MIR-429 Associated With Drug Resistance Of Epithelial Ovarianc Cancer

Chapter1 Research progress of miRNA and multidrug resistance in epithelial Epithelial ovarian cancerEpithelial ovarian cancer is a primary gynecological tumor of the ovarian is the fourth most common cause of death for women in the world. Currently the standard treatment protocol used in the initial management of OC is primary, followed by a platinum and taxane combination chemotherapy. But drug resistance is an obstacle to the treatment of epithelial ovarian cancer. Understanding the molecular dysregulation underlying chemoresistance is important for enhancing therapeutic outcome. Previous studies have shown that multidrug resistance in epithelial ovarian cancer is related to various factors. This chapter is summarized about the miRNA in ovarian epithelial carcinoma and its role in the development and molecular mechanism of multi-drug resistance.Chapter 2 Analysis of microarrays identified genes and miRNAs associated with drug resistance in epithelial ovarian cancerObjective:The aim of this study was to excavate the potential miRNAs and genes associated with drug resistance in epithelial ovarian cancer through microarray expression profiling data analysis and bioinformatics approaches.Methods:MiRNA microarray profile GS54665 and gene expression profile GSE33482, GSE28646, GSE15372 were downloaded from Gene Expression Omnibus database and the dysregulated mirRNAs/genes were screened with the GE02R. Subsequently, the correlation between the differentially expressed miRNAs/genes and the regulation of drug resistance in epithelial ovarian cancer was investigated through comprehensive bioinformatic methods, including pathway enrichment, biological process annotation, text mining, protein-protein interaction analysis and mRNA-miRNA interaction analysis and then miRNAs and genes were further identified in cisplatin-resistant SKOV3（SKOV3/DDP） and A2780 （A2780/DDP） cells by Real-time quantitative PCR（QRT-QPCR）.Results:1 One miRNA expression microarray dataset and three mRNA expression microarray datasets that were standards-compliant and related to drug resistance in epithelial ovarian cancer were obtained from the GEO database. Through analysis of the microarray data, we found nine differentially expressed miRNAs and 38 genes, including seven mRNA that exhibited exactly the same expression trends in all three sets of microarrays.2 Pathway enrichment analysis of the nine miRNAs identified multiple signaling pathways associated with drug resistance in epithelial ovarian cancer, including focal adhesion, the phosphatidylinositol 3-kinase （PI3K）-Akt signaling pathway, the avian erythroblastosis oncogene B （ErbB） signaling pathway, and the mitogen-activated protein kinase （MAPK） signaling pathway. Biological process annotation and pathway enrichment analysis showed that the above-described 38 genes were significantly related to the pathways regulating drug resistance in epithelial ovarian cancer, such as cytokine-cytokine receptor interaction pathways, cell adhesion and immunomodulation.3 In addition, miRNA-mRNA interaction analysis revealed the existence of a targeted regulatory relationship between the nine miRNAs and most of the 38 genes. Overall, the results demonstrated the correlation between the differentially expressed miRNAs/genes identified in the present study and the regulation of drug resistance in epithelial ovarian cancer.4 In the following QRT-PCR experiment, the relative expression levels of almost all the nine mirRNAs in the SKOV3（SKOV3/DDP） or A2780 （A2780/DDP） cells were coincided with the chips. And the relative expression levels of the seven genes（namely NHS-like 1 （NHSL1）, ephrin type-A receptor 3 （EPHA3）, synuclein alpha （SNCA）, ubiquitin specific peptidase 51 （USP51）, zinc finger and SCAN domain-containing protein 4 （ZSCAN4）, ephrin type-A receptor 7 （EPHA7） and peptidase inhibitor 15 （PI15）） in the A2780 （A2780/DDP） cells were coincided with the chips.5 MiR-141 is one of the members of the miR-200 family, EPHA7 and PI15 were predicted as its potential target genes, the expression level of miRNA-141 was negatively correlated with that of EPHA7 and PI15. Conclusions:1. We found 9 miRNAs and 38 mRNA that associated with drug resistance of epithelial ovarian cancer through analyzing microarray data, including seven mRNA that exhibited exactly the same expression trends in all three sets of microarrays.2. The expression level of miRNA-141 was negatively correlated with the expression levels of EPHA7 and PI15. miRNA-141, EPHA7 and PI15 may jointly participate in the regulation of drug resistance in epithelial ovarian cancer and serve as potential targets in targeted therapies for epithelial ovarian cancerChapter 3 Preliminary analysis of lncRNA expression profile related to multidrug resistant ovarian epithelial cancer and ceRNA screening that related to miRNAChemotherapy is the preferred therapetic approach for the therapy of advanced epithelial ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs in cancers. LncRNAs （Long non-coding RNA）, accounting for a major propotion of mamalian transcriptomes, are generally considered as non-protein coding transcripts longer than 200 nucleotides. RNA polymerase II is responsible for the transcription of most lncRNAs. Due to the absence of effective "Open Reading Frame", lncRNAs barely encode any proteins. LncRNAs modulate various aspects of cellular regulation thus playing important roles in epigenetics, transcriptional regulation of gene expression. LncRNAs have been reported to play critical regulatory roles in variety of disease, especially in neurodegenerative disease and cancer, but arely associated with drug resistance.Objective:To analyse the multidrug resistance of epithelial ovarian cancer associated lncRNAs and genes and obtain a primary list of multidrug resistance associated lncRNAs and genes, with the aim to provide resources for clarifying the mechanism of multidrug resistance and identifying effective molecular targets for multidrug resistance of epithelial ovarian cancer.Methods:1. The differentially expressed lncRNAs and genes between the drug-resistant tissues and drug-sensitive tissues of the epithelial ovarian cancer were identified by the lncRNA expression profiling using lncRNA microarray for 5 samples respectively.2. The co-expressed mRNAs for each differentialed lncRNAs were analysed using Pearson correlation with r （correlation）>0.7, p<0.05.Then we conduct a functional enrichment analsyis of this set of co-expressed mRNAs by Hypergeometric cumulative distribution function with FDR<0.01, P<0.05.3. We screened out the lncRNA with ceRNA regulation through ceRNA_score analyse, then we analysised the target genes by GO enrichment analysis and Pathway bioinformatics analysis; and we also screening the ceRNA_netword related to the miR-200 family.4. For each lncRNA, we searched the mRNAs loci that were within 300 k windows up- and downstream of the given lncRNA,and with the Pearson correlation of lncRNA-mRNA expression is significant （P<0.01）, and the mRNAs were identified as "cis-regulated mRNAs".5. we calculated the overlap of co-expressed mRNA set of given lncRNAs with transcriptional factor target genes and calculated the signifiant by using hypergeometric distribution. If co-expressed mRNAs significantly overlaped with target genes of given TF, suggests that this TF might interact with given lncRNAs. We use transcriptional factor target set identifed by Encyclopedia of DNA Elements （ENCODE）.Results:1. Of the 738 dysregulated expression lncRNA detected in drug-resistant tissues when compared to drug-sensitive tissues,311 lncRNA were up-regulated and 427 genes were down-regulated （fold change≥2 p≤0.05）. While NONHSAT076042 （Fold change:8.95） was the largest up-regulated expression lncRNA, TCONS₁2₀0021133 （Fold change:7.29） was the largest down-regulated expression lncRNA;At the same time we also detected 983 dysregulated mRNA, with 678 mRNA were up-regulated and 308 genes were down-regulated （fold change≥2 p≤0.05）, of which SFRP2 （Fold change:28.48） is the largest up-regulated mRNA, LDLRAD1 （Foldchange:16.44） is the largest down-regulated mRNA.2. We calculated the co-expression mRNA of each differentialed lncRNA by the Pearson Correlation with r（correlation）>0.7, p<0.05 and listed out the top 300 for up-and down-regulation respectively, then we used Hypergeometric cumulative distribution function to conducte the enrichment of functional terms of this set of co-expressed mRNAs, and listed out top500. The reslults showed that the up-regulated lncRNA were mainly consistant with leukocyte transendothelial migration, extracellular matrix, DNA packaging, focal adhesion, ECM-receptor interaction and so on （p<0.05）;while the down-regulated lncRNA were major related with extracellular matrix, cytoskeleton, DNA packaging, focal adhesion, ECM-receptor interaction and so on （p<0.05）.3. We then screened out the lncRNA through ceRNA_network analyse, and found 25 lncRNAs with 335 ceRNA_crosstalk, and we also found 5 ceRNAs of miR-200 famaliy. And the lncRNA with ceRNA function mainly involved in biological processes and KEGG pathways as follows:cell adhesion, negative regulation of NIK/NF-kappaB signaling, regulaion of mesenchymal cell proliferation, Apoptosis, Ras signal pathway, ErbB signaling pathways etc.4. cis regulation analysis showed there were 64 of lncRNA with "cis-regulated mRNAs".5. Through trans analysis, we identified the target genes of lncRNAs and transcription factors（IF） that auxiliary regulate target gene transcription, and we screened 29 transcription factors that constitute the lncRNA control network, including E2F4, CTCF and so on. Described in this regulation network, the target gene expression with lncRNA significantly, and they were the target genes of transcription factors.Conclusions:1. We obtained a significant number of commonly dysregulated lncRNA and mRNAs by comparing the lncRNA and mRNA expression profiles of between the five drug-resistant samples and drug-sensitive amples. We further integrated the lncRNA and mRNA profiling results, followed by the construction of a primary multidrug resistance network and identification of functional lncRNA-mRNA pairs that may be involved in multidrug resistance of epithelial ovarian cancer.2. The lncRNAs that associated with multidrug resistance of epithelial ovarian cancer may be involved in through the cis adjacent genes, transcription factors effect on target genes regulated by trans, or by ceRNA network with mirRNA, or by participating in a variety of signaling pathways and biological processes involved in epithelial ovarian cancer drug resistance. 4.3 There were 5 lncRNAs （NONHSAT059750 NONHSAT129265 NONHSAT128169 ENST00000529924 TCONS₁2₀0003421） had ceRNA function with miR-429 and its target genes.Chapter 4 Biological function study of miR-429 in the epithelial ovarian cancer SKOV3/DDP cell in vitroObjective:Epithelial ovarian cancer（EOC） is gynecological tumor with the highest mortality rates of the malignant tumor, drug resistance is one of the most serious problems in the process of tumor therapy. Tumor drug resistance is associated with a variety of factors, miRNA may be closely related to drug resistance. MiR-429 belongs to miR-200 family, miR-200 family is associated with a variety of tumor drug resistance. In this part, we will verify the miR-429 impact on the biology function of ovarian cancer cell SKOV3/DDP through the experiments in vitroMethods:In our study, transfection with miRNAs was performed according to the protocol to establish the overexpressed stable cell lines （SK/DDP-429 or SK/DDP-NC, respectively）. The miR-429 expression level further identified by real-time quantitative PCR （qRT-PCR）. Cell viability and IC50 values （drug concentration causing 50% inhibition of cell growth）were analyzed with the. CCK8 assay. Apoptosis was analysied with Flow cytometry.Results:1 The 50% inhibitory concentration （IC50） of cisplatin in the SKOV3/DDP cells was about 2.5-fold than the parental SKOV3 cells. Quantitative real-time PCR revealed that miR-429 was significantly downregulated in SKOV3/DDP cells compared with parental SKOV3 cells.2 The level of miR-429 was increased significantly in cells transfected with LV-miR-429 overexpression lentivirus compared with the negative control（LV-miR-NC）. We found that the IC50 of cisplatin was decreased in cells that had been transfected with LV-miR-429 compared with the negative control （LV-miR-NC）.3 The effect of miR-429 on the viability of ovarian cancer cells was detected by the CCK8 assay.The cell proliferation was significantly inhibited at 48 h after recombinant lentivirus LV-miR-429 infection （P<0.05） The ability of colony formation of the SKOV3/DDP cells transfected with miR-429 cells than decreased obviously （P<0.05）.4. The FCM analysis showed that the apoptosis rate miR-429 group of SKOV3/DDP cells was significantly higher than the other two groups of cells （P<0.05）.5. We used Western blots to determine the effect of miR-429 on levels of endogenous autophagy protein, and we found that the protein leves of Atg-7 and LC3A/B were decreased significantly. Conclusions:1. Transfect miR-429 into the SKOV3/DDP cells Contrast the proliferation ability of SKOV3/DDP cell between before and after transfection; and increase the sensitive of the SKOV3/DDP cells to cis-platinum.2. Over-expressing miR-429 may suppress the autophagy pathway and increase the sensitive to cis-platinum of ovarian cancer.Chapter 5 The verification of the target genes of miR-429 and Dual-Luciferase report system authentication target genes ZEB1 （NM₀30751） binding sitesObjective:Epithelial ovarian cancer（EOC） is one of the most major malignancies of the female reproductive system. The mortality rate of epithelial ovarian cancer is the highest among all gynecological malignancies, and the 5-year overall survival of the patiens is only 30%. So far although initial is successful for 80-90% of patients will have a complete clinical response to this initial treatment, but unfortunately, the majority of these responders will eventually develop platinum-resistant. Several studies have showed that miRNAs regulate the biology of cancer cells including their multiple drug resistance, by regulating the expression of target genes involved in signaling pathways, to form a complicated and huge regulatory networks.Methods:In our study, transfection with miRNAs was performed according to the protocol to establish the overexpressed stable cell lines （SK/DDP-429 or SK/DDP-NC, respectively）. we predict target gene of miR-429 by biological software including miRBase、TargetScan databases and verified the protein expression of target genes of the cells after transfected miR-429 by Western Blot. Finally through the dual luciferase report system to validate ZEB1 exist on the role of miR-429 targets.Results:1. We predict target gene of miR-429 by biological software and choose ZEB1 as the target gene. We found that the ZEB1 protein expression level of miR-429 group was lower than the other two groups by Western Blot.2. The dual luciferase reporter vectors containing 3’UTR of ZEB1 were constructed by using Dual-Luciferase Reporter Assay System, and then the relative activity of firefly luciferase was detected to confirm the binding site of miR-429 on ZEB1.Conclusions:1. ZEB1 is the target gene of miR-429, and overexpression of miR-429 could rerpessed the ZEB1 expression.2. miR-429 could regulate ZEB1 may mainly depend on the combination with base pairs of the first prediction site area （369-376）.