[Plasmid-mediated miRNA-1-2 specifically inhibits Hand2 protein expression in H9C2 cells].
OBJECTIVE
To construct an eukaryotic expression vector for miRNA-1-2 that can be expressed in rat H9C2 cardiomyocytes.


METHODS
The precursor miRNA (pre-miRNA) DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4.1-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase (RT)-PCR was performed to determine miRNA-1-2 precursor expression.


CONCLUSIONS
DNA sequencing indicated that the miR-1-2 expression plasmid was correctly constructed. The precursor miRNA-1-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.
Shan
Zhi-xin
ZX
Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou, China. zhixinshan@yahoo.com.cn
Lin
Qiu-xiong
QX
Deng
Chun-yu
CY
Zhou
Zhi-ling
ZL
Zhang
Xu-chao
XC
Fu
Yong-heng
YH
Yu
Xi-yong
XY
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D049452:Green Fluorescent Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C498767:Hand2 protein, rat