ESCC组织SNHG17表达及转染si-SNHG17质粒的人食管鳞癌细胞株增殖、凋亡、放射敏感性观察

Expression of SNHG17 in ESCC tissues and observation of proliferation,apoptosis,and radiosensitivity of human ESCC cell line transfected with SNHG17 plasmid

目的观察食管鳞癌(ESCC)组织小核仁RNA宿主基因17(SNHG17)表达情况及转染si-SNHG17质粒的人ESCC细胞株ECA109增殖、凋亡、放射敏感性。方法取ESCC肿瘤组织与癌旁组织标本各89例份;另取ECA109细胞,并随机分为对照组(Control组,空白培养基处理)、si NC组(转染si NC质粒)、si-SNHG17组(转染si-SNHG17质粒)、si-SNHG17+inhibitor NC组(si-SNHG17与inhibitor NC共转染)、si-SNHG17+miR-375 inhibitor组(si-SNHG17与miR-375 inhibitor共转染);采用RT-qPCR法检测ESCC组织、癌旁组织和各组细胞SNHG17、miR-375、USP14 mRNA,CCK-8法检测各组细胞增殖能力(OD值),流式细胞术及Western blot法检测各组细胞凋亡能力[凋亡率及凋亡相关蛋白(Bax、Bcl-2、Caspase3、USP14蛋白)],克隆形成实验检测各组细胞放射敏感性。双荧光素酶报告基因实验分别验证SNHG17、miR-375及miR-375、USP14的靶向性关系。结果与癌旁组织比较,肿瘤组织中SN⁃HG17、USP14 mRNA表达升高,miR-375表达降低(P均<0.05)。与Control组、si-NC组比较,si-SNHG17组SNHG17表达、OD值、Bcl-2及USP14表达降低,miR-375表达、细胞凋亡率、放射敏感性、Bax及Caspase3蛋白表达升高(P均<0.05)。与si-SNHG17组、si-SNHG17+inhibitor NC组比较,si-SNHG17+miR-375 inhibitor组OD值、Bcl-2及USP14表达升高(P均<0.05),miR-375表达、细胞凋亡率、放射敏感性、Bax及Caspase3蛋白表达降低(P均<0.05)。SN⁃HG17靶向负调控miR-375表达,miR-375靶向负调控USP14表达。结论ESCC组织中SNHG17表达较癌旁组织升高;转染si-SNHG17质粒可抑制ECA109细胞增殖,促进细胞凋亡,增强细胞的放射敏感性,机制可能是上调miR-375来抑制USP14蛋白的表达。

Objective To observe the expression of small nucleolar RNA host gene 17(SNHG17)in esophageal squamous cell carcinoma(ESCC)tissues and the proliferation,apoptosis and radiosensitivity of human ESCC cell line ECA109 transfected with si-SNHG17 plasmid.Methods Eighty-nine cases of ESCC tissues and 89 cases of adjacent tis⁃sues were selected;ECA109 cells were randomly divided into the Control group(Control group,blank medium treat⁃ment),si NC group(transfected with si NC plasmid),si-SNHG17 group(transfected with si-SNHG17 plasmid),si-SN⁃HG17+inhibitor NC group(co-transfected with si-SNHG17 and inhibitor NC),and si-SNHG17+miR-375 inhibitor group(co-transfected with si-SNHG17 and miR-375 inhibitor),respectively.The expression levels of LncRNA SNHG17,miR-375 and USP14 mRNA in the ESCC tissues,adjacent tissues,and cells were detected by RT-qPCR;CCK-8 method was used to detect the proliferation ability of cells in each group;apoptosis was detected by flow cytometry;Western blotting was used to detect the expression of apoptosis-related proteins Bax,Bcl-2,Caspase3 and USP14.After the cells in each group were irradiated with X ray,the radiosensitivity of the cells in each group was detected by clone formation test.Dou⁃ble luciferase reporter gene experiment was applied to verify the relationship between SNHG17 and miR-375,and be⁃tween miR-375 and USP14,respectively.Results Compared with the adjacent tissues,the expression levels of SN⁃HG17 and USP14 mRNAs increased,while miR-375 expression decreased in tumor tissues(all P<0.05).Compared with the Control group and si NC group,the expression of SNHG17,OD450nm,the expression levels of Bcl-2 and USP14 in ECA109 cells of the si SNHG17 group decreased(all P<0.05),but the expression of miR-375,apoptosis rate,radiosensi⁃tivity,and the expression levels of Bax and Caspase3 proteins increased(all P<0.05).Compared with the si-SNHG17 group and si-SNHG17+inhibitor NC group,the expression of SNHG17,OD450nm,the expression levels of Bcl-2 and USP14 in the si-SNHG17+miR-375 inhibitor group increased(all P<0.05),but the expression of miR-375,apoptosis rate,radio⁃sensitivity,the expression levels of Bax and Caspase3 proteins decreased significantly(all P<0.05).SNHG17 negatively regulated the expression of miR-375,and miR-375 negatively regulated the expression of USP14.Conclusion The ex⁃pression of SNHG17 in the ESCC tissues is higher than that in the adjacent tissues;transfection of si-SNHG17 plasmid can inhibit the proliferation of ECA109 cells,promote apoptosis,and enhance cell radiosensitivity,possibly by up-regulat⁃ing miR-375 to inhibit the expression of USP14 protein.