| Objectives: Epithelial ovarian cancer(EOC)is the most lethal malignant tumor in female reproductive system.The standard treatment is cytoreductive surgery combined with platinum-based first-line chemotherapies.Paclitaxel is the first-line chemotherapy drug for EOC and resistance for paclitaxel is an important cause of relapse and death,but the mechanism has not been fully elucidated.In order to explore the mechanism of paclitaxel resistance,we used siRNA library to screen paclitaxel resistant epithelial ovarian cancer cells quickly,and used the cell model to explore the new mechanism of paclitaxel resistance through high-throughput sequencing,data mining and other means,in order to seek the new therapeutic targets.Methods: Molab Laboratory of University of Chicago Medical Center in the United States have successfully constructed the siRNA library of pSOK-N29,and have verified it's diversity and coverage,which can cover the whole human genome.We packaged the library plasmid into retrovirus,infected paclitaxel sensitive epithelial ovarian cancer cell line OVCAR8,and then established the drug-resistant cell model through lethal dose paclitaxel screening and verify it's drug-resistant phenotype.In order to find enrichment fragments for further study,the genomic DNA of drug-resistant cells with different concentrations of paclitaxel was extracted,and the 29 base fragments were amplified specifically for sequencing on Illumina Hi Seq platform.Tq-PCR was used to detect the expression of drug resistance related genes in drug-resistant cells,and abnormal activation of MAPK signaling pathway was found.In order to explore the mechanism of drug resistance,we tested the therapeutic effect of paclitaxel combined with vemurafenib,a BRAF inhibitor inMAPK signaling pathway,on drug-resistant ovarian cancer cells in vivo and in vitro.Data mining was used to predict and screen 11 genes which were related to paclitaxel resistance.Tq-PCR technology was then used to detect the m RNA expression level of the above genes in parental,resistant cells and drug-resistant animal tissues,and the expression level of IL13RA1 was increased in both cells and tissues.After further silence IL13RA1,we explored it's paclitaxel resistance phenotype and mechanism on epithelial ovarian cancer in vitro and in vivo.Results:1.Under the 2% serum cultured,the lethal dose of paclitaxel on OVCAR8 was 20 nm.2.Using lethal dose of paclitaxel 20 nm and BSD for double screening,and the drug-resistant cells were successfully obtained,then gradually increased paclitaxel dose.Compared with the parent cell OVCAR8,the chemoresistance of resistant cell line OVCAR8-N29 increased 200 times.3.Compared with the parental cells,the resistant cells were more resistant to paclitaxel,and had stronger migration and proliferation ability.After treated with paclitaxel,the G2 / M phase ratio and apoptosis rate of were decreased in vitro.4.Tq-PCR was used to detect the expression level of drug resistance related genes of OVCAR8 and OVCAR8-N29 after DMSO and paclitaxel treatment.Compared with parental cell line,the expression levels of MDR1,VIM,TIMP1,BRAF,ULK1,p53 and Bcl-2 genes were significantly increased,while the expression levels of CDH1,ZEB1,PTEN,PI3 KCA and Vps34 were significantly decreased in resistant cells treated with paclitaxel.5.The genomic DNA of drug-resistant cell lines was extracted after screening with different concentrations of paclitaxel and the 29 base fragments were amplified specifically for sequencing on Illumina Hi Seq platform.6.In vitro experiments showed that paclitaxel combined with vemurafenib could increase the sensitivity of resistant ovarian cancer cells OVCAR8-N29 and Hey A8-MDR to paclitaxel,and the apoptosis rate of drug-resistant cells increased significantly.7.Animal experiment indicated that the tumor proliferation rate and tumor quality of the drug-resistant cell line Hey A8-MDR treated with paclitaxel and vemurafenib were lower than those of other groups.8.Data mining predicted 11 genes related to paclitaxel resistance:LAMB1,FAM114A1,CUEDC 1,TM4SF1,PPIC,SNX7,IL13RA1,BRCA1,SLC35F5,TNFRSF12 A,TRNP1.The expression level of IL13RA1 gene was significantly increased in cell and animal tumor tissues by Tq-PCR.9.After IL13RA1 was silenced,crystal violet staining and WST-1cytotoxicity experiments showed that the drug-resistant ovarian cancer cells OVCAR8-N29 and Hey A8-MDR were more sensitive to paclitaxel,and cell cycle analysis showed that the G2 / M phase ratio of Hey A8-MDR cells increased,apoptosis analysis showed that apoptosis rate increased significantly in OVCAR8-N29 and Hey A8-MDR cells.At the same time,scratch test and WST-1 proliferation test showed that the migration ability and proliferation ability of OVCAR8 and Hey A8 cells decreased significantly after silencing IL13RA1.10.In vivo,after silencing IL13RA1,the tumorigenicity and proliferation activity of drug-resistant Hey A8-MDR cells decreased,while the weight of tumor mass for Hey A8 cells decreased compared with the control group.11.For resistance mechanism test we found that after IL13RA1 was silenced,the m RNA expression level of STAT6 and p53 decreased,and14-3-3 increased in resistant cells compared with the parental cells.Conclusions : In this study,the retroviruses containing pSOK-N29 siRNA library were used to transfect the paclitaxel sensitive ovarian cancer cell OVCAR8.Then the lethal dose of paclitaxel was used for rapid screening and establishing resistant cell line.Crystal violet staining,WST-1assay,flow cytometry,apoptosis analysis were used to verify the resistance phenotype of the resistant cell and Tq-PCR found that MDR1 overexpression,abnormal activation of MAPK / ERK signaling pathway,EMT,IL13RA1 involved JAK/STAT6 signaling pathway which may regulate downstream apoptosis and cell cycle may be involved in the paclitaxel resistant mechanism.Vemurafenib combined with paclitaxel has synergistic effect on the treatment of resistant epithelial ovarian cancer. |
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