[Small interfering RNA inhibits the expression of connexin43 in the human corpus cavernosum penis smooth muscle cells].
OBJECTIVE
To inhibit the expression of connexin43 (Cx43) in the human corpus cavernosum penis smooth muscle cells by small interfering RNA (siRNA) and detect the gap junction intercellular communication (GJIC), and to investigate the application of siRNA technology in the gap junction of corpus cavernosum penis smooth muscle cells and its role in the penile erection process.


METHODS
With the help of the software of Ambion Corporation, the specific recombinant plasmids with siRNA targeting human Cx43 gene were constructed. The recombinant plasmids having been stably transferred into human corpus cavernosum penis smooth muscle cells for 48 hours, semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques were used to examine the inhibitory effects of siRNA on the expressions of the Cx43 gene and protein, in comparison with the siRNA negative control and the blank control group, respectively. The GJIC was detected by scrape-loading and fluorescence dye transfer experiments through the fluorescence microscope.


RESULTS
The results of enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pSilencer 1.0-U6-siRNA-Cx43 was successfully constructed. The relative levels of Cx43 mRNA and protein expression in the smooth muscle cells were (0.45 +/- 0.08)% and (0.56 +/- 0.06)% after successful transfer of the recombinant plasmid. However, the expression levels of mRNA and protein were (0.72 +/- 0.04)% and (0.80 +/- 0.08)% in the negative siRNA transfer group, and (0.74 +/- 0.09)% and (0.77 +/- 0.11)% in the blank control, respectively, with a significant difference (P < 0.05). The GJIC also decreased significantly.


CONCLUSIONS
siRNA can significantly inhibit the expression of Cx43 and block the GJIC in the human corpus cavernosum penis smooth muscle cells. siRNA technology plays an important role in penile erection and flaccidity.
Cao
Zheng-guo
ZG
Department of Urology, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui 230001, China. zgcao2002@126.com
Zhu
Yu-ping
YP
Sun
You-wen
YW
Dong
Xiao-cheng
XC
Qi
Lin
L
Xiao
Jun
J
Chen
Hao
H
Ren
Wei-hua
WH
Zou
Lian
L
D015152:Blotting, Northern; D002478:Cells, Cultured; D018031:Connexin 43 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007365:Intercellular Junctions; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D010413:Penis / Q000166:cytology / Q000378:metabolism*; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018031:Connexin 43; D034741:RNA, Small Interfering