Chen Gang, Mou Lun-pan, Yao Jin, You Ting-ting, Shen Xiao-yan, Zhu Xiang-qing, Qiao Yu-fang, Lin Miao, Fang Xiao-wen, Zou Xin, Lin Li-xiang
Department of Endocrinology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou 350001, China.
Zhonghua Yi Xue Za Zhi. 2008 Nov 4;88(40):2821-5.
To construct RNAi recombinant adenoviral expressive vectors specific to glycogen synthase kinase-3beta (GSK-3beta) and to observe its gene knockdown effect on the expression of GSK-3beta, and to explore the effect of Wnt/beta-catenin pathway on the proliferation of human thyrocytes using the RNAi adenovirus vector.
An adenovirus plasmid that contained the RNAi cassette targeting the GSK-3beta gene was constructed by homologous recombination and cloning techniques, transfected into human embryo kidney (HEK) 293A cells to product adenovirus, and then was used to infect the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. Normal human thyrocytes fart from thyroid adenoma were obtained during resection of adenoma, cultured, and infected by the GSK-3beta specific RNAi adenovirus. The GSK-3beta gene silencing effect induced by the RNAi adenovirus was detected by Western blotting 0, 24, 48, 72, 120, and 144 hours later. BrdU method was used to detect the cell proliferation. Another HEK293A cells were divided into 3 groups: infected with recombinant adenovirus plasmid Ad-1457, infected with un-recombinant framework plasmid pAd-DEST, and un-infected. 72 hours later Western blotting was used to examine the level of beta-catenin.
The GSK-3beta expression of the thyrocytes infected with the recombinant adenovirus plasmid Ad-1457 were significantly lower than those of the thyrocytes infected with Ad-DEST (all P<0.05). The expression of beta-catenin of the thyrocytes infected with Ad-DEST was significantly higher than those of the Ad-DEST group and un-infected group (both P<0.05). BrdU assay suggested that the proliferation rates 1, 3, 5, and 7 days after infection of the thyrocytes infected with Ad1457 plasmid were significantly higher than those of the thyrocytes infected with the plasmid pAd-DEST (all P<0.05).
RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently. The Wnt/beta-catenin pathway plays an important role in the regulation of proliferation of human thyrocytes.
构建糖原合酶激酶-3β（GSK-3β）特异性RNA干扰重组腺病毒表达载体，观察其对GSK-3β基因表达的沉默效应，并利用RNA干扰腺病毒载体探讨Wnt/β-连环蛋白信号通路对人甲状腺细胞增殖的影响。
采用同源重组和克隆技术构建含靶向GSK-3β基因RNA干扰盒的腺病毒质粒，转染人胚肾（HEK）293A细胞包装腺病毒，再用其感染HEK293A细胞扩增病毒原液。采用空斑形成试验测定病毒原液滴度。在甲状腺腺瘤切除术中获取远离腺瘤的正常人甲状腺细胞，进行培养，并感染GSK-3β特异性RNA干扰腺病毒。于感染后0、24、48、72、120和144小时，采用蛋白质印迹法检测RNA干扰腺病毒诱导的GSK-3β基因沉默效果。采用BrdU法检测细胞增殖情况。另将HEK293A细胞分为3组：分别感染重组腺病毒质粒Ad-1457、感染非重组骨架质粒pAd-DEST、未感染。72小时后，采用蛋白质印迹法检测β-连环蛋白水平。
感染重组腺病毒质粒Ad-1457的甲状腺细胞中GSK-3β表达明显低于感染Ad-DEST的甲状腺细胞（均P<0.05）。感染Ad-DEST的甲状腺细胞中β-连环蛋白表达明显高于Ad-DEST组和未感染组（均P<0.05）。BrdU检测显示，感染Ad1457质粒的甲状腺细胞在感染后1、3、5和7天的增殖率明显高于感染质粒pAd-DEST的甲状腺细胞（均P<0.05）。
RNA干扰腺病毒是有效抑制靶基因表达的重要工具。Wnt/β-连环蛋白信号通路在人甲状腺细胞增殖调控中起重要作用。
