qq.com

摘要/Abstract

摘要： 目的探究miR-150-5p靶向SIRT1提高肝癌细胞放射敏感性的作用机制。方法用分次放疗放射递增法诱导建立放射抵抗型细胞株(RR-HepG2);RT-qPCR检测HepG2和RR-HepG2在不同放射剂量下miR-150-5p的表达水平;细胞克隆实验检测相同放射剂量下两种细胞的放疗敏感性;流式细胞计量术和Western blot检测过表达miR-150-5p对HepG2凋亡的影响;双荧光素酶报告基因法检测miR-150-5p与SIRT1的关联;细胞克隆实验和Western blot检测过表达SIRT1后,细胞的放疗敏感性和凋亡蛋白的变化。结果与亲代HepG2比较,相同放射剂量下,RR-HepG2组miR-150-5p的表达量均显著降低(P＜0.05);放射处理后,与control、agomiR-NC组比较,agomiR-150-5p组的细胞存活分数显著降低,敏感性高(P＜0.05),Bax、caspase-9蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低,细胞凋亡率显著升高;双荧光素酶报告基因法验证miR-150-5p靶向调控SIRT1。与agomiR-NC组比较,野生型(WT)agomiR-150-5p荧光素酶活性显著降低,SIRT1蛋白水平显著降低(P＜0.05);与anta-agomiR-NC比较,野生型(WT)anta-agomiR-150-5p荧光素酶活性显著升高,SIRT1蛋白水平显著升高(P＜0.05);放射处理后,与agomiR-NC组比较,agomiR-150-5p组的细胞存活分数显著降低,Bax、caspase-9蛋白表达水平显著升高,Bcl-2蛋白表达水平显著降低(P＜0.05);与agomiR-150-5p+vector组比较,agomiR-150-5p+SIRT1组的细胞存活分数显著升高,Bax、caspase-9蛋白表达水平显著降低,Bcl-2蛋白表达水平显著升高(P＜0.05)。结论 miR-150-5p靶向SIRT1,下调其表达,提高肝癌细胞放射敏感性,可为临床肝癌放射治疗增敏提供靶点。

关键词: miR-150-5p, SIRT1, 肝癌, 放疗敏感性

Abstract: Objective To investigate the effect on the radiosensitivity enhancement of hepatocellular carcinoma cells by targeting SIRT1 with miR-150-5p. Methods The establishment of radioresistant cell strain RR-HepG2 was induced by fractional incremental radiotherapy. RT-qPCR was used to detect the expression of miR-150-5p in HepG2 and RR-HepG2 at different radiation doses. Cell cloning assay was used to detect the radiosensitivity of the two groups cell at the same radiation dose. Flow cytometry and Western blot were used to detect the effect of over-expression of miR-150-5p on apoptosis of HepG2 cells. Double luciferase assay was used to detect the association between miR-150-5p and SIRT1.Cell cloning and Western blot were used to detect the changes of radiosensitivity and apoptotic protein after over-expression of SIRT1. Results The expression of miR-150-5p in RR-HepG2 group decreased significantly with the same radiation dose(P＜0.05). The survival fraction and sensitivity of cells in agomiR-150-5p group decreased significantly(P＜0.05), the expression of Bax and caspase-9 increased significantly, the expression of Bcl-2 decreased significantly, and the apoptotic rate was significantly increased. The wild type (WT)agomiR-150-5p luciferase activity decreased significantly and SIRT1 protein level decreased significantly(P＜0.05); wild type (WT)anta-agomiR-150-5p luciferase activity increased significantly and SIRT1 protein level increased significantly (P＜0.05); WT-anta-agomiR-150-5p luciferase activity increased significantly and SIRT1 protein level increased significantly (P＜0.05). The survival fraction of cells in the agomiR-150-5p group decreased significantly, the expression of Bax and caspase-9 increased significantly, and the expression of Bcl-2 decreased significantly(P＜0.05). The survival fraction of cells in the agomiR-150-5p+SIRT1 group increased significantly, the expression levels of Bax and caspase-9 decreased significantly, and the expression of Bcl-2 increased significantly(P＜0.05). Conclusions The down-regulation of SIRT1 expression by miR-150-5p improves the radiosensitivity of hepatocellular carcinoma cells and provides a potential target for clinical radiotherapy of hepatocellular carcinoma.

Key words: miR-150-5p, SIRT1, hepatocellular carcinoma, radiosensitivity

中图分类号:

R735.7

吴大平, 徐璐, 吴焕良, 郑文宏, 郑小妹, 谢文蕊. miR-150-5p 靶向 SIRT1 提高肝癌细胞系HepG2放疗敏感性[J]. 基础医学与临床, 2020, 40(3): 321-327.

WU Da-ping, XU Lu, WU Huan-liang, ZHENG Wen-hong, ZHENG Xiao-mei, XIE Wen-rui. miR-150-5p enhances radiotherapy sensitivity of hepatocellular carcinoma cells line HepG2 through targeting SIRT1[J]. Basic & Clinical Medicine, 2020, 40(3): 321-327.

参考文献

[1]Dionisi F, Guarneri A, Dell'Acqua V, et al. Radiotherapy in the multidisciplinary treatment of liver cancer: a survey on behalf of the Italian Association of Radiation Oncology[J]. La Radiologia Medica, 2016, 121:735-743.
[2]孙伟. miR-150在肝癌增殖和转移中的作用及相关机制的研究[D]. 西安:第四军医大学, 2016,45-100.
[3]Wu SJ, Chen J, Wu BY, et al. MicroRNA-150 enhances radiosensitivity by inhibiting the AKT pathway in NK/T cell lymphoma[J]. J Exp Clin Cancer Res, 2018, 37:18-27.
[4]Wu Q, Wang Y, Qian M, et al. Sirt1 suppresses Wnt/βCatenin signaling in liver cancer cells by targeting βCatenin in a PKAα-dependent manner[J]. Cell Signal, 2017, 37:62-73.
[5]Cheng J, Liu C, Liu L, et al. MEK1 signaling promotes self-renewal and tumorigenicity of liver cancer stem cells via maintaining SIRT1 protein stabilization:[J]. Oncotarget, 2016, 7:20597-20611.
[6]王伟, 柯善保, 刘明博,等. CCR4对肝癌细胞索拉非尼放射敏感性及裸鼠成瘤的影响[J]. 中华放射肿瘤学杂志, 2019, 28:136-139.
[7]Liu C, Wang C, Wang J, et al. miR-1297 promotes cell proliferation by inhibiting RB1 in liver cancer[J]. Oncol Lett, 2016, 12:5177-5182.
[8]Sugita BM, Zabala Y, Fonseca A, et al. Abstract 3431: The oncogenic role of miR-150-5p in triple-negative breast cancer[J]. Cancer Res, 2017, 77:3431-3431.
[9]吴少杰, 黄宇贤, 陈俊, 等. MicroRNA-150对NK/T细胞淋巴瘤组织和NK-92细胞辐射敏感性的影响[J]. 中国肿瘤生物治疗杂志, 2017, 24:838-844.
[10]房佳惠, 杨巍. Bcl-2家族蛋白对心肌细胞凋亡作用的研究进展[J]. 现代医学, 2017,45:104-108.
[11]蒋参, 熊慧生, 巫桁锞,等. 金丝桃苷介导P53/Caspase通路对人肝癌HepG2细胞增殖和凋亡的调节作用[J]. 中国免疫学杂志, 2018, 34:78-82.
[12]党树伟,李国东,刘明. 沉默信息调节因子1在肝细胞癌中的研究进展[J]. 中华肝胆外科杂志, 2018, 24:781-785.
[13]康议心, 高社干, 郭艳珍,等. SIRT1基因沉默对DLBCL细胞放射敏感性影响[J]. 中华放射肿瘤学杂志, 2017, 26:687-690.
[14]Jiang G, Wen L, Zheng H, et al. miR-204-5p targeting SIRT1 regulates hepatocellular carcinoma progression[J]. Cell Biochem Funct, 2016, 34:505-510.
[15]Zhang X, Li, YH, Wang D, et al. miR-22 suppresses tumorigenesis and improves radiosensitivity of breast cancer cells by targeting Sirt1[J]. Biol Res, 2017, 50:27-36.