Effects Of CXCR4 On Epithelial Ovarian Cancer Growth Characteristics As Well As Invasion, Metastasis

Objective:Chemotatic factors are single strand protein superfamilyand cytokines that make cells develop chemotatic movement.The function of chemotatic factors is mediated by their receptors.CXCR4 is one of chemotatic recepors,and educes important effect in proliferation,migration,invasion and adhension of many cancer cells.In ovarian cancer investigation, they observed fourteen chemotatic receptors,and only CXCR4 was positive in ovarian cancer cell.In the prophase study,we found that CXCL12 and CXCR4 were negative in nomal ovarian tissiue,and CXCR4 was hypsi-expressed in ovarian cancer tissue.The study of effect of CXCR4 in ovarian cancer is fewer,so we want to explore that the effect of CXCL12-CXCR4 biological axis in peritoneal metastatic of ovarian cancer.Methods:1 Establishment of the eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 we gained PCR production of CXCR4 by PCR.We got linear vector and the fragment of CXCR4 by renaturation,enzyme-cutting,conjunction and gained the eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4.Recombiant plasmid that identification was correct was sequenced and the result was analyzed by Sequence3.0 Microsoft.2 Establishment and identification of the epithelial ovarian cancer cell strain which expresses CXCR4 protein stably and highly The eukaryotic expression recombinant plasimid pReceiver-M02-CXCR4 and vector pReceiver-M02 were transfected into SKOV3 which did not express CXCR4 protein by lipofectamine-mediated gene transfection method,and gained positive cell clone by G418 screening.Cells that are transfected with were examined by RT-PCR,Western Blot and immunocytochemistry methods.3 Study of CXCL12-CXCR4 in tumor proliferation,migration, invasion of ovarian cell lines in vitro SKOV3 transfected with plasmid,SKOV3 transfected with vector,SKOV3 were cultured in vitro.Methylthiazolyltetrazolium was used to analyze effect of different concentration of CXCL12 on three cell lines proliferation in serum-free growing conditions,and to determine inhibition of neutralizing CXCR4 antibody or CXCR4 antagonist AMD3100.Transwell and Matrigel were used to evaluate effect of various concentrationof CXCL12 on three ovarian cell lines' migration and invasion,as well as inhibition of neutralizing CXCR4 antibody or antagonist AMD3100.Results:1 The PCR production of CXCR4 CXCR4 was amplified by PCR We gained a specific strap that was 1059 bp and was to match with the fragment of CXCR4. 2 Extraction and sequencing of eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 Recombinant plamids were sequenced after construction.The result confirmed that the order of connection of gene and vector was correct and the sequence of gene was coincidence with CXCR4 of GeneBank.3 The screening of the epithelial ovarian cancer cell strain which expresses CXCR4 protein stably and highly Screening concentration of G418 is 600μg/ml.After fourteen days,most of ovarian cancer cells tansfected with plasmid and vector died and formed cell sites.We gained several single cells by limiting dilution assay.The shape and growth velocity of the cells that were transfected had no marked change,but their stretch speed became slow.We named SKOV3 transfected with pReceiver-M02-CXCR4 and pReceiver-M02 as SKOV3/CXCR4 and SKOV3/neg cells respectively.4 Identification of the epithelial ovarian cancer cell which expresses CXCR4 protein highly and stably We identified the epithelial ovarian cancer cells which express CXCR4 protein stably by RT-PCR,Western blot,immunocytochemistry methods.All these indicate that CXCR4 is positive in ovarian cancer cells transfected with plasmid.Transfection efficiency is about 73%.5 The effect of CXCL12-CXCR4 on ovarian cancer cells proliferation Under serum-free condition,100ng/ml CXCL12 can promote the proliferation of SKOV3/CXCR4 cell (F=256.89,P＜0.05),and this effect can be inhibited by 10μg/ml neutralizing CXCR4 antibody or CXCR4 antagonist AMD3100.100ng/ml CXCL12 can not promote the proliferation of SKOV3/neg and SKOV3 cell(F=0.31,P＞0.05) (F=0.92,P＞0.05).6 The effect of CXCL12-CXCR4 on ovarian cancer cells migration With regard to SKOV3/CXCR4 cell,100ng/ml CXCL12 can promote cell migration(F=393.21,P＜0.05),and is strongly inhibited by treatment with 10μg/ml neutralizing CXCR4 antibody or CXCR4 antagonist AMD3100.With regard to SKOV3/neg and SKOV3 cell,CXCL12 can not promote celt migration,regardless of the concentration of CXCL12(F=0.41, P＞0.05)(F=0.62,P＞0.05).7 The effect of CXCL12-CXCR4 on ovarian cancer cell invasion Matrigel is reconstituted artificial basel membrane, which have the shape and function of internal basel membrame. Under serum-free condition,with regard to SKOV3/CXCR4 cells,they displayed minimal invasiveness through Matrigel when no CXCL12 was present in the lower chamber of transwell.After the effect of CXCL12,the number of invading cells through Matrigel was significantly higher than that of control group,and was strongly inhibited by treatment with 10μg/ml neutralizing CXCR4 antibody or CXCR4 antagonist AMD3100(F=16.65,P＜0.05).With regard to SKOV3/neg and SKOV3,CXCL12 can not promote cell invasion,regardless of the concentration of CXCL12.Every group had no significant difference(F=0.04,P＞0.05)(F=0.05,P＞0.05).Conclusion:1 Establishment of eukaryotic expression recombinant plasmid pReceiver-M02-CXCR4 and the epithelial ovarian cancer cell which expresses CXCR4 protein stably is successful, and which is identified by RT-PCR,Western blot, immunocytochemistry.2 CXCL12-CXCR4 interreaction can promote proliferation, migration,invasion of ovarian cancer cells in vitro. CXCL12-CXCR4 biological axis has important effect in peritoneal metastasis of epithelial ovarian cancer.