Research On RDX, ITGA5 And Their Signal Transduction Pathways In Multidrug Resistance Of Ovarian Cancer

SCREENING NODE PROTEIN IN MULTI-RESISTANT SIGNALING PATHWAYS AND ANALYSIS IT RELATIONSHIP WITH CLINICAL PROGNOSIS IN OVARIAN CANCERObjective: Using bioinformatics method and combining previous research results,screening of signaling pathways key gene associated with multi-resistant in ovarian cancer,and analyze its relationship with ovarian cancer clinical pathology.Methods: Using Pathway Pathway enrichment and GO bioinformatics methods analysis and forecast of potential target genes.Using fluorescence quantitative polymerase chain reaction(PCR)to detect 9 potential genes expression in ovarian epithelial carcinoma.Using immunohistochemical method to detect protein expression in four screening genes,and analyze its clinical pathology with malignant ovarian tumors and multi-resistant.The expression level of RDX and ITGA5 in the tumor tissues with different times of cisplatin intervention were detected by QRT-PCR and WB.Results:(1)Screening nine genes potential associated with ovarian cancer multi-resistant,including ezrin(EZR),radixin(RDX),moesin(MSN),1(PDK1)isozyme,ras homolog family member(RHOA),A SDC1,SDC2,ITGA5,FN1.(2)QRT-PCR detect nine potential drug resistance genes m RNA expression in ovarian cancer tissue,EZR,FN1,MSN,PDK1,RHOA,SDC1 gene are decreased 0.65 times,0.62 times,increased 1.09 times,1.69 times,1.42 times and 1.69 times,respectively,there was no statistically significant difference(P > 0.05).ITGA5,SDC2 and RDX raised 2.03 times,2.38 times and 2.38 times,respectively.(P< 0.05);(3)Immunohistochemistry(IHC)was detected the protein of FN1,ITGA5,RDX and SDC2 in 122 cases of ovarian malignant tumor.The expression level of FN1,ITGA5 and RDX increased successively in platinum drugs resistant ovarian tissue then sensitive ovarian tissue(P?0.05);while the expression of SDC2 decreased orderly(P?0.05)(4)The expression of RDX was correlated with FIGO clinical stage,postoperative residual(P<0.05),The expression of ITGA5 was correlated with postoperative residual(P<0.05),But there was no difference with SDC2,FN1,clinical stages,histological type,cell differentiation,tumor size,lymph nodes,peritoneal metastasis(P?0.05);(5)The cumulate survival rate of patient with RDX,ITGA5 positive expression was significantly lower than that of RDX,ITGA5 negative expression.In multivariate analyse,RDX and histological type involvement remained statistically independent prognostic factors.Conclusion: RDX,SDC2,ITGA5 and FN1 may be multi-resistant signaling pathways regulate gene in ovarian cancer,and RDX,ITGA5 platinum drugs resistant ovarian expression in malignant tumor is significantly higher than platinum drug sensitive ovarian malignant tumors,and has correlation with survival prognosis.THE BIOLOGICAL FUNCTION OF RDX,ITGA5 IN DRUG RESISTANCE CELLS OF OVARIAN CANCERObjective: To construct and identify the lentiviral vector expressing the gene of RDX?ITGA5,and explore the biological effect and the functional pathways after down regulating the RDX?ITGA5 gene on SKOV3-GFP/DDP? cells.Methods: Screening the sh RNA with the best interference effect,transfection with sh RNA was performed according to the protocol to establish the downexpressed stable cell lines.The RDX ? ITGA5 expression level further identified by QRT-PCR)and WB.Detecting the proliferation ability,the IC50 value to cisplatin,the growth curve under different concentrations of cisplatin and different time,and the colony forming ability in 4 groups cells:SKOV3-GFP/DDP?,SKOV3-GFP/DDP?-NC,SKOV3-GFP/DDP?-RDXi,SKOV3-GFP/DDP?-ITGA5 i.Meanwhile,flow cytometry was used to detect cell cycle distribution in different cisplatin concentrations,transwell was used to detect cell migration and invasion.Results: The RDX ? ITGA5 lentiviruses interfere vector was successfully constructed.Compared the SKOV3-GFP/DDP?-RDXi,SKOV3-GFP/DDP?-ITGA5 i group to the other two control groups:1)the SKOV3-GFP/DDP ?-RDXi,SKOV3-GFP/DDP ?-ITGA5 i group showed significantly dcreased growth rate as well as the IC50 values to cisplatin.2)The growth inhibition rate of the experimental group increased,meanwhile the sensitivity to cisplatin was increased after down regulation RDX?ITGA5.3)Cisplatin-induced cell cycle was arrested in G2/M phase,the rate was significantly increased.4)Cells migration and invasion ability decreased obviously.Conclusion: RDX?ITGA5 silencing could reduce the growth ability,migration and invasion of SKOV3-GFP/DDP? cells,Cell cycle arrested in G2/M phase.EFFECT OF RDX,ITGA5 ON APOPTOSIS AND PATHWAYS IN MULTI-RESISTANT EPITHELIAL OVARIAN CANCER SKOV3-GFP/DDP?Objective: To explore the effect of down-regulated RDX,ITGA5 on the apoptosis in cisplatin-resistant SKOV3-GFP/DDP ? cells,so that offering a possible approach for the RDX,ITGA5 to reverse the multi-resistant ovarian cancer,providing a beneficial evidence for clinic.Methods: The four groups cell were acted by different cisplatin concentrations after 48 h,flow cytometry was used to detect cell apoptosis.Using Path Scan ? Antibody Kit,Western blot method to detect four groups(SKOV3-GFP/DDP??SKOV3-GFP/DDP?-NC?SKOV3-GFP/DDP?-RDXi,SKOV3-GFP/DDP?-ITGA5i)of cell signaling pathways key protein expression and change.Results:(1)In SKOV3-GFP/DDP?-RDXi,SKOV3-GFP/DDP?-ITGA5 i cells cisplatin-induced apoptosis rate was increased;(2)Compared with NC group,the protein expression SKOV3GFP/DDP ?-RDXi Smad2 and TAK1 decreased significantly;P44/42MAPK(ERK1/2),Akt,Bad,HSP27,p53,JNK/SAPK,PARP,Caspase 3,Caspase-7,Chk1,Ik Ba,EGFR/Erb B1,HER2 / Erb B2,HER3 / Erb B3,FGFR1,FGFR3,FGFR4,Ins R,Trk A/NTRK1,Trk B/NTRK2,Met/HGFR,Ron/MST1 R,Ret,c-Kit/SCFR,Akt/PKB/Rac and protein expression level rise significantly(P < 0.05);(3)compared with NC group,the protein expression levels of SKOV3 GFP/DDP ?-ITGA5 i EGFR/Erb B1,HER2 / Erb B2,FGFR1,Trk A/NTRK1,ALK,P44/42 MAPK(ERK1/2),Akt,Bad,HSP27,p53,p38 lightning MAPK,JNK/SAPK,PARP,Caspase 3,Caspase-7,Ik Ba,Chk1,Ik Ba,Eph B4 and Akt/PKB/Rac rise significantly;IGF-IR protein expression level significantly lowered(P < 0.05);(4)In the RAS-PI3K-AKT1 pathway,PI3K?MEK1/2 ? Erk1/2 were significantly increased,but Ras was significantly descreased(P < 0.05).Conclusion: RDX,ITGA5 silencing significantly increased cisplatin-induced apoptosis induced by cisplatin in SKOV3-GFP/DDP? cells.Down-regulation of RDX,ITGA5 enhanced the chemosensitivity of cisplatin-resistant ovarian cancer cells by activating caspase and inhibiting the Ras-PI3K-AKT1 pathway,which had provided an important guideline for us to further research in the mechanism of signal transduction pathways in the development of multi-resistant ovarian cancer and for the application in clinic.