膀胱癌组织JARID1B表达及其慢病毒表达载体的构建及功能研究

目的研究膀胱癌组织及人膀胱癌细胞株T24中组蛋白去甲基化酶JARID1B的表达。构建JARID1B慢病毒表达载体并进行相关鉴定;观察经包装后慢病毒感染的T24中JARID1B表达情况。方法 Western blotting检测T24和6例膀胱癌及其癌旁组织JARID1B蛋白表达。利用真核表达质粒pcDNA3.1（-）-JARID1B,将JARID1B基因连接入含增强型绿色荧光蛋白（EGFP）的慢病毒表达载体pLv-EGFP中,构建重组慢病毒表达载体pLv-EGFP-JARID1B,将经酶切和DNA测序鉴定的重组质粒通过脂质体转染至T24,荧光显微镜观察GFP表达,RT-PCT检测JARID1B mRNA表达。包装慢病毒并感染T24,Western blotting检测目的蛋白JARID1B表达。结果膀胱癌及其癌旁组织JARID1B蛋白表达阳性率分别为16.7%和66.7%,且前者表达强度较弱;T24 JARID1B蛋白表达阴性。重组质粒经酶切和DNA测序证实目的基因插入正确;pLv-EGFP-JARID1B转染T24,荧光显微镜观察发现细胞表达GFP,RT-PCR显示T24表达JARID1B mRNA。包装慢病毒再感染T24后Western blotting分析显示,细胞表达目的蛋白JARID1B。结论膀胱癌组织JARID1B表达下调。成功构建JARID1B慢病毒表达载体,包装慢病毒感染T24后细胞过表达JARID1B。

Expression of JARID1B in bladder cancer tissues and construction and function of lentiviral expression vector

Objective To investigate the expression of histone demethyltransferases JARID1B in bladder cancer tissues and human bladder cancer cell line T24, construct JARID1B lentiviral expression vector and perform identification, and investigate the expression of JARID1B infected by packed lentivirus. Methods The expression of JARID1B protein in 6 cases of bladder cancer tissues, tissues adjacent to cancer, and human bladder cancer cell line T24 was detected by Western blotting. With eukaryotic expression plasmid pcDNA3.1（-）-JARID1B, JARID1B gene sequence was incorporated into lentiviral vectors pLv-enhanced green fluorescent protein (EGFP) which contained EGFP, and recombinant lentivirus expression vector pLv-EGFP-JARID1B was constructed. After DNA sequence analysis, pLv-JARID1B plasmid was transfected into T24 by Lipofectamine, the expression of GFP was observed by fluorescence microscopy, and the expression of JARID1B mRNA was detected by RT-PCR. Lentivirus was packed and T24 was infected, and the expression of JARID1B protein wes determined by Western blotting. Results The positive expression rates of JARID1B protein in bladder cancer tissues and tissues adjacent to cancer were 16.7% and 66.7%, respectively, and the expression was weaker for the former. The expression of JARID1B protein in T24 was negative. JARID1B sequence was verified to be inserted into lentiviral vectors pLv-EGFP. After transfection of T24 with pLv-EGFP-JARID1B, the expression of GFP was observed by fluorescence microscope, and the expression of JARID1B mRNA in T24 was detected by RT-PCR. After infection of T24 by packed lentivirus, Western blotting analysis revealed that the expression of JARID1B protein was detected.ConclusionThe expression of JARID1B is down-regulated in bladder cancer tissues. JARID1B lentiviral expression plasmid can be successfully constructed, and there is overexpression of JARID1B in T24 infected with packed lentivirus.