[Construction of a recombined adenovirus vector carrying pri-miR-21 gene  and research on it's target gene TLR4].
OBJECTIVE
To construct the recombined adenovirus vector carrying pri-miR-21 gene,  which can express mature miR-21 efficiently,  and to study the interaction of miR- 21 with its target gene TLR4.


METHODS
Using healthy mouse's gDNA as template,  the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting.


RESULTS
Through the PCR,  restriction enzyme digestion,  DNA sequencing and expression of GFP,  recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioinformatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels.


CONCLUSIONS
The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.
Institute of Laboratory Medicine, General Hospital, Ningxia Medical University, Yinchuan, China.
Jing
J
Zhao
Guang-xian
GX
Xu
Wei
W
Jia
Hui
H
Dong
Yi-lin
YL
Zhang
Zhi-jun
ZJ
Zhao
Jun
J
Wei
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000378:metabolism
C531950:MIRN21 microRNA, mouse; D035683:MicroRNAs; D051197:Toll-Like Receptor 4