[Inhibition of MYH9 expression by short hairpin RNA expression vector in human umbilical vein endothelial cells (HUVEC)].
OBJECTIVE
To construct the expression vector of small hairpin RNA (shRNA) and to investigate its efficacy in silencing nonmuscle myosin heavy chain IIA (MYH9)gene.


METHODS
According to the MYH9 cDNA sequence in GenBank, three shRNA expression vectors targeting MYH9 gene were constructed and were confirmed by digestion with restriction enzymes and DNA sequencing. The recombinant plasmids were transfected into HUVEC cells. The expression of MYH9 was determined by semi-quantitative RT-PCR at mRNA level and by western blotting at protein level at 24 h, 48 h and 72 h after transfection.


RESULTS
The decrease of MYH9 expression at mRNA and protein levels gradually became more evident from 24 h to 48 h, and achieved the maximal degree at 48 h, then became weakened and restored at 72 h. The efficiency of pGCsi-MYH9-3 was the best among these three plasmids. The introduction of pGCsi-MYH9-3 was showed to efficiently and specifically inhibit the expression of MYH9 with inhibitory rate at 71.2% according to results of Western blot. RT-PCR results showed that mRNA transcription of MYH9 gene was reduced by 86.5% at 48 h after introduction of pGCsi-MYH9-3.


CONCLUSIONS
Plasmid pGCsi-MYH9-3 could inhibit MYH9 expression in HUVEC cells successfully and effectively, which promised its further application in related function analysis of MYH9.
Huang
Ya-Lin
YL
Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing 210038, China. huangyalin228@yahoo.com.cn
Kou
Jun-Ping
JP
Song
Jia-Xi
JX
Yu
Bo-Yang
BY
D015153:Blotting, Western; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins / Q000166:cytology*
C103814:MYH9 protein, human; D020409:Molecular Motor Proteins; D034741:RNA, Small Interfering; D018995:Myosin Heavy Chains