沉默MKRN1基因表达对人胚胎肾细胞HEK-293T增殖及凋亡的影响  

Effect of MKRN1 silencing on proliferation and apoptosis of human embryonic kidney cells HEK-293T

作　　者：张艺 吴道秋 汤弘婷 李梦醒 刘虹麟 陈忠良 李琴山 Zhang Yi;Wu Daoqiu;Tang Hongting;Li Mengxing;Liu Honglin;Chen Zhongliang;Li Qinshan(Department of Clinical Biochemistry,School of Medical Laboratory Science,Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Department of Hematology,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Institute of Clinical Medical Sciences,China-Japan Friendship Hospital,Beijing 100000,China;Tissue Engineering and Stem Cell Experimental Center,Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Guizhou Provincial Prenatal Diagnosis Center,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学医学检验学院临床生物化学教研室,贵州省贵阳市550004 [2]贵州医科大学附属医院血液科,贵州省贵阳市550004 [3]北京中日友好医院临床医学研究所,北京市100000 [4]贵州医科大学组织工程与干细胞实验中心,贵州省贵阳市550004 [5]贵州医科大学附属医院贵州省产前诊断中心,贵州省贵阳市550004

出　　处：《中国组织工程研究》2023年第24期3838-3844,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81960476,81460365),项目负责人:李琴山;国家自然科学基金(81760039),项目负责人:李梦醒;国家自然科学基金(81402451,82173378),项目负责人:刘虹麟;贵州省科技厅资助项目([2019]1270),项目负责人:李琴山;贵州省科技厅资助项目([2020]4Y160),项目负责人:李梦醒;贵州省卫生健康委基金(gzwkj2021-160),项目负责人:李梦醒。

摘　　要：背景:研究报道敲低MKRN1可以抑制肿瘤细胞的生长,但MKRN1是否作为肿瘤相关蛋白在机体发挥作用还需进一步研究。由于siRNA介导的基因沉默是瞬时表达,持续时间短且常规慢病毒技术存在脱靶效应,利用四环素调控(Tet-on)慢病毒系统构建稳定沉默MKRN1基因的细胞系有着可逆的优势,且可规避上述缺点,对于深入研究MKRN1的生物学功能有重要的意义。目的:构建Tet-on慢病毒系统介导的MKRN1基因沉默载体,研究沉默MKRN1基因表达对人胚胎肾细胞HEK-293T增殖、凋亡的影响。方法:采用RNAi技术,针对MKRN1基因设计3条shRNA(命名为sh1,sh2,sh3),将3条shRNA序列连入四环素诱导表达载体pTripz中验证连入序列;通过三质粒系统转染HEK-293T细胞,并用含有1 mg/L强力霉素的完全培养基培养,进行慢病毒包装,收取48 h及72 h病毒液,将其浓缩后感染HEK-293T细胞,感染48 h后用荧光显微镜观察病毒感染效率。通过实时荧光定量PCR、Western blot检测MKRN1的敲减效率;Western blot检测四环素诱导表达系统是否构建成功;通过集落形成实验及Western blot检测沉默MKRN1表达后对HEK-293T细胞增殖、凋亡能力的影响。结果与结论:(1)设计的3条MKRN1-短发夹RNA(sh1、sh2、sh3),均成功连入Tet-on载体,且各组载体序列正确;通过慢病毒成功感染HEK-293T细胞;(2)实时荧光定量PCR及Western blot检测发现3条shRNA序列中1号序列敲低MKRN1水平明显降低(P<0.05),且沉默组不加入强力霉素诱导后目的基因的表达得到回复;(3)集落形成实验发现沉默MKRN1后HEK-293T细胞增殖能力下降(P<0.05);Western blot检测敲低MKRN1后,增殖细胞核抗原表达下调,细胞凋亡相关指标BAX表达增加,Bcl2表达下调,Bcl2/BAX的比值下调(P<0.001);(4)结论:采用Tet-on慢病毒载体系统构建了稳定沉默MKRN1的HEK-293T细胞株,且沉默MKRN1后能够抑制HEK-293T细胞的增殖,并促进其凋亡。通过构建稳定沉默MKRN1的HEK-293T�BACKGROUND:It has been repo rted that makorin ring finger protein 1(MKRN1)knoc kdown can inhibit the growth of tumor cells,but whether MKRN1 plays a role in the body as a tumor-related protein needs further study.The siRNA-mediated gene silencing is transient,and therefore,the duration is short and the conventional lentivirus technique also has an off-target effect.A cell line stably silencing MKRN1 gene constructed using Tet-on lentivirus system has reversible advantages and can avoid the above shortcomings,which is of great significance for the further study on the biological function of MKRN1.OBJECTIVE:To construct a MKRN1 gene silencing vector mediated by the Tet-on lentivirus system in order to study the effect of MKRN1 gene silencing on the proliferation and apoptosis of human embryonic kidney cells HEK-293T.METHODS:Using RNAi technique,three short hairpin RNAs(shRNAs)were designed for MKRN1 gene,namely sh1,sh2,and sh3,and three shRNA sequences were ligated into tetra cycline-induced expression vector pTripz.The correct sequence was verified by sequencing.HEK-293T cells were transfected with the three-plasmid system and cultured in the complete medium containing 1 mg/L doxycycline for lentivirus packaging.The virus solution was collected at 48 and72 hours and concentrated to infect HEK-293T cells.Fo rty-eight hours after infection,the virus infection efficiency was observed by fluorescence microscope.The knocko ut efficiency of MKRN1 was detected by real-time fluorescence quantitative PCR and western blot assay.The successful construction of tetracyclineinduced expression system was detected by western blot assay.Colony formation test and western blot assay were used to detect the effect of silencing MKRN1expression on the proliferation and apoptosis of HEK-293T cells.RESULTS AND CONCLUSION:(1)Three MKRN1-shRNAs,named sh1,sh2,and sh3,were successfully inserted into the Tet-on vector,and the sequence of each vector was correct.HEK-293T cells were successfully transfected with lentivirus.(2)RT-qPCR and wester

关 键 词：MKRN1基因 四环素诱导调控表达系统 基因表达调控 慢病毒 

分 类 号：R446[医药卫生—诊断学] R318[医药卫生—临床医学] R496