[Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells].
OBJECTIVE
To construct THY1 eukaryotic expression plasmid and study its effects on the growth of epithelial ovarian cancer cell line SKOV3.


METHODS
THY1 gene fragment was obtained from normal human ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1(+)-THY1, which was transformed into E. coli JM109 followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analyzed by PCR, restriction endonucleases digestion and DNA sequencing. SKOV3 cells divided into SKOV3-THY1, SKOV-3-Null and SKOV3 groups were transfected via liposome with the recombinant plasmid pcDNA3.1(+)-THY1, empty plasmid, or not transfected, respectively. The expression of THY1 mRNA and its protein were examined by RT-PCR and Western blot methods. The cell growth and apoptosis were evaluated by MTT assay and flow cytometry.


RESULTS
The gene fragment of exogenous THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1(+) as verified by PCR, restriction endonucleases digestion and DNA sequencing, and recombinant expression plasmid pcDNA3.1(+)-THY1 transfection resulted in stable expression in SKOV3 cells as shown by RT-PCR and Western blotting. The cell growth inhibition rate of SKOV3-THY1 group (56.6% at the fifth day) was significantly higher than that of the SKOV3-Null group (12.5%, P<0.05), and the cell apoptosis rate in SKOV3-THY1 group (31.8%) was significantly higher than those in SKOV3-Null group (10.5%) and SKOV3 group (9.8%, P<0.05), but the apoptosis rate between the latter two groups was similar (P>0.05).


CONCLUSIONS
The recombinant plasmid pcDNA3.1(+)-THY1 can be expressed stably in human ovarian cancer cell line SKOV3. THY1 transfection can inhibit the growth of SKOV3 cells in vitro, suggesting the important role of THY1 gene in pathogenesis and development of ovarian cancer.
Zeng
Li-Qin
LQ
Department of Gynecology and Obstetrics, West China Second Hospital, Sichuan University, Chengdu 610041, China. zenglq89@163.com
Peng
Zhi-Lan
ZL
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D012333:RNA, Messenger; D011994:Recombinant Proteins; D018800:Thy-1 Antigens