[Pirh2 shRNA mediated by psiRNA-hH1 vector plasmid effectively inhibits the proliferation of lung carcinoma cells: in vitro and in vivo experiments].
OBJECTIVE
To investigate the role of P53 induced RING-H2 protein (pirh2) for proliferation of lung cancer cell in vitro and vivo.


METHODS
Immunohistochemistry was applied to detect the expression of pirh2 protein in 53 specimens of lung cancer resected during operation. Human lung adenocarcinoma cells of the line A549 were cultured. Three kinds of plasmid, two of which were designed targeting two sites of pirh2 and named psiRNA-Pirh2A and psiRNA-Pirh2B, and one of which was designed targeting nothing as negative-control and was named psiRNA-Con. A549 cells were transfected with these plasmids respectively. Real-time PCR and Western-blotting were applied to detect the expression of pirh2 in the A549 cells. The ability of cell proliferation was analyzed with cell counting kit-8 (CCK-8) method. According to the result of CCK-8 testing, the better one in suppressing the growth of A549 cells was chosen to be transplanted in nude rats. Eighteen nude rats were randomly divided into 3 equal groups to be inoculated subcutaneously with normal A549 cells, A549 stably expressing psiRNA-pirh2, and A549 cells expression psiRNA-Con. Thirty days later the rats were killed and the tumors were taken out to be examined.


RESULTS
Forty-two of the 53 C lung cancer specimens (79.2%) showed expression of pirh2 protein. A549 cells expressing psiRNA-Pirh2A and psiRNA-Pirh2B grew much slower than the normal A549 cells (q = 6.82 and q = 5.63, P < 0.05). However, there was difference in growth speed among the normal A549 cells, A549 cells transfected with psiRNA-Con, and those transfected with psiRNA-hH1. Eleven days after inoculation tumors could be touched in the rats of pirh2 shRNA group, obviously later than in the other two groups. The weight of the tumors taken pout of the rats of the pirh2 shRNA group was 0.59 +/- 0.16 g, significantly lower than that of the normal A549 cell group (1.66 g +/- 0.21 g, t = 6.27, P < 0.05). But there was no significant difference in the tumor weight between the normal A549 cell group and psiRNA-Con group.


CONCLUSIONS
pirh2 may be a critical factor in lung cancer formation. The successfully constructed shRNA targeting pirh2 efficiently decreases its expression in lung cancer cells and inhibits the growth of the lung cancer cells.
Department of Respiratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Yuan
Y
Su
Ming
M
Bai
Li-ping
LP
Zhu
Yang
Y
Jin
Xiao-ju
XJ
Zhang
Qiong
Q
Zhou
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D011923:Rats, Nude; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D047368:Tumor Burden; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D023041:Xenograft Model Antitumor Assays / Q000379:methods; D016335:Zinc Fingers
D034741:RNA, Small Interfering; C485369:RCHY1 protein, human; D044767:Ubiquitin-Protein Ligases