Inhibitory effect of shRNA targeting GPR48 on invasion and metastasis of human cervical carcinoma cell line HeLa.
OBJECTIVE
GPR48 regulates invasion and metastasis of cancer cells by linking Gs protein to stimulate intracellular signaling pathway and affect actin filament aggregation and the activity of matrix metalloproteinase. This study was to explore the effects of GPR48 short hairpin RNA (shRNA) on GPR48 expression, and invasion and metastasis of cervical carcinoma cell line HeLa.


METHODS
Plasmids containing two different sequences of human GPR48 mRNA coding region were constructed and transfected into HeLa cells. HeLa cell clones with stable small interfering RNA (siRNA) expression were screened by neomycin resistance. The expression of GPR48 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. In vitro invasive activity was measured by Boyden chamber experiment. GPR48 shRNA-transfected or untransfected HeLa cells were inoculated subcutaneously into nude mice to observe tumor growth and lung metastasis.


RESULTS
Compared with that in negative shRNA-transfected cells, GPR48 mRNA expression in GPR48 shRNA-transfected cells was downregulated by more than 80% and the number of HeLa cells infiltrated Boyden chamber membrane was decreased (94.4 +/- 15.7 vs. 28.3 +/- 1.5 and 17.6 +/- 1.5, p < 0.01). The depletion of endogenous GPR48 suppressed in vivo metastasis: the number of lung metastases was significantly larger in negative shRNA group than in GPR48 shRNA groups (7.3 +/- 1.8 vs. 1.3 +/- 0.3 and 1.5 +/- 0.4, p < 0.01).


CONCLUSIONS
GPR48 shRNA can inhibit in vitro invasion and in vivo metastasis of HeLa cells efficiently.
Gao
Yun
Y
Institute of Endocrinology, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning, PR China. gaoyungao@hotmail.com
Shan
Zhong-Yan
ZY
Wang
Hong
H
Zhang
Hong-Mei
HM
Teng
Wei-Ping
WP
D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000556:secondary; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009374:Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014183:Transplantation, Heterologous; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
C411562:LGR4 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled