Overexpressed CRABP2 Genes In Platinum Resistance Reverse Ovarian Cancer And Influence The Tumor Microenvironment;Preliminary Explore The 3D Co-Culture Ovarian Cancer Cell

Objective On the basis of our previous studies, we has been established SKOV3 GFP/DDP Ⅱ by acquired cisplatin resistance ovarian cancer cell, then successfully constructed CRABP2 overexpression of cisplatin resistance ovarian cancer cell SKOV3 GFP/DDP Ⅱ-CRABP2. We detect the cytokines of CRABP2 overexpression in ovarian cancer microenvironment influence and reverse ovarian cancer cells of platinum resistance CRABP2 gene expression validation in animal models, explore CRABP2 overexpression on downstream signaling pathways related to drug resistance. To establish 3D co-culture model will be better simulated the growth of ovarian cancer cells microenvironment, to make a preliminary study of the 3D cocultivation method, Look for the reverse ovarian cancer cisplatin resistance of foundation for new targets.Methods We had been successfully established the early stage of the research of acquired cisplatin-resistant ovarian cancer cell SKOV3 GFP/DDP Ⅱ and cisplatin sensitive cell SKOV3-GFP, planting subcutaneously in nude mice after cisplatin intervention by several times. Cytokines expression was dynamicly detected by real-time PCR. Using SKOV3 GFP/DDP II CRABP2 and control SKOV3-GFP/DDP II-Mcherry, planting subcutaneously in nude mice after cisplatin intervention by several times. Western Blot detected the CRABP2 overexpression transplanted tumor on Fas apoptosis signaling pathways downstream of node gene protein expression and cell cycle regulation related protein expression. At the same time the real-time PCR detected the dynamic expression of cytokines. People mentum samples, was separateed and purifed to get mesothelial cells and fibroblasts. Using Transwell Chambers, mesothelial cells, fibroblasts and SKOV3/DDPⅢ structured 3D co-culture model,fibroblasts at the bottom, mesothelial cells between the middle,SKOV3/ DDP Ⅲ on the upper. Using the method of Clarity embedded into to fat transparent high permeability of the three-dimensional model, using immunofluorescence observated 3D co-culture model imaging.Result QRT-PCR results showed that:after cisplatin injected there were 22 cytokines that sensitive group expression was higher than the resistance. After the sensitive group injected cisplatin there were 5 cytokines which the expression was lower than the resistance. In CRABP2 overexpression and its control group, after cisplatin injected, there were 20 cytokines that control expression was lower than CRABP2 overexpression group. After cisplatin injected there were 7 cytokines that the control group was higher than that of CRABP2 overexpression group. After cisplatin injected there were 4 cytokines that sensitive group expression was lower than the resistance, the same as the CRABP2 overexpression, the expression is higher than the controls group. There were 19 cytokines that the sensitive group expression was higher than the resistance, the same as overexpression CRABP2.In a word, it was influencing by the tumor microenvironment to reverse the platinum resistance of ovarian cancer.SKOV3 GFP/DDP Ⅱ and its control group SKOV3-GFP/DDP Ⅱ McHerry were subcutaneous transplantation tumor cell of nude mice, SKOV3 GFP/DDP Ⅱ-CRABP2 in nude mice subcutaneous transplantation tumor cell cycle was suppressed, apoptotic pathways were activated:the dependence of cell cycle protein kinase CDK2, CDK4, CDK6 and cyclin D1 expression in SKOV3 GFP/DDP Ⅱ-CRABP2 transplanted tumor was significantly reduced (P< 0.05), and p27 kip1 protein expression was enhanced (P< 0.05); Fas apoptosis signaling pathway genes of nodes on the Fas, FADD and Caspase9 and Caspase3 protein expression were increased (P< 0.05), while apoptosis signaling pathways downstream of DNA repair genes PARP1 protein expression was lower (P< 0.05), namely CRABP2 gene reversed cisplatin resistance of ovarian cancer cells restored sensitivity to cisplatin.3D co-culture model immunofluorescence was observated that in the upper was SKOV3/DDP III, intermediate was mesothelial cells, bottom was matrix mixed fibroblasts.Conclusion CRABP2 Overexpression can make the resistance of ovarian cancer cells to restore their sensitivity to cisplatin, affect the tumor microenvironment inhibiting cell cycle, activate the Fas apoptosis signaling pathways to reverse the effect of ovarian cancer drug resistance. Platinum resistance is reversed ovarian cancer clinical treatment provides a new target and theoretical basis. Using 3D co-culture model can simulate the growth of ovarian cancer cells microenvironment to study the influence of the tumor microenvironment of platinum resistance.