人泛素特异性蛋白酶42在肿瘤组织中的表达及其shRNA干扰重组质粒的构建  

Expression of human ubiquitin⁃specific protease 42 in tumor tissues andconstruction of its shRNA interfering recombinant plasmid

作　　者：郭紫婵 魏媛 王秀丽[1] 赵蓉[1] 王晓霞[1] GUO Zi-chan;WEI Yuan;WANG Xiu-li;ZHAO Rong;WANG Xiao-xia(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室,山西太原030001

出　　处：《中国生物制品学杂志》2022年第11期1338-1341,共4页Chinese Journal of Biologicals

基　　金：山西省自然科学基金(201901D111214);细胞生理学教育部重点实验室(山西医科大学)开放基金(KLMEC/SXMU-201906).

摘　　要：目的评价人泛素特异性蛋白酶42(ubiquitin-specific protease 42,USP42)在不同肿瘤组织中的表达水平,并构建其shRNA干扰重组质粒。方法通过网络数据库GEPIA和Ualcan分析USP42在不同肿瘤组织中的表达水平。以人USP42 mRNA为靶序列设计合成含有shRNA序列的寡核苷酸,克隆至真核表达载体pGPU6/Neo,构建重组质粒USP42-shRNA。采用Neofect®DNA转染试剂将重组质粒转染至人结肠癌细胞HCT116(干扰组),同时设对照组(转染NC-shRNA),Western blot法检测USP42表达的干扰效果,Transwell小室试验检测USP42对结肠癌细胞迁移和侵袭功能的影响。结果经GEPIA和Ualcan数据库分析发现,USP42在胆管癌、胃癌、胸腺癌和结肠癌等肿瘤组织中呈高表达。测序鉴定证明重组质粒USP42-shRNA构建正确。与对照组比较,干扰组HCT116细胞中USP42蛋白表达水平明显降低(t=16.97,P<0.001),且能显著抑制HCT116细胞的侵袭和迁移能力(t分别为4.974和7.787,P<0.01)。结论USP42可能在多种肿瘤的发生发展中发挥重要作用。建立的重组质粒USP42-shRNA可在HCT116细胞中有效干扰USP42的表达,并能抑制肿瘤细胞的侵袭和迁移。Objective To evaluate the expression levels of human ubiquitin-specific protease 42(USP42)in various tumor tissues and construct its shRNA interfering recombinant plasmid.Methods The expression levels of USP42 in various tumor tissues were analyzed by using GEPIA and Ualcan online databases.Oligonucleotides containing shRNA targeting humanUSP42 mRNA were synthesized and cloned into eukaryotic expression vectorpGPU6/Neo.Theconstructedrecombinant plasmid USP42-shRNA was transfected to HCT116 cells(interference group)with Neofect®DNA transfection reagent,using the NC-shRNA as control.The interference effect of USP42 expression was determined by Western blot,while the effect of USP42 on migration and invasion of colon cancer cells by Transwell assay.Results GEPIA and Ualcan databases showed that USP42 was highly expressed in tumor tissues such as cholangiocarcinoma(CHOL),stamoch adenocarcinoma(STAD),thymoma(THYM)and colon adenocarcinoma(COAD).Recombinant plasmid USP42-shRNA was constructed correctly as proved by sequencing.Compared with that in control group,the expression level of USP42 in HCT116 cells of interference group decreased significantly(t=16.97,P<0.001),and the protein significantly inhibited the invasion and migration abilities of human COAD HCT11 cells(t=4.974 and 7.787,P<0.01).Conclusion USP42 may play an important role in occurrence and development of various tumors.The constructed recombinant plasmid USP42-shRNA effectively interfered with the expression of USP42 in HCT116 cells and inhibited the invasion and migration of tumor cells.

关 键 词：泛素特异性蛋白酶42 shRNA序列 HCT116细胞 细胞迁移 细胞侵袭 

分 类 号：R34[医药卫生—基础医学]