miR-122对肝癌细胞HepG2放疗敏感性影响及其机制研究

发布时间：2018-03-23 00:13

本文选题：miR-　切入点：肝癌　出处：《中华肿瘤防治杂志》2017年16期 　论文类型：期刊论文

【摘要】：目的 miR-122是一种肝脏特异性miRNA,miR-122过表达可以降低肝脏肿瘤细胞的相关特性,并增强化疗药物的敏感性,但是在放疗方面研究较少。本研究探讨miR-122对肝癌细胞放疗敏感性的影响及其可能的机制。方法通过转染miR-122类似物构建HepG2-miR-122mimic细胞,利用Q-PCR检测miR-122的表达,通过成克隆分析观察miR-122对肝癌细胞放疗敏感性的影响,ELISA方法检测X射线照射后不同时间点细胞内乏氧因子HIF1-α及ROS的表达;蛋白质印迹法检测X射线照射后48h各细胞凋亡相关蛋白Bcl-2、Bax及Bim的表达变化。结果 Q-PCR检测显示,HepG2-miR-122mimic细胞中miR-122的表达较HepG2细胞增加了约8.2倍。成克隆分析显示,HepG2细胞转染miR-122mimic后放疗敏感性增高,与HepG2组相比,准域剂量Dq的放疗增敏比(SER)为1.52。ELISA分析显示,HepG2细胞在0hHIF-1α表达量为5.20ng/mL。X射线照射后HepG2细胞中HIF-1α升高,至48h升至最高,为6.69ng/mL。而HepG2-miR-122mimic细胞在0h的表达量为4.7ng/mL,X射线照射后HIF-1α的含量进一步下降,至48h下降至3.41ng/mL。48h时miR-122 mimic转染情况对HIF-1α的表达差异有统计学意义,F=70.819,P0.001;X射线暴露对HIF-1α的表达差异有统计学意义,F=132.436,P0.001;miR-122mimic的转染情况及X射线暴露之间存在交互作用,F=4.290,P=0.039。ROS含量在X射线照射后表现为轻度升高,在0h HepG2组和HepG2-miR-122mimic组ROS表达量分别为116.21和121IU/mL,X射线照射后ROS进一步升高,至48h升至最高,分别为130和192IU/mL。48h时miR-122mimic转染情况对ROS的表达差异有统计学意义,F=98.189,P0.001;X射线暴露对ROS的表达差异有统计学意义,F=145.373,P0.001;miR-122mimic的转染情况及X射线暴露之间存在交互作用,F=6.548,P=0.012。蛋白质印迹法检测结果显示,HepG2-miR-122mimic细胞中BCL-2的表达量为HepG2的56.32%,X射线照射后48hHepG2细胞中BCL-2的表达量降至原来的55.01%,而HepG2-miR-122mimic细胞则降至23%。HepG2-miR-122mimic细胞中Bax和Bim的表达量分别为HepG2细胞的170.21%和140.35%,X射线照射后,HepG2细胞中Bax和Bim的表达量分别升至HepG2的220.12%和178.34%,而HepG2-miR-122mimic细胞中,Bax和Bim的表达量则分别升至HepG2细胞的240%和260%。miR-122mimic转染情况对BCL-2、Bax和Bim的表达差异均有统计学意义,均P0.01。X射线暴露对BCL-2、Bax及Bim的表达差异均有统计学意义,均P0.01。miR-122mimic转染情况及X射线暴露对Bcl-2、Bim及Bax的表达均不存在交互作用。结论 miR-122可以上调肝癌细胞HepG2的放射敏感性,其机制可能与调节HIF-1α和ROS及凋亡相关蛋白有关。
[Abstract]:Objective miR-122 is a liver-specific miRNA-miR-122 overexpression that can reduce the related characteristics of liver tumor cells and enhance the sensitivity of chemotherapeutic drugs. In this study, we studied the effect of miR-122 on the radiosensitivity of hepatoma cells and its possible mechanism. Methods HepG2-miR-122mimic cells were constructed by transfection of miR-122 analogues, and the expression of miR-122 was detected by Q-PCR. The effects of miR-122 on the radiosensitivity of hepatoma cells were observed by clone analysis. The expression of hypoxia factor HIF1- 伪 and ROS were detected by Elisa at different time points after X-ray irradiation. The expression of apoptosis-related protein Bcl-2 Bax and Bim in HepG2-miR-122 mimic cells was detected by Western blotting at 48 h after irradiation. Results the expression of miR-122 in HepG2-miR-122 mimic cells was increased by about 8.2-fold compared with that in HepG2 cells. Clone analysis showed that miR-122 expression in HepG2 cells was about 8.2-fold higher than that in HepG2 cells. After transfection of miR-122mimic, the sensitivity of radiotherapy was increased. Compared with HepG2 group, 1.52.ELISA analysis showed that the expression of 0hHIF-1 伪 in HepG2 cells increased after 5.20ng/mL.X irradiation, and reached the highest level at 48 h. The expression of HIF-1 伪 in HepG2-miR-122mimic cells was 4.7ng / mLX irradiation at 0 h, and the content of HIF-1 伪 decreased further after X-ray irradiation. There was significant difference in the expression of HIF-1 伪 between miR-122 mimic transfection and 3.41ng/mL.48h at 48h. There was a significant difference in the expression of HIF-1 伪 after X-rays exposure to miR-122 mimic. There was a significant difference in the expression of HIF-1 伪. There was a significant difference in the transfection of HIF-1 伪 and the interaction between F4.290P0.039.Ros content in X-ray exposure. A slight increase was observed after irradiation. The expression of ROS in 0 h HepG2 group and HepG2-miR-122mimic group were 116.21 and 121 IU / mL X ray irradiation respectively, and ROS increased further, and reached the highest level at 48 h. There were significant differences in the expression of ROS between miR-122mimic transfection and 192IU/mL.48h respectively. There was a significant difference in the expression of ROS by X-Ray exposure to FN 98.189 and P0.001. There was a significant difference in the transfection of FN145.373 and P0.001miR-122 mimic and the interaction between ROS and X-ray exposure. Western blotting was used to detect the expression of ROS. The results showed that the expression of BCL-2 in HepG2-miR-122 mimic cells was 56.32% of that of HepG2. After X-ray irradiation, the expression of BCL-2 in 48hHepG2 cells decreased to 55.01%, while that of Bax and Bim in HepG2-miR-122mimic cells decreased to 170.21% and 140.35% of HepG2 cells, respectively. The expression of Bax and Bim in HepG2 cells increased to 220.12% and 178.34% of HepG2, respectively, while the expression of HepG2-miR-122mimic cells increased to 240% of HepG2 cells and 240% of 260%.miR-122mimic transfection, respectively. There were significant differences in the expression of BCL-2nBax and Bim in HepG2 cells. There was significant difference in the expression of BCL-2nBax and Bim by P0.01. There was no interaction between P0.01.miR-122mimic transfection and X-ray exposure to Bcl-2mBim and Bax. Conclusion miR-122 can up-regulate the radiosensitivity of hepatoma cells to HepG2. The mechanism may be related to the regulation of HIF-1 伪 and ROS and apoptosis-related proteins.
【作者单位】：江苏大学附属人民医院肿瘤放疗科;
【基金】：[基金项目]镇江市科技支撑计划-社会发展(SH2014040)
【分类号】：R730.55;R735.7

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