沉默CHL1基因表达促进放射抗拒性食管癌KYSE-R细胞的放射敏感性  

CHL1 gene silencing promotes the radiosensitivity of radioresistant esophageal cancer KYSE-R cells

作　　者：孙蕊格 柯青 邓鑫州 柴晶晶 丁燕鹏 徐姝珺 骆志国[1] SUN Ruige;KE Qing;DENG Xinzhou;CHAI Jingjing;DING Yanpeng;XU Shujun;LUO Zhiguo(Cancer Center,Shiyan Taihe Hospital Affiliated to Hubei University of Medical,Shiyan 442000,Hubei Province,China;Graduate School,Jinzhou Medical University,Jinzhou 121001,Liaoning Province,China;Department of Chemoradiotherapy,Zhongnan Hospital of Wuhan University,Wuhan 430071,Hubei Province,China)

机构地区：[1]湖北医药学院附属十堰市太和医院肿瘤防治中心,湖北十堰442000 [2]锦州医科大学研究生学院,辽宁锦州121001 [3]武汉大学中南医院放化疗科,湖北武汉430071

出　　处：《肿瘤》2020年第12期809-820,共12页Tumor

基　　金：国家自然科学基金资助项目(编号:81602391,81802997,81502666);湖北省自然科学基金资助项目(编号:2019CFA034,2018CFB405,2017CFB456,2017CFB167);湖北省教育厅基金资助项目(编号:D20172102,Q20162109,Q20202106);湖北医药学院自由探索基金资助项目(编号:FDFR201802);湖北医药学院研究生科技创新项目(编号:YC2019025);湖北省科技局基金资助项目(编号:19Y40)。

摘　　要：目的:探讨沉默细胞黏附分子CHL1(close homolog of L1)基因表达对食管癌KYSE-R细胞放射敏感性的影响。方法:选取食管癌KYSE细胞,采用连续照射的方法构建放射抗拒性的KYSE-R细胞;分别采用克隆形成实验和FCM法检测放射抗拒KYSE-R与亲本细胞KYSE细胞间的放射敏感性差异;蛋白质印迹法检测KYSE和KYSE-R细胞中CHL1蛋白的表达水平。采用脂质体法将3条针对CHL1基因不同靶点的siRNA-CHL1(siCHL1-1、siCHL1-2和siCHL1-3)分别转入食管癌放射抗拒细胞KYSE-R,利用实时荧光定量PCR和蛋白质印迹法确定干扰效果最佳的siRNA。将siCHL1-3转染至KYSE-R细胞中,经X-照射后再分别用克隆形成实验、FCM法和CCK-8法检测沉默CHL1基因表达后对KYSE-R细胞放射敏感性的影响,并进一步采用蛋白质印迹法检测KYSE-R细胞中凋亡相关蛋白Bax、Bcl-2和Bcl-XL的表达情况。结果:构建获得具有更强的放射抗拒性的KYSE-R细胞。KYSE-R细胞中CHL1蛋白的表达水平明显高于亲本KYSE细胞(P<0.05),siCHL1-3的干扰效果最佳(P<0.05)。沉默CHL1基因表达后,经X-射线照射后CHL1干扰组KYSE-R细胞的存活曲线明显降低(P=0.0001),细胞凋亡的凋亡率明显提高,被阻滞在G2/M期细胞所占的百分比明显增加(P<0.05),细胞的增殖能力被明显抑制(P值均<0.05)。蛋白质印迹法结果显示,经X-射线照射后,CHL1干扰组KYSE-R细胞中Bax蛋白表达水平明显增高,而Bcl-2和Bcl-XL蛋白的表达水平明显降低(P值均<0.05)。结论:干扰CHL1基因表达可增强食管癌放射抗拒KYSE-R细胞对放射的敏感性。Objective:To investigate the effect of silencing cell adhesion molecule CHL1(close homolog of L1)gene on the radiosensitivity of esophageal cancer KYSE-R cells.Methods:Select esophageal cancer KYSE cells,and use continuous irradiation to construct radioresistant KYSE-R cells.The difference in radiosensitivity between the radioresistant KYSE-R and the parental KYSE cells was detected by colony formation assay and FCM,respectively.The expression level of CHL1 protein in KYSE and KYSE-R cells was detected by Western blotting.Three siRNA-CHL1(siCHL1-1,siCHL1-2 and siCHL1-3)targeting different targets of CHL1 gene were transfected into KYSE-R by liposome,and the best CHL1 interference sequence was determined by real-time fluorescent quantitative PCR and Western blotting.siCHL1-3 was transfected into KYSE-R cells,and the radiosensitivity of KYSE-R cells was determined by clone formation assay.FCM was used to detect apoptosis and cell cycle distribution of KYSE-R cells after irradiation.The proliferation of KYSE-R cells was detected by CCK-8 method.The expression level of Bax,Bcl-2 And Bcl-XL proteins were detected by Western blotting.Results:The radioresistant KYSE-R cells were constructed.The expression level of CHL1 protein in KYSE-R cells was significantly higher than that in KYSE cells,and the interference effect of siCHL1-3 was the best.The result of clone formation experiment showed that the survival curve of cells in the CHL1 interference group decreased significantly after radiation exposure(P=0.0001).The results of FCM showed that CHL1 gene silencing could enhance radiation-induced apoptosis rate and G2/M cell cycle distribution arrest(both P<0.05).After radiation irradiation,CHL1 gene silencing could significantly inhibit cell proliferation(P<0.05).The expression level of Bax protein was significantly increased,and the expression level of Bcl-2 and Bcl-XL proteins were significantly decreased in the CHL1 interference group after radiation exposure(all P<0.05).Conclusion:CHL1 gene silencing can enhance radiose

关 键 词：食管肿瘤 细胞黏附分子 放射肿瘤学 细胞增殖 细胞凋亡 

分 类 号：R739.65[医药卫生—肿瘤]