| Background and ObjectiveOvarian cancer is one of the three malignant tumors of gynecology. Its mortality is far beyond cervical cancer and endometrial carcinoma, ranking first in gynecologic tumors. It has the feature of onset conceals, short duration, and it is easy to early inva-sion and migration. Though application surgery combination chemotherapy and pre-operatie chemotherapy as treatment method, the long-term survival of ovarian cancer has no significant improve. Now looking for a effective gene target to suppress the pr-ogression of human ovarian carcinoma is a hot research point. Deleted in liver cancer 1(DLC1) locus at human chromosome 8p21.3-22. Its expression was lost or downreg-ulated in various cancers due to either genomic deletion or aberrant promoter methyl-ation. DLC1 protein is a Rho GTPase activatiog protein (Rho GAP), and its RhoGAP domains can negatively regulated Rho proteins(RhoA,Cdc42) and their downstream effectors to restraint tumor cells invasion and migration ability. DLC1 protein also can make focal adhension kinase(FAKY925) dephosphorylation, in order to suppress the proliferation and migration ability of human liver cancer cells. c-JUN N-terminal kin- ase(JNK) protein can regulate the expression of nuclear transcription factor AP-1, to participate in the occurrence and development of ovarian cancer. At present the res-earch about the relationship with DLC1 gene and the proliferation and migration ability of ovrian cancer, and the relation with DLC1 FAK and JNKprotein are less reported.In our research a recombinant plasmid pEGFP-C3-DLC1 was transfected into OVCAR-3 cells by lipofectamine. And observe the effect and the mechanism of DLC1 gene on cell proliferation, cell cycle and migration ability of human ovarian cancer cell line OVCAR-3. To investigate the effects of DLC1 gene on carcinogenesis and developing in ovarian cancer. To find a new gene therapy taget of ovarian cancer.Methods1 The purification of recombined plasmid pEGFP-C3-DLCl, and verified by double-enzyme cleavage method, ovarian cancer cell line OVCAR-3 was transfected with recombinant plasmid pEGFP-C3-DLC1 and empty plasmid pEGFP-C3 by lipofectamine. Three groups were divided:OVCAR-3 group, OVCAR-3-EGFP group and OVCAR-3-DLC1 group.Transfected cells were seleted by G418. The expression of DLC1 mRNA and protein were examined by RT-PCR and Western blotting respectively,in order to observe the transfect effects.2 The cell viability and, cell cycle and migration ability were detected by MTT assay, flow cytometry and Transwell chamber. To analyse the effects of DLC1 gene on the proliferation and migration ability of human ovarian cancer OVCAR-3 cells.3 The protein expressions of FAK, JNK, p-FAK(Y925) and p-JNK(Thrl 83/Tyr 185) were detected by western blotting. To analyse the action mechanism of DLC1.4 Statistical software SPSS 16.0 were used to analyse the data results. One-way analysis of variance and bonferronitest were used to measurement differences between groups. Pearson product-moment correlation analysis were used to analyse the correlation between p-FAK, p-JNK and the proliferation, cell cycle and migration abilities of OVCAR-3 cells. Test level for the bilateral a=0.05.Results1 Double enzyme cut prove insert sequence correct by that restructuring plasmid appear two specific bands 4.7 Kb and 3.9 Kb, respectively stand for pEGFP-C3 and DLC1cDNA. There is no expression of DLC1mRNA and DLC1 protein in OVCAR-3 cells. But DLC1 mRNA and protein were expressed in transfected cells.2 After DLC1 gene transfected, from the second day the proliferation ability of OVCAR-3 cells were decreased. And it has significant difference(P<0.05). At sixth day the A492 for OVCAR-3,OVCAR-3-EGFP,OVCAR-3-DLC1 three groups was 1.132±0.045,1.106±0.031,0.503±0.022 respectively, it was significantly less than those of two control groups(F= 1604.000, P= 0.000).3 The cell cycle tested by flow cytometry shows that:The cells of G0/G1 phase in OVCAR-3,OVCAR-3-EGFP,OVCAR-3-DLC1 three groups was (55.06±0.7 3)%, (56.22±0.51)%, (61.40±0.75)% respectively,it was significantly more than those of OVCAR-3 group and OVCAR-3-EGFP group(F=126.151, P=0.000). The cells of S phase in OVCAR-3,OVCAR-3-EGFP,OVCAR-3-DLC1 three groups was (23.24±0.42)%, (18.96±0.35)%, (17.10±0.44)% respectively. OVCAR-3-DLC1 group was significantly less than those of another two groups(F=1619.000, P=0.000). The cells of G2/M phase in three groups was (23.60±0.46)%, (23.77±0.57)%, (21.50±0.20)% respectively. OVCAR-3-DLC1 group was significantly less than those of another two groups(F=25.052, P=0.001).4 The Transwell chamber assay shows that:The number of migration cells in OVCAR-3-DLC1, OVCAR-3-EGFP, OVCAR-3 groups was (40.27±2.15), (56.80±2.04), (57.07±3.01), OVCAR-3-DLC1 group was significiantly less than another two groups (F=233.13, P=0.00).5 In OVCAR-3-DLC1, OVCAR-3-EGFP, OVCAR-3 three groups, the expression level of p-FAK(Y925),p-JNK(Thr183/Tyr185) proteins was 0.63±0.06,0.70±0.05 VS 0.72±0.04; 1.78±0.11,2.37±0.11 VS 2.41±0.15 respectively. OVCAR-3-DLC1 group were significiantly less than another two groups(F=9.186, P=0.001; F=93.729, P=0.000). But in three groups, the the expression level of FAK,JNK proteins has no significiantly changes(P>0.05).6 Pearson product-moment correlation analysis shows that: The expression level of p-FAK,p-JNK protein is correlated with the proliferation(r=0.41, P=0.02; r=0.39, P=0.03) and migration(r=0.36, P=0.04; r=0.37, P=0.03) abilities of OVCAR-3 group cells. But it isn't correlated with cell cycle(r=-0.08, P=0.77; r=-0.00, P=1.00).Conclusions1 DLC1 gene may inhibit the proliferation ability in vitro of ovarian cancer,by suppress the G|/S check point.2 DLC1 gene can suppress the migration in vitro of ovarian cancer.3 DLC1 protein may suppress the proliferation and migration abilities in vitro, through make p-FAK(Y925), p-JNK(Thr183/Tyr185) proteins dephosphory-lation. But it participate in cell cycles by the other cell signal ways. |
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