circLPAR3靶向miR-1238对食管癌细胞放射敏感性的影响

doi:10.3760/cma.j.cn113030-20200919-00473

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摘要目的探讨circLPAR3对食管癌细胞放射敏感性的影响及作用机制。方法收集37例食管癌患者的癌组织和癌旁组织,体外培养食管癌细胞系Eca-109、EC9706和KYSE30及食管上皮细胞HET-1A,RT-qPCR法检测组织和细胞中circLPAR3和miR-1238的表达水平。以Eca-109细胞为研究对象,转染circLPAR3 siRNA、miR-1238模拟物或共转染circLPAR3siRNA与miR-1238抑制剂。用细胞克隆实验检测沉默circLPAR3、过表达miR-1238或同时沉默circLPAR3和miR-1238对Eca-109细胞放射敏感性的影响。4Gy照射沉默circLPAR3、过表达miR-1238或同时沉默circLPAR3和miR-1238的Eca-109细胞,CCK-8法(A值)、流式细胞术、蛋白印迹法分别检测沉默circLPAR3、过表达miR-1238或同时沉默circLPAR3和miR-1238联合4Gy照射对Eca-109细胞增殖、凋亡及相关蛋白CyclinD1、p21、Bcl-2和Bax表达的影响。双荧光素酶报告基因实验和RNA Pull down实验验证circLPAR3和miR-1238调控关系。结果与癌旁组织比较,食管癌组织中circLPAR3表达升高(P<0.05),miR-1238表达降低(P<0.05)。与HET-1A细胞比较,Eca-109、EC9706和KYSE30细胞中circLPAR3表达升高(均P<0.05),miR-1238表达降低(均P<0.05)。沉默circLPAR3、过表达miR-1238降低了Eca-109细胞存活分数(均P<0.05),放射增敏比分别为1.21、1.75。沉默circLPAR3、过表达miR-1238降低了Eca-109细胞A值、CyclinD1和Bcl-2蛋白表达(均P<0.05),而提高了Eca-109细胞凋亡率、p21和Bax蛋白表达(均P<0.05)。沉默circLPAR3、过表达miR-1238联合4Gy照射后Eca-109细胞A值、CyclinD1和Bcl-2蛋白表达降低(均P<0.05),Eca-109细胞凋亡率、p21和Bax蛋白表达升高(均P<0.05)。circLPAR3在Eca-109细胞中靶向结合并负调控miR-1238的表达。同时沉默miR-1238、circLPAR3表达后Eca-109细胞存活分数较只沉默circLPAR3时升高,放射增敏比为0.59。沉默miR-1238逆转了沉默circLPAR3联合4Gy照射对Eca-109细胞增殖和凋亡的影响。结论 circLPAR3在食管癌组织和细胞系中呈高表达,沉默其表达可靶向上调miR-1238抑制食管癌Eca-109细胞增殖,促进其凋亡,并增强细胞放射敏感性。

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	袁小笋
	张蕾
	饶石磊
	张凯
	马慧利
	李长生
	张敬伟
	任中海

关键词 ： circLPAR3基因, miR-1238基因, 细胞增殖, 细胞凋亡, 放射敏感性, 食管癌组织, 食管癌细胞系

Abstract：Objective To evaluate the effect of circLPAR3 on the radiosensitivity of esophageal cancer cells and investigate its mechanism. Methods The cancer tissues and and adjacent tissues of 37 patients with esophageal cancer were collected, and esophageal cancer cell lines Eca-109, EC9706 and KYSE30 and esophageal epithelial cells HET-1A were cultured in vitro. The expression levels of circLPAR3 and miR-1238 in the tissues and cells were measured by RT-qPCR. Eca-109 cells were transfected with circLPAR3siRNA and miR-1238 mimics or co-transfected with circLPAR3siRNA and miR-1238 inhibitor. Cell cloning experiment was conducted to evaluate the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 on the radiosensitivity of Eca-109 cells. After Eca-109 cells that silenced circLPAR3, overexpressed miR-1238 or silenced both circLPAR3 and miR-1238 were exposed to 4Gy irradiation, CCK-8 assay (A value), flow cytometry and Western blot were employed to assess the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 combined with 4Gy irradiation on the proliferation and apoptosis of Eca-109 cells and the expression levels of CyclinD1, p21, Bcl-2 and Bax proteins. Dual luciferase reporter gene experiment and RNA pull down experiment were performed to verify the regulatory relationship between circLPAR3 and miR-1238. Results Compared with adjacent tissues, the expression level of circLPAR3 was up-regulated in the esophageal cancer tissues (P<0.05), while that of miR-1238 was down-regulated (P<0.05). Compared with HET-1A cells, the expression levels of circLPAR3 were up-regulated in the esophageal cancer cell lines Eca-109, EC9706 and KYSE30(all P<0.05), whereas those of miR-1238 were down-regulated (all P<0.05). Silencing circLPAR3 or overexpressing miR-1238 reduced the survival fraction of Eca-109 cells (all P<0.05), and the sensitization ratio was 1.21 and 1.75, respectively. Silencing circLPAR3 or overexpressing miR-1238decreased the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins (all P<0.05), while increased the apoptosis rate of Eca-109 cells and the expression levels of p21 and Bax proteins (all P<0.05). After silencing circLPAR3 or overexpressing miR-1238 combined with 4Gy irradiation, the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins were decreased (all P<0.05), while Eca-109 cell apoptosis rate and the expression levels of p21 and Bax proteins were increased (all P<0.05). circLPAR3 targeted and negatively regulated the expression level of miR-1238 in Eca-109 cells. After silencing miR-1238 and circLPAR3simultaneously, the survival fraction of Eca-109 cells was higher than that when only silencing circLPAR3, and the sensitization ratio was 0.59. Silencing miR-1238 reversed the effects of silencing circLPAR3 combined with 4Gy irradiation on the proliferation and apoptosis of Eca-109 cells. Conclusion circLPAR3 is highly expressed in esophageal cancer tissues and cell lines, and silencing the expression of circLPAR3 can inhibit the proliferation of esophageal cancer Eca-109 cells, promote their apoptosis, and enhance cell radiosensitivity by up-regulating miR-1238.

Key words： circLPAR3 gene miR-1238 gene Cell proliferation Cell apoptosis Radiosensitivity Esophageal cancer tissue Esophageal cancer cell line

收稿日期: 2020-09-19

引用本文:

袁小笋,张蕾,饶石磊等. circLPAR3靶向miR-1238对食管癌细胞放射敏感性的影响[J]. 中华放射肿瘤学杂志, 2022, 31(1): 71-78.

Yuan Xiaosun,Zhang Lei,Rao Shilei et al. Effect of circLPAR3 on radiosensitivity of esophageal cancer cells by targeting miR-1238[J]. Chinese Journal of Radiation Oncology, 2022, 31(1): 71-78.

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http://journal12.magtechjournal.com/Jweb_fszlx/CN/10.3760/cma.j.cn113030-20200919-00473 或 http://journal12.magtechjournal.com/Jweb_fszlx/CN/Y2022/V31/I1/71

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