孙思洁,张海兵,耿晓如,章杭,胡阳阳,周东亚
SUN Sijie,ZHANG Haibing,GENG Xiaoru,ZHANG Hang,HU Yangyang,ZHOU Dongya

• 1:南京中医药大学沭阳附属医院肿瘤科

摘要(Abstract)：

目的探讨miR-21对食管癌(EC)细胞放疗敏感性的作用及其机制。方法将处于指数生长期的人食管癌KYSE150细胞置于培养瓶中培养,使用X放射线照射KYSE150细胞,得到放疗抵抗株KYSE150R;通过基因编辑技术合成hsa-miR-21 mimic和miR-21 inhibitor,使用Lipo3000进行细胞转染,分为KYSE-150细胞对照组(miR-21 inhibitor NC转染KYSE-150细胞)、KYSE-150细胞实验组(miR-21 inhibitor转染KYSE-150细胞)、KYSE-150R细胞对照组(miR-21 mimic NC转染KYSE-150R细胞)及KYSE-150R细胞实验组(miR-21 mimic转染KYSE-150R细胞);克隆形成实验检测放射敏感性,计算细胞存活分数,根据单靶多击模型模拟存活曲线;增殖实验,用酶标仪在450 nm处测定吸光度;Western blot分析p-ATM (Ser1981)、p-ATR (Ser428)、p-BRCA1(Ser1524)、p-Chk1(Ser345)、p-Chk2(Thr68)、p-H2AX(Ser139)、p-p53 (Ser15)、p53的表达。结果克隆形成实验显示,在亲本株KYSE150细胞中,inhibitor-KYSE150组细胞存活分数低于阴性对照组inhibitor NC组(P<0.05);在放疗抵抗KYSE150R细胞中,mimic-KYSE150R在细胞存活分数高于阴性对照组mimic NC-R组(P<0.05);细胞增殖实验显示,inhibitor-KYSE150组细胞增殖曲线高于阴性对照组inhibitor NC组,mimic-KYSE150R在细胞增殖曲线低于阴性对照组mimic NC-R组(P<0.05);蛋白质印迹分析显示,inhibitor-KYSE150组细胞中p-ATM、p-ATR、p-H2AX、p-BRCA1、p-CHK1、p-CHK2及p-p53表达水平低于inhibitor NC组细胞(P<0.05);mimic-KYSE150R组细胞中p-ATM、p-ATR、p-H2AX、p-BRCA1、p-CHK1、p-CHK2以及p-p53表达水平高于mimic NC-R组细胞(P<0.05)。结论 miR-21过表达可降低EC对放疗敏感性,其机制可能与肿瘤细胞中DNA损伤有关。
Objective To investigate the effect and mechanism of miR-21 on the radiosensitivity of esophageal cancer( EC) cells and its mechanism.Methods Human EC KYSE150 cells in the exponential growth stage were cultured in culture bottles to simulate the radiotherapy process of EC patients. X-ray radiation was used to irradiate KYSE150 cells to obtain the radiotherapy resistant KYSE150 R. hsa-miR-21 mimic and miR-21 inhibitor were synthesized by gene editing technology,transfected cells with Lipo3000, and divided into 4 groups: KYSE-150 cell control group(KYSE-150 cells transfected with miR-21 inhibitor NC), KYSE-150 cell experimental group( KYSE-150 cells transfected with miR-21 inhibitor), KYSE-150R cell control group(KYSE-150R cells transfected with miR-21 mimic NC) and KYSE-150R cells experiment group(KYSE-150R cells transfected with miR-21 mimic). The radiosensitivity was detected by cloning formation experiment, the cell survival score was calculated, and the survival curve was simulated according to the single-target multi-strike model.In proliferation experiment, absorbance was measured at 450 nm by microplate analyzer. Expression of p-ATM(Ser1981), p-ATR(Ser428), p-BRCA1(Ser1524), p-Chk1(Ser345), p-Chk2(Thr68),p-H2AX( Ser139), p-p53( Ser15), and p53 were analyzed by Western blotting.Results In parental strains KYSE150 cells of clone formation experiment, the survival score of cells in the inhibitor-KYSE150 group was lower than that in the inhibitor NC group(P< 0. 05); the survival score of mimic-KYSE150R in radiotherapy resistant KYSE150R cells was lower than that of mimic NC-R group(P< 0. 05). Cell proliferation assay showed that the cell proliferation curve of inhibitorKYSE150 group was higher than that of inhibitor-NC group, and that of mimic-KYSE150R group was lower than that of mimic NC-R group(P< 0. 05). Western blot analysis showed that the expression levels of p-ATM, p-ATR, p-H2AX, p-BRCA1, p-CHK1, p-CHK2, and p-p53 in inhibitor KYSE150 group were lower than those in inhibitor NC group(P< 0. 05). The expression levels of p-ATM,p-ATR, p-H2AX, p-BRCA1, p-CHK1, p-CHK2, and p-p53 in mimic-KYSE150R group were higher than those of mimic NC-R group(P< 0. 05).Conclusion Overexpression of miR-21 can reduce the radiosensitivity of EC, and its mechanism may be related to DNA damage in carcinoma cells.

关键词(KeyWords)： 食管肿瘤;放射治疗;敏感性;DNA损伤;调节机制
esophageal cancer(EC);radiotherapy;sensitivity;DNA damage;regulatory mechanism

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金（11672332）

作者(Author): 孙思洁,张海兵,耿晓如,章杭,胡阳阳,周东亚
SUN Sijie,ZHANG Haibing,GENG Xiaoru,ZHANG Hang,HU Yangyang,ZHOU Dongya

DOI: 10.19367/j.cnki.2096-8388.2022.01.004

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