摘要
目的研究环状带有FG重复序列1(CircAGFG1)通过miR-375/泛素特异性蛋白酶22(USP22)轴对食管鳞癌(ESCC)细胞放射敏感性的影响。方法采用实时荧光定量PCR和免疫印迹法检测人ESCC细胞株KYSE150及人ESCC放射性抵抗细胞KYSE150-R中CircAGFG1、miR-375、USP22表达。将体外培养的KYSE150-R细胞随机分为对照组、辐照组、辐照+阴性对照组、辐照+CircAGFG1敲低组、辐照+CircAGFG1敲低+miR-375 inhibitor组,分组转染后,4Gy 6MV-X线辐照后24 h采用实时荧光定量PCR和免疫印迹法检测各组KYSE150-R细胞中CircAGFG1、miR-375、USP22表达;采用CCK-8法和平板集落形成实验检测各组KYSE150-R细胞增殖;采用流式细胞术检测各组KYSE150-R细胞凋亡;采用免疫荧光染色检测各组KYSE150-R细胞凋亡相关蛋白Bax与Bcl-2比值(Bax/Bcl-2)。将各组细胞接种在裸鼠右后肢腋下来构建ESCC移植瘤模型,饲养3周后检测各组裸鼠肿瘤体积及重量。采用双荧光素酶报告基因实验检测KYSE150-R中CircAGFG1对miR-375的靶向调节与miR-375对USP22的靶向调节。结果与KYSE150细胞相比,KYSE150-R细胞中CircAGFG1 mRNA表达、USP22 mRNA与蛋白表达升高(P<0.05),miR-375 mRNA表达降低(P<0.05)。与对照组相比,辐照+CircAGFG1敲低组CircAGFG1 mRNA表达、USP22 mRNA及蛋白表达、细胞增殖率、集落生成率、裸鼠肿瘤体积与肿瘤重量降低(P<0.05),细胞凋亡率、Bax/Bcl-2、miR-375 mRNA表达升高(P<0.05);辐照组、辐照+阴性对照组细胞各指标比较无明显差异(P>0.05)。与辐照组相比,辐照+CircAGFG1敲低组CircAGFG1 mRNA、USP22 mRNA及蛋白表达、细胞增殖率、集落生成率、裸鼠肿瘤体积与肿瘤重量降低(P<0.05),细胞凋亡率、Bax/Bcl-2、miR-375 mRNA表达升高(P<0.05)。与辐照+CircAGFG1敲低组相比,辐照+CircAGFG1敲低+miR-375 inhibitor组USP22 mRNA及蛋白表达、细胞增殖率、集落生成率、裸鼠肿瘤体积与肿瘤重量升高(P<0.05),细胞凋亡率、Bax/Bcl-2、miR-375 mRNA表达降低(P<0.05);辐照+阴性对照组与对照组细胞各指标无明显变化(P>0.05)。CircAGFG1可靶向下调KYSE150-R细胞miR-375表达,而miR-375可靶向下调KYSE150-R细胞USP22表达。结论敲低CircAGFG1可通过上调miR-375来减弱USP22表达,从而增强ESCC细胞的放射敏感性,提升放射性对ESCC细胞的杀伤力,并促使其凋亡。
Objective To study the effect of circular ArfGAP with FG repeats 1(CircAGFG1)on radiosensitivity of esophageal squamous cell carcinoma(ESCC)cells via miR-375/ubiquitin specific protease 22(USP22)axis.Methods The expression of CircAGFG1,miR-375 and USP22 in human ESCC cell line KYSE150 and human ESCC radiation resistant cell KYSE150-R was detected by real-time fluorescent quantitative PCR and Western blot.The KYSE150-R cells cultured in vitro were randomly divided into control group,radiation group,radiation+negative control group,radiation+CircAGFG1 knockdown group,and radiation+CircAGFG1 knockdown+miR-375 inhibitor group,after transfection,the expression of CircAGFG1,miR-375 and USP22 in KYSE150-R cells of each group was detected by real-time fluorescent quantitative PCR and Western blot;the proliferation of KYSE150-R cells in each group was detected by CCK-8 method and plate colony formation test;the apoptosis of KYSE150-R cells was detected by flow cytometry;the ratio of apoptosis related protein Bax to Bcl-2(Bax/Bcl-2)of KYSE150-R cells in each group was detected by immunofluorescence staining.The cells of each group were inoculated under the armpit of the right hind limb of nude mice to construct an ESCC transplanted tumor model,the tumor volume and weight of nude mice in each group were measured after 3 weeks of feeding.Double luciferase reporter gene experiment was used to detect the targeting regulation of CircAGFG1 on miR-375 and the targeting regulation of miR-375 on USP22 in KYSE150-R.Results Compared with KYSE150 cells,the expression of CircAGFG1,USP22 mRNA and protein in KYSE150-R cells increased(P<0.05),the expression of miR-375 decreased(P<0.05).Compared with the control group,the cell proliferation rate,colony generation rate,CircAGFG1 expression,USP22 mRNA and protein expression,tumor volume and tumor weight of nude mice in the radiation+CircAGFG1 knockdown group reduced(P<0.05),the apoptosis rate,Bax/Bcl-2,miR-375 expression increased(P<0.05);there was no obvious change in the cell indexes of the radiation group and the radiation+negative control group(P>0.05).Compared with the radiation group,the cell proliferation rate,colony generation rate,CircAGFG1 expression,USP22 mRNA and protein expression,tumor volume and tumor weight of nude mice in the radiation+CircAGFG1 knockdown group reduced(P<0.05),the apoptosis rate,Bax/Bcl-2,miR-375 expression increased(P<0.05).Compared with the radiation+CircAGFG1 knockdown group,the cell proliferation rate,USP22 mRNA and protein expression,tumor volume and tumor weight of nude mice in the radiation+CircAGFG1 knockdown group increased(P<0.05),the apoptosis rate,Bax/Bcl-2,miR-375 expression reduced(P<0.05);in the radiation+negative control group,there was no significant change in the cell indexes(P>0.05).CircAGFG1 was able to target and down regulate the expression of miR-375 in KYSE150-R cells,while miR-375 was able to target and down regulate the expression of USP22 in KYSE150-R cells.Conclusion Knocking down CircAGFG1 can reduce the expression of USP22 by up regulating miR-375,thereby enhancing the radiosensitivity of ESCC cells,enhancing the lethality of radiation to ESCC cells,and promoting their apoptosis.
作者
易琼
邰国梅
钱红燕
王锋
谭程
倪峰
葛琴
YI Qiong;TAI Guomei;QIAN Hongyan;WANG Feng;TAN Cheng;NI Feng;GE Qin(Department of Radiotherapy,Nantong University Cancer Hospital,Nantong Cancer Hospital,Nantong 226361,Jiangsu,China;Central Laboratory of Cancer Research Institute,The Affiliated Cancer Hospital of Nantong University,Nantong 226361,Jiangsu,China)
出处
《西部医学》
2024年第8期1123-1130,共8页
Medical Journal of West China
基金
江苏省南通市科技局指令课题(JC22022093)。
