[Construction of GSK-3beta-targeting RNAi adenovirus vector and the influence of Wnt/beta-catenin pathway in proliferation of human thyrocytes].
OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Chen
Gang
G
Department of Endocrinology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou 350001, China. chengangfj@163.com
Mou
Lun-pan
LP
Yao
Jin
J
You
Ting-ting
TT
Shen
Xiao-yan
XY
Zhu
Xiang-qing
XQ
Qiao
Yu-fang
YF
Lin
Miao
M
Fang
Xiao-wen
XW
Zou
Xin
X
Lin
Li-xiang
LX
D000256:Adenoviridae; D002460:Cell Line; D049109:Cell Proliferation; D005822:Genetic Vectors; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics*; D006801:Humans; D034622:RNA Interference; D013961:Thyroid Gland / Q000166:cytology*; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism*
D051153:Wnt Proteins; D051176:beta Catenin; D038362:Glycogen Synthase Kinase 3