miR-616通过靶向TIMP-2增强肺癌细胞放射敏感性的机制研究

doi:10.11904/j.issn.1002-3070.2020.01.006

摘要/Abstract

摘要： 目的探讨miR-616通过靶向TIMP-2增强肺癌细胞放射敏感性的机制。方法肺癌HCC827细胞组常规培养;RI组肺癌细胞使用X射线发生器进行X射线照射,以4 Gy/min的固定剂量率发射;用于照射细胞的X射线的能量分级为8 Gy,照射孵育时间为24 h;miR-616 inhibitor组肺癌细胞用miR-616-shRNA靶向转染;miR-616 inhibitor+RI组肺癌细胞用miR-616-shRNA靶向转染,随后使用X射线发生器进行X射线照射。以上细胞培养72 h,设6个平行样,试验结束后,MTT法测定细胞增殖、吉姆萨染色测定集落百分比、流式细胞仪测定细胞凋亡及细胞周期水平、Transwell室法测定细胞侵袭水平、RT-PCR法及Western blot法测定细胞miR-616基因、TIMP-2基因和蛋白水平。结果与HCC827细胞组比较,RI组、miR-616 inhibitor组和miR-616 inhibitor+RI组的OD值、存活率水平、克隆形成数目、穿膜数、miR-616 mRNA表达水平降低(P＜0.05);与RI组、miR-616 inhibitor组比较,miR-616 inhibitor+RI组的OD值、存活率水平、克隆形成数目、穿膜数、miR-616 mRNA表达水平降低(P＜0.05);与HCC827细胞组比较,RI组、miR-616 inhibitor组、miR-616 inhibitor+RI组的凋亡率、G₁期、TIMP-2 mRNA、蛋白表达水平升高(P＜0.05);与RI组、miR-616 inhibitor组比较,miR-616 inhibitor+RI组的凋亡率、G₁期、TIMP-2 mRNA、蛋白表达水平升高(P＜0.05)。结论 miR-616的表达下调可以在体外增强肺癌细胞放射敏感性;进而增强辐射介导的肿瘤细胞抑制;细胞凋亡和生长停滞。其机制可能与抑制miR-616可促进TIMP-2基因和蛋白的表达有关。

关键词: miR-616, TIMP-2, 肺癌细胞, 放射敏感性

Abstract: Objective The aim of this study was to investigate the mechanism of miR-616 enhancing the radiosensitivity of lung cancer cells by targeting TIMP-2.Methods Lung cancer HCC827 cells were routinely cultured and HCC827 cells were irradiated with X-rays as the RI group,which emitted at a fixed dose rate of 4 Gy/min;the energy classification of X-rays used to irradiate the cells was 8 Gy,and the incubation time was 24 h.HCC827 cells were transfected with miR-616-shRNA as the miR-616 inhibitor group.HCC827 cells were transfected with miR-616-shRNA and then were used X-ray irradiation by X-ray generator as the miR-616 inhibitor + RI group.The above cells were cultured for 72 hours,and six replicates were set in each dose.At the end of the experiment,MTT assay was used to determine cell proliferation,Giemsa staining was used to determine the colony percentage,flow cytometry was used to determine apoptosis and distribution of cell cycle,Transwell chamber was used to determine cell invasion,RT-PCR and Western blot were used to determine the expression miR-616 and TIMP-2 genes at levels of mRNA and protein.Results Compared with the HCC827 cells group,the OD values,survival rate,number of clone formation,number of transmembrane cells,the level of miR-616 mRNA in the RI group,miR-616 inhibitor group,and miR-616 inhibitor + RI group were reduced(P＜0.05).Compared with the RI group and the miR-616 inhibitor group,the OD values,survival rate,number of clone formation,number of membrane cells,and the level of miR-616 mRNA in the miR-616 inhibitor + RI group were decreased(P＜0.05).Compared with the HCC827 cells group,apoptotic rates,the cell cycle at the G1 phase,and the expression of TIMP-2 at levels of mRNA and protein in the RI group,miR-616 inhibitor group,and miR-616 inhibitor + RI group were increased(P＜0.05).Compared with the RI group and miR-616 group,apoptotic rates,the cell cycle at the G1 phase,and the expression of TIMP-2 at levels of mRNA and protein in the miR-616 inhibitor + RI group were increased(P＜0.05).Conclusion The down-regulated expression of miR-616 can enhance the radiosensitivity of lung cancer cells in vitro,and then enhance the radiation-mediated inhibition of tumor viability,cell apoptosis induced and cell cycle arrested.The mechanism may be related to the inhibition of miR-616,which can promote the related expression of TIMP-2 gene and protein.

Key words: MicroRNA-616, TIMP-2, Lung cancer cells, Radiosensitivity

中图分类号:

R734.2

范庞双, 庞先琼. miR-616通过靶向TIMP-2增强肺癌细胞放射敏感性的机制研究[J]. 实用肿瘤学杂志, 2020, 34(1): 30-36.

FAN Pangshuang, PANG Xianqiong. The mechanism of miR-616 enhancing the radiosensitivity of lung cancer cells by targeting TIMP-2[J]. Journal of Practical Oncology, 2020, 34(1): 30-36.

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