[Effects of COX-2 gene silencing by shRNA on biological characteristics of human hepatocellular carcinoma cell line HepG2].
OBJECTIVE
Cyclooxygenase-2 (COX-2) protein is highly expressed in hepatocellular carcinoma (HCC). It may be involved in tumorigenesis and development of HCC. This study was to explore the effects of COX-2 short hairpin RNA (shRNA) on COX-2 expression in HCC cell line HepG2 and on adhesiveness and invasiveness of HepG2 cells in vitro.


METHODS
Plasmids WBH1 and WBH2 containing 2 different sequences of human COX-2 mRNA coding region were constructed, and transfected into HepG2 cells, respectively. The expression of COX-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at 24, 48, 72 and 96 h after transfection. At 48 h after transfection, the adhesiveness of HepG2 cells to extracellular matrix matrigel was detected using MTT assay. Invasiveness was measured by Transwell experiment.


RESULTS
The transfection rate in HepG2 cells was about 60%. The inhibition rates of COX-2 mRNA expression in HepG2 cells were 18.5%, 88.6%, 52.8%, and 42.4% at 24, 48, 72 and 96 h after transfection of plasmid WBH1 (P<0.01); the inhibition rates of COX-2 protein expression were 10.2%, 80.5%, 45.3%, and 39.0%, respectively (P<0.01). Plasmid WBH2 had no significant inhibitory effect on COX-2 expression in HepG2 cells (P>0.05). The adhesion rate of WBH1-transfected cells was obviously reduced by 47.4% of control cells [(6.0+/-0.4)% vs. (11.4+/-0.2)%, P<0.01]. The cell number that infiltrated Transwell membrane in WBH1-transfected group was significantly reduced by 63.7% of control group (8.25+/-1.50 vs. 22.75+/-1.70, P<0.01). WBH2 had no obvious effects on adhesiveness and invasiveness of HepG2 cells.


CONCLUSIONS
COX-2 shRNA can inhibit the adhesiveness and invasiveness of HepG2 cells through intervening the expression of COX-2.
Xu
Ke-Shu
KS
Department of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, P. R. China. xuzou@medmail.com.cn
Wang
Hua-Feng
HF
Qian
Wei
W
Wang
Bu-Hai
BH
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D041681:NIH 3T3 Cells; D009361:Neoplasm Invasiveness; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human