| BACKGROUND AND OBJECTEpithelial ovarian cancer is a leading cause of death in women around the world. Due to the lack of obvious symptoms, the majority of patients is only identified in the advanced stages of the disease. The prognosis for ovarian cancer patients is very poor. Although new chemotherapeutic agents have significantly improved five-year survival rate, the overall mortality of ovarian cancer has remained unchanged. The tumorigenesis of ovarian cancer is a polygenic disease and a multistep complicated process involved, which includes the activation of pro-oncogenes and inactivation of suppressor genes. However, most cancer related deaths are not due to the growth of the primary tumor but result from its invasive spread to secondary sites. Though some candidate genes were found to be related to metastasis, neither of them could be interpreted the mechanism of invasion and metastasis completely. Consequently, it is significantly necessary to identify the novel genes related to the tumorigenesis, development and progress to improve the early diagnosis and treatment of ovarian cancer, and PLA2G2A is one of them we found.Phospholipase A2(PLA2) is a esterase that cleave glycerophospholipids at the sn-2ester bond to release a fatty acid andlysophospholipid. PLA2G2A belongs to a family of phospholipases and is a low molecular weight (16kDa) secreted phospholipases. The major known functions of PLA2G2A are mostly related to inflammatory responses, antimicrobial defense, anticoagulation, cell proliferation, ischemic injury and allergic diseases.PLA2G2A is expressed in various types of cancers, including prostate cancer, colorectal cancer and so on, which plays different roles in these cancers. Although PLA2G2A has been proposed as a potential tumor suppressor, evidence supporting this model is conflicting and it does not behave like a classical oncogene or tumor suppressor gene. For example, PLA2G2A is upregulated in prostate, gastric, intestinal human tumors. Several studies of human prostate cancer support an oncogenic role for sPLA2-IIA, particularly in its progression to advanced cancer. In contrast, in human gastric cancer, patients expressing high levels of sPLA2-IIA showed a highly significant survival advantage. A recent study reported that PLA2G2A may function as an inhibitor of gastric cancer invasion in vitro and β-catenin-dependent Wnt-signaling may serve as its up-stream regulator.Till now, the biological functions in tumor development of PLA2G2A in ovarian cancer have not been discussed. Consequently, PLA2G2A was overexpressed in ovarian cancer cells to identify how PLA2G2A might contribute to ovarian cancer development and progression and provide the potential therapeutic target for ovarian cancer.METHODPLA2G2A mRNA expression level was examined in ovarian serous cancer cell lines A2780, HO8910, HO8910pm, SKOV3and OVCAR3.The cDNA representing the complete open reading frame of PLA2G2A was amplified from the cDNA of fresh human placenta tissue using the specific primers as follows:forward,5’-CCGGAATTCGCCACCATGAAGACCCTCCTACTGT-3’;revers-e,5’-CCGCTCGAGTCAGCAACGAGGGGTGCT-3’,and then was cloned into the EcoRI-XhoI vector fragment derived from the pcDNA3.1vector (Invitrogen) to generate pcDNA3.1-PLA2G2A(3.1-PLA2G2A) cells. The expression plasmid was verified by sequencing of both strands and was used to transfect the HO8910pm cells using lipofectamine2000transfection reagent (Invitrogen) according to the manufacturer’s protocol. After24h, the cells for stable transfection cells were passaged at a1:10dilution into the fresh growth medium. After24h,750ug/mL G418(Invitrogen) was added in order to select the stable transfected cell lines for2weeks and individual colonies were isolated, expanded and maintained in375ug/mL G418. The overexpression of PLA2G2A in these clones was confirmed by RT-PCR and western blot analysis. The empty pcDNA3.1plasmid was used similarly to establish pcDNA3.1-vector (3.1-vector).The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to assess cellular proliferative ability between3.1-Vector and3.1-PLA2G2A. The viability of cells was assessed by clonogenic assays between3.1-Vector and3.1-PLA2G2A. To assess whether PLA2G2A overexpression affects migration and invasion of HO8910pm cells, both cell migration and invasion ability were evaluated by a two-chamber assay. To assess the effect of PLA2G2A overexpression on cell cycle, a cell cycle assay was performed using a FACSCalibur flow cytometer (BD Biosciences). In order to evaluate the ability of conditional medium to regulate the migration of HUVEC, the HUVEC migration ability was evaluated by a two-chamber assay. At last, we evaluated correlations among overexpression of PLA2G2A β-Catenin, Bcl-2, p65and AKT, western blot was performed to assess their relationship in ovarian cancer to discuss the possible related pathway proteins.Methylation-specific PCR (MS-PCR)Genomic DNA was extracted from epithelial ovarian cancer cells according to the manufacturer’s instructions of DNA extraction kit. Sodium bisulfite treatment of the genomic DNA was performed the CpGenome DNA modification kit according to the manufacturer’s instructions. The locations of CpG sites within the PLA2G2A gene promoter have been described previously. Primers and PCR conditions for MSP are available on request. M standard, U standard and3DW were used as positive and negative control.RESULTSThe mRNA expression levels of all the five types of ovarian cancer cell lines A2780, HO8910, HO8910pm, SKOV3and OVCAR3were very low and almost cannot be detected by RT-PCR. The overexpression of PLA2G2A in transfected HO8910pm cells was confirmed by RT-PCR and western blot analysis. Cells transfected with3.1-PLA2G2A showed significant increased PLA2G2A in both mRNA and protein levels compared with the control empty vector cell lineMTT assay showed that3.1-PLA2G2A inhibited celluar proliferation compared to the3.1-vector cells (P<0.01). Overexpression of PLA2G2A in HO8910pm cells dramatically reduced colony formation efficiency (168±60vs824±97, P<0.01).3.1-PLA2G2A cells showed significant decreased invasive ability in comparison with the control cells (184±22.86vs312±23.55, P<0.01).Similarly, migration of3.1-PLA2G2A cells was greatly decreased compared with the empty vector control cells (126±17.74vs281±38.97,P<0.01).In the cell cycle assay performed using a FACSCalibur flow cytometer, overexpression of PLA2G2A caused a moderately cell cycle arrest in the G1phase in3.1-PLA2G2A cells (66.9±3.6%vs79.1±1.0%, P<0.05).Migration ability of HUVECs accompanied with overexpression of PLA2G2A was obviously decreased than that of empty vector control (165±17.40vs393±51.99, P<0.01).Western blot was performed to assess the relationship between overexpression of PLA2G2A and β-Catenin. It showed that overexpression of PLA2G2A would decrease the expression of phosphorylation of β-Catenin, leading to the accumulation of β-Catenin and consequent Wnt pathway hyperactivation which promoted the expression of downstream PLA2G2A gene. Additional, Bcl-2,p65and AKT were also evaluated the different expression in transfected ovarian cancer cells, but the results didn’t appear obvious differences.A number of potential binding sites were found in sPLA2-IIA promoter region from-260to+20, which included those for signal transducer and activator of transcription. methylation-specific PCR (MSP) was used to verified whether the three CpG sites(-186,-111,-82) were methylated as shown in Jurkat Leukemia Cells, of which site-186is a potential binding site for the y-IRE binding domain, site-111is part of the DNA binding region for NF-Y/CCAAT box-binding factor-82is within the potential Spl and site-82is within the potential Sp1.As shown in figure(9), three CpG sites were all in presence of methylation,but site-82was less methylation to some extent.CONCLUSIONPLA2G2A mRNA expression was decreased in ovarian serous cancer cells, and the methylation in the specific binding site of promoter might be one of the reasons of low expression of PLA2G2A in ovarian cancer. Overexpression of PLA2G2A inhibited proliferation, invasion, migration and induced G1arrest of ovarian cancer cells. It also could decrease migration of HUVEC. All the above processes might be mediated by degrading expression of phosphalation β-Catenin. |
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