The Effect Of Matrix Metalloproteinase-2Gene Knocking Down On The Metastasis Ability And Angiogenesis Of Ovarian Cancer Cells

BackgroundOwing to the paucity of symptoms and their insidious onset, most ovarian cancerpatients present with advanced disease. Although surgical cytoreduction followed byplatinum-based chemotherapy is the standard treatment for these patients,, the5-yearsurvival rate remains only around30%. Thus, novel approaches to improve diseaseoutcome are urgently needed. Invasion and metastasis is the principal cause of morta-lity. Previous studies indicated that matrix metalloproteinases2(MMP-2) play impor-tant roles in the process of ovarian cancer invasion and metastasis. MMP-2coulddegrade basement membrane components, result in tumor cell migration and releaseangiogenic factors, including VEGF, which modulate angiogenesis of tumor. MMP-2and VEGF have been associated with increased tumor spread and poor prognosis inovarian cancer. In recent years, studies suggested that the knocking down of MMPsgene could inhibited metastasis and angiogenesis in many tumors. The agent againstVEGF ligands, Bevacizumab, have been admitted to advanced ovarian cancertreatment. However, RNA interference technology, a potent gene silencing strategy,has not been used to study in VEGF expression, metastasis and angiogenesis of ovarian cancer after knocking down of MMP-2gene. Here, we will study about this.ObjectiveTo investigate the effect and potential mechanism of MMP-2gene on the humanovarian cancer cells, we suppressed MMP-2expression with RNA interference andthen observed the changes on the growth, adhesion, invasion and migration of thehuman ovarian cancer cell line OVCAR-3, and detected the ovarain cancer cell-induced angiogenesis after RNAi in vitro.Methods1. The culture of human ovarian cancer cell line OVCAR-3and human umbilical veinendothelial cells (HUVECs): OVCAR-3cells and HUVECs cells were cultured inDulbecco’s Modified Eagle’s Medium (DMEM) supplemented with10%fetal bovineserum(FBS),100units/mL penicillin and100units/mL streptomycin. Cells wereincubated at37℃in a humidified5%CO2atmosphere.2. The designation and synthesis of siRNAs targeted MMP-2gene: three siRNAs targetedMMP-2gene and one scrambled siRNA were designed and synthesized. When tranfec-ting the equal amount of DMEM was used as blank control group. Using Real-Timequantitative PCR technique to detect the expression of MMP-2mRNA, and identifythe siRNA for the best effect of gene silencing，and use it in the following study.3. After transfection with the optimal siRNA, we detected the expression of mRNA andprotein of MMP-2and VEGF at diferent time by Real-Time quantitative PCRtechnology and Western Blot analysis.4. Methyl-thiazolyl tetrazolium (MTT) assay was conducted to detect OVCAR-3cellssurvival rate after MMP-2gene knocked down.5. Adhesion assay was performed to assess the adhesive ability at60min and90min ofOVCAR-3cells after the knock-down of MMP-2gene, and calculated inhibition ratio.6. Cell invasion assay was performed using Transwell chambers to detect the invasionability of OVCAR-3cells after the knock-down of MMP-2gene.7. Wound healing assay was carried out to determine the cell protrusion and migrationability of OVCAR-3cells after the knock-down of MMP-2gene. 8. In vitro angiogenic assay was used to detect the degree of angiogenesis ability ofHUVECs induced by OVCAR-3cells. The formation of tube-like structures wasobserved and measure the total tube length and branches per field.9. Statistical Analysis: Software SPSS13.0was used for all statistical evaluation. All datawere expressed as mean±standard error.Data were analyzed for statistical significanceby using one-way ANOVA. P <0.05was considered statistically significant.Results1. The expression of MMP-2was inhibited after transfection and the inhibitory effects indifferent sequence varied. The most efficient inhibitory rate was77.8%. This optimalsiRNA was used in the following assaies.2. By contrast to the NC group, MMP-2mRNA expression was decreased by73.8%,78.8%and78.4%in24h,48h and72h after transfection, the changes were statisticallysignificant, P <0.01. During the experimental groups, the changes only between48hand72h groups were not significant(P=0.654). While the VEGF mRNA expressionwas decreased by75.5%in48h after transfection.Such change was statisticallysignificant, P <0.01.3. By contrast to the NC group, MMP-2protein expression was decreased by72.6%,81.2%and76.4%in24h,48h and72h after transfection, the changes were statisticallysignificant, P <0.01. Between the experimental groups, the changes all significantlyP<0.01. While the VEGF protein expression was decreased by78.3%in48hr aftertransfection. Such change was statistically significant, P <0.01.4. MTT assay demonstrated that the knock-down of MMP-2gene by RNAi did not causeany significant inhibitory effect in tumor cell growth.5. Cell adhesion assay in vitro revealed that adhesiive ability of OVCAR-3cell signific-ant decreases at60min and90min. The inhibition ratio were42.1%and50.4%respectively (P <0.01).6. Cell invasion assay shown that the average OVCAR-3cell number of infiltratedtranswell membranemigrated in experimental group(99.2±8.3) were observed to besignificantly lower than the number of negative control group (141.2±22.7) and blankcontrol groups (140.1±24.1)(P <0.01). 7. Wound healing assay in vitro shown that the mobility of OVCAR-3cells wassignificantly inhibited after RNAi against MMP-2gene. The cells of experimentalgroup migrating into the wounding region was120±14μm, much less than that ofnegative group (187±23μm) and blank group (186±17μm), such changes weretatistically significant, P <0.01.8. In vitro angiogenic assay shown that the ability of capillary network formation ofHUVECs induced by tumor conditioned medium was significantly reduced after RNAiagainst MMP-2gene in OVCAR-3cells. Compare with the negative group, the totaltube length and branch number of experimental group were reduced52.9%and47.5%respectively. Such changes were statistically significant, P <0.01.Conclusion1. RNAi against MMP-2successfully inhibited the mRNA and protein expression ofMMP-2in the ovarian cancer cell line, OVCAR-3, leading to a potent suppression oftumor cell adhesion invasion and migration without affecting cell growth.2. RNAi targeting MMP-2could inhibited ovarian cancer cell-induced tube formation ofendothelial cells in vitro; MMP-2transcriptional suppression decreased VEGF inovarian cancer cells. These data suggest that MMP-2might mediated VEGFexpression and modulated angiogenesis of ovarian cancer.