Study On The Mechanism Of MiR-429/ZEB1 Axis Regulating The Sensitivity Of Platinum-resistant Ovarian Cancer Cell

Part 1 Explored the Potential Mechanism of ZEB1 Mediating Multi-drug Resistance in Epithelial Ovarian Cancer Based on BioinformaticsObjective: Ovarian cancer is an early insidious disease with poor prognosis and easy resistance to conventional treatment.The reason why easily causing the low survival rate is mainly maybe due to the chemoresistance and the easy invasion and metastasis of epithelial ovarian cancer cells.Mi RNA-429 is an endogenous non-coding small RNA that can be bound to the 3’UTR of target genes and regulate their translation to display its function.ZEB1 plays a critical role in chemotherapy resistance and invasion of tumors,but its potential mechanism in multi-drug resistance of ovarian cancer remains unclear.In this paper,we studied the potential mechanism of ZEB1 affecting mul-tidrug resistance of ovarian cancer through bioinformatics method,providing a new idea for further treatment of ovarian cancer resistance and searching for tumor markers with high sensitivity and specificity.Methods: Firstly,the expression of ZEB1 m RNA in ovarian cancer and normal ovarian epithelium,ovarian drug-resistant tissue and sensitive tissue were analyzed by bioinformatics method.Secondly,Pearson Text was used to detect the relationship between miR-429 and ZEB1/2.Next,bioinformatics was used to search for ZEB1-related ovarian cancer resistance genes and proteins,and KEGG pathway enrichment was performed using David database,and the prognosis of ovarian cancer patients with ZEB1/2 was analyzed by Kaplan Meier Potter database.The correlation between ZEB1 expression level and risk clinical factors was analyzed based on TCGA database,and the influence of pathological factors on the prognosis of ovarian cancer was analyzed by multivariate Cox regression.Finally,the expression of miR-429 and ZEB2 m RNA level in clinical tissues of our hospital(29 cases of drug-resistant samples vs 41 cases of sensitive ones)was retrospectively analyzed.The correlation between miR-429 and ZEB2 m RNA expression levels and clinicopathological factors was analyzed using chi-square test,the prognosis of ovarian cancer was analyzed by the Kaplan Meier method,and independent risk factors were evaluated by univariate Cox proportional risk model.In addition,ROC curve analysis of their significance as multidrug resistance diagnosis of ovarian cancer.Results: The expression of ZEB1 m RNA in OC tissues was higher than that in normal tissues.Results based on TCGA database analysis demonstrated that the expression of miR-429 in drug-resistant samples was lower than that in sensitive ones,and the expression of ZEB1/2 m RNA in drug-resistant ones was higher than that in sensitive,however both all of them being no statistical significance(P >0.05).It was found that miRNA-429/miRNA-200 c was a predictive target miRNA of ZEB1,and ZEB1 was a predictive target gene of miRNA-429/miRNA-200 c.Pearson Text analysis of ovarian data from TCGA database demonstrated that miR-429 was negatively regulated with ZEB1/2.ZEB1/2 also negatively regulated by miR-429(P <0.05).Tissue samples from OC patients(41 cases of platinum-sensitive tissues vs 29 cases of platinumresistant tissues)were analyzed in the Affiliated Tumor Hospital of Guangxi Medical University.QRT-PCR detected the expression of miR429 in drugresistant tissues,which were lower than that in sensitive ones,and the difference was statistically significant(P =0.0002,P <0.05),while the expression of ZEB2,which belongs to the ZEB family,was higher in drug-resistant tissues than in sensitive ones,which did not reach statistical significance(P =0.1635,P >0.05),but the expression trend was consistent with bioinformatics analysis.In addition,we screened 3 candidate genes TIMP3,COMP and PTGER3 moderately associated with ZEB1 for OC resistance and 10 key proteins interacting with ZEB1(AKT1,CD44,FN1,TP53,CDH1,SOX2,Notch1,VEGFA,MYC,GAPDH),KEGG pathway mainly enriched in cancer proteoglycan,cancer pathway,mirco-RNA in cancer,PI3K-Akt signaling pathway,focal adhesion,ECM-receptor interaction,MAPK signal pathway,HIF-1 signal pathway,signal pathway regulating pluripotency of stem cells,RAP1 signal pathway,HIPPO signal pathway,etc.The upregulation of ZEB1/2 m RNA level was significantly associated with the decrease of OS and PFS in ovarian cancer patients.And multivariate Cox regression analysis of TCGA clinical data suggested that advanced age,drug resistance,stage III,stage IV,and complete response(CR)was independent influencing factors for the prognosis of patients.Finally,our clinical follow-up data showed that the upregulation of miR429 was beneficial to the prognosis of ovarian cancer patients,but not statistically significant(OS: P =0.18;PFS: P = 0.157).Ovarian cancer patients with upregulated ZEB2 expression showed a poor prognosis and was statistically significant(OS: P =0.001;PFS: P= 2.677e-04);Mi R-429 expression was associated with ovarian cancer drug resistance and treatment response(P <0.05),drug resistance.ROC curve also showed that miRNA-429 combined with ZEB2(AUC:77.3%,P <0.0001)the accuracy of diagnosis of multidrug resistance in ovarian cancer was higher than that of miR-429(AUC:76.2%,P <0.0001)or ZEB2(AUC: 70.6%,P <0.001).Conclusion: Mi R-429 and ZEB1 play an important role in the occurrence,development and drug resistance of ovarian cancer.In the DNA level,three candidate genes(TIMP3,COMP,PTGER3)moderately associated with ZEB1 resistance to OC were screened.At the m RNA level,there may be a double feedback loop between miR-429 and ZEB1/2,which are negatively regulated.In the protein level,10 key proteins interacting with ZEB1 were screened(AKT1,CD44,FN1,TP53,CDH1,SOX2,Notch1,VEGFA,MYC,GAPDH).During the gene expression process of ZEB1,it may be involved in the multidrug resistance mechanism of ovarian cancer by regulating the above genes,miRNAs and proteins,and ZEB1 may be significantly associated with poor prognosis in ovarian cancer patients.Finally,our clinical follow-up data also showed that miR-429 was associated with drug resistance and treatment response of ovarian cancer.Moreover,ZEB2 has a certain indicator effect on the prognosis of ovarian cancer patients,and MIR429 +ZEB2 combined index has a certain significance in the diagnosis of multidrug resistance of ovarian cancer.Part 2 Biological Effects of ZEB1 on Platinum-resistant Ovarian Cancer CellsObjective: To investigate the changes in the proliferation,invasion,apoptosis and senescence of cisplatin-resistant OC cell lines SKOV3/DDP and A2780/DDP at a certain cisplatinum concentration,so as to clarify the biological role of ZEB1 in ovarian cancer drug resistance.Methods: Targeted cells were infected with lentivirus.The expression of ZEB1 m RNA in SKOV3,SKOV3/DDP,SKOV3/DDP+ LV-si ZEB1,SKOV3/DDP+LV-NC,A2780,A2780/DDP,A2780/DDP+ LV-si ZEB1 and A2780/DDP+LV-NC were detected by q RT-PCR.The IC50 value and proliferation condition of all above cells was detected by CCK8 assay.Transwell assay was applied to detect the invasion ability of the above groups of cells,under cisplatin treatment.Cell apoptosis was detected by flow cytometry.And β-galactosidase kit was utilized to detect cell aging in each group.Results: q RT-PCR showed that the mRNA level of ZEB1 in SKOV3/DDP and A2780/DDP was higher than that in SKOV3 and A2780 cell lines.The expression of ZEB1 m RNA in SKOV3/DDP+ LV-si ZEB1 and A2780/DDP+ LVsi ZEB1 was lower than that in the negative control group and drug-resistant group,respectively.CCK8 assay showed that the IC50 value of SKOV3/DDP+ LVsi ZEB1 group was lower than that of SKOV3/DDP and SKOV3/DDP+LV-NC group,and that of A2780/DDP+ LV-si ZEB1 group was lower than that of the blank group and the negative control group.In addition,proliferation ability of SKOV3/DDP+ LV-si ZEB1 and A2780/DDP+ LV-si ZEB1 was decreased compared with the negative control group under the intervention of cisplatin at a certain concentration.Transwell assay demonstrated that the invasion ability of SKOV3/DDP+ LV-si ZEB1 group was weakened compared with the negative control group and the blank group when treated with cisplatin at a certain concentration.And the results of flow cytometry suggested that the apoptosis of SKOV3/DDP+ LV-si ZEB1 and A2780/DDP+ LV-si ZEB1 group was enhanced compared with the negative control group under the intervention of certain cisplatin concentration.In addition,the results of β-galactosidase kit revealed that the senescence condition of A2780/DDP+ LV-SIZEB1 was improved compared with the negative control group.Conclusion: Downregulation of ZEB1 at a certain concentration of cisplatin can inhibit proliferation,invasion and other biological processes of drug-resistant ovarian cancer cells,and promote apoptosis and senescence.Therefore,ZEB1 may affect the sensitivity of ovarian cancer cisplatin-resistant cells to cisplatin drugs by regulating the above biological effects.Part 3 Mi R-429/ZEB1 Axis Regulation of ferroptosis in Platinum-resistant Ovarian Epithelial Cancer CellsObjective: miRNA-429 is an endogenous non-coding small RNA that regulates target genes after transcription and is involved in the occurrence,development,migration,invasion and drug resistance of ovarian cancer.Zinc finger E-box homologous binding protein 1(ZEB1),a member of the ZEB protein family,plays a major role in epithelial-mesenchymal transition,DNA repair,stem cell biology and tumor-induced immunosuppression.In recent years,a newly discovered non-apoptotic form of cell death characterized by iron-dependent aggregation of lethal lipid reactive oxygen species is called ferroptosis.The use of drugs to induce it of ovarian cancer cells has become a new direction of ovarian cancer treatment,but the mechanism has not been clarified yet.Relevant literature predicted that ZEB1 would be involved in the construction of ferroptosis model of ovarian cancer,but its function and mechanism in ferroptosis of ovarian cancer cells have not been reported.The former studies have confirmed that ZEB1 is the target gene of miRNA-429,and ZEB1 can be negatively regulated by miR429.This study aims to explore the biological function and mechanism of the miR429/ZEB1 axis in the ferroptosis model of platinum-resistant ovarian epithelial cancer cells,so as to provide a new idea of thinking for the study of drug resistance mechanism of ovarian cancer.Methods: CCK-8 method,Gemissa staining,flow cytometry and Western Blot were used to verify the successful establishment of ferroptosis model of platinum-resistant ovarian cancer cells.The regulation effect of over-expression of 429 and interference with ZEB1 on ferroptosis of platinum-resistant ovarian cancer was detected by CCK-8 method,Gimassa staining and flow cytometry.Results: In this study,we first demonstrated that Erastin induced ferroptosis in platinum-resistant EOC cells of SKOV3/DDP and A2780/DDP.Results of Gemusa staining revealed that the whole cell induced by Erastin had cytoplasmic shrinkage,being like spherical.Flow cytometry suggested that Erastin can induce the production of lipid ROS associated with intracellular iron death,however,which was mitigated by Ferrostatin-1,an iron death inhibitor.Meanwhile,during Erastin induction,the expression of ferroptosis related protein GPX4 was downregulated with the increase of dose.Therefore,the morphological,biological and proteomics results are in line with the characteristics of ferroptosis.Secondly,on the basis of the former study,we demonstrated that miR-429 can accelerate the iron death process of platinum-resistant ovarian epithelial cancer cells induced by Erastin by CCK-8 and Gimsa staining experiments.Owing to we have previously confirmed that zinc finger E-box homologous binding protein 1(ZEB1)is the target gene of miRNA-429,which can be downregulated by miR429.Furthermore,we found that zinc finger E-box homologous binding protein 1(ZEB1),through which lenti-cirus mediated down-regulation of it,can enhance the occurrence of ferroptosis in platinum resistant ovarian epithelial cancer cells induced by Erastin using CCK-8 and Gemusa staining assay.Conclusion: The ferroptosis model of platinum-resistant ovarian epithelial cancer cells was successfully constructed.Based on the iron death of platinumresistant ovarian epithelial cancer cells,the promoting effect of miR-429 on ferroptosis of platinum-resistant ovarian epithelial cancer cells was proved.It is suggested that miR-429 promotes ferroptosis process induced by Erastin in platinum-resistant ovarian cancer cells could be through a novel mechanism of down-regulation of ZEB1,which provides a new strategy and the theoretical basis for the treatment of drug resistance in ovarian cancer cells.