Oncogenic microRNA-181d binding to OGT contributes to resistance of ovarian cancer cells to cisplatin.
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ABSTRACT: Ovarian cancer (OC), a common gynecological cancer, is characterized by a high malignant potential. MicroRNAs (miRNAs or miRs) have been associated with the chemo- or radiotherapeutic resistance of human malignancies. Herein, the current study set out to explore the regulatory mechanism of miR-181d involved in the cisplatin (DDP) resistance of OC cells. Firstly, in-situ hybridization method was performed to identify miR-181d expression in ovarian tissues of DDP-resistant or DDP-sensitive patients. In addition, miR-181d expression in A2780 cells and A2780/DDP cell lines was determined by RT-qPCR. Gain- and loss-of-function experiments were then performed to characterize the effect of miR-181d on OC cell behaviors. We probed the miR-181d affinity to OGT, as well as the downstream glycosylation of KEAP1 and ubiquitination of NRF2. Further, in vivo experiments were performed to define the role of miR-181d in tumor resistance to DDP. miR-181d was highly expressed in the ovarian tissues of DDP-resistant patients and the A2780/DDP cell line. Ectopic expression of miR-181d augmented DDP resistance in OC cells. In addition, miR-181d was found to target the 3'UTR of OGT mRNA, and negatively regulate the OGT expression. Mechanistic results indicated that OGT repressed NRF2 expression through glycosylation of KEAP1, thereby inhibiting the DDP resistance of OC cells. Furthermore, miR-181d negatively orchestrated the OGT/KEAP1/NRF2 axis to enhance the OC resistance to DDP in vivo. Overall, these findings suggest that miR-181d-mediated OGT inhibition restricts the glycosylation of KEAP1, and then reduces the ubiquitination and degradation of NRF2, leading to DDP resistance of OC. This study provides new insights for prevention and control of OC.
Project description:This study is to investigate transcription factors involved in cisplatin resistance in ovarian cancer cells. The transcriptome of cisplatin resistant and sensitive A2780 epithelial ovarian cancer cells was obtained from GSE15372. Ovarian transcriptome data GSE62944 was downloaded from TCGA and applied for transcription regulatory network analysis. The analysis results were confirmed using quantitative polymerase chain reaction. The roles of SREBP2 in cisplatin-resistant cells were investigated by RNA inference and cell viability analysis. Transcription regulatory network analysis found that 12 transcription factors and their targets were involved in cisplatin resistant in A2780 cells. Among these factors, the targets of EZH2 and SREBP2 revealed by Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining were also enriched in differentially expressed genes between cisplatin resistant and cisplatin sensitive cells. Their targets were enriched mainly in cell cycle and cholesterol metabolic process, respectively. Bioinformatic analysis illustrated three known targets of SREBP2, namely LDLR, FDFT1, and HMGCR were increased in A2780-resistant cell lines. Additionally, the three genes and SREBP2 were also elevated in live cells after cisplatin treatment via quantitative polymerase chain reaction. Importantly, RNA inference of SREBP2 in A2780 cell line resulted in a decrease of cell viability after cisplatin treatment. SREBP2 played important roles in cisplatin resistance and cholesterol metabolic process might be a novel target for cancer therapy. Impact statement Transcriptome of cisplatin resistant and sensitive A2780 epithelial ovarian cancer cells was obtained from GSE15372 and TCGA. Twelve transcription factors and their targets were involved in cisplatin resistant. Among these factors, the targets of EZH2 and SREBP2 revealed by Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining were also enriched in differentially expressed genes. Their targets were enriched mainly in cell cycle and cholesterol metabolic process. Three targets of SREBP2, namely LDLR, FDFT1, and HMGCR were increased in A2780-resistant cell lines and were found elevated in live cells after cisplatin treatment via qPCR. RNAi of SREBP2 in A2780 cell line resulted in a decrease of cell viability after cisplatin treatment. SREBP2 played important roles in cisplatin resistance and might be a novel target for cancer therapy.
Project description:MicroRNAs (miRs) have been reported to be associated with the development of numerous types of cancer. However, the function of miRs in human ovarian carcinoma chemoresistance remains largely undefined. In the present study, cell chemotherapy combined with a Cell Counting Kit-8 assay demonstrated that miR-20a performed important roles in ovarian cancer cells chemoresistance. Flow cytometry, cellular proliferation assays and Transwell assays results revealed that the proliferation and migration rates of OVCAR3/DDP cells were increased in comparison with parental cells. Western blot analysis results suggested that epithelial-mesenchymal transition (EMT) activated by miR-20a contributed to OVCAR3/DDP cell migration. The present study highlighted the importance of miR-20a in regulating the chemoresistant properties of OVCAR3 cells and promoting cisplatin-resistant cell migration by activating EMT. The results of present study may therefore provide novel insights into reversing the chemoresistance of ovarian cancer and improving its treatment.
Project description:Cisplatin resistance significantly affects the survival rate of patients with ovarian cancer. However, the main mechanism underlying cisplatin resistance in ovarian cancer remains unclear. Methods: Immunohistochemistry was used to determine the expression of OGT, OGA and O-GlcNAc in chemoresistant and chemosensitive ovarian cancer tissues. Functional analyses (in vitro and in vivo) were performed to confirm the role of OGT in cisplatin resistance. Autophagy-related proteins were tested by western blot. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus reporter were used for autophagy flux analysis. Immunoprecipitation assay was utilized to detect protein-protein interactions. Results: We found that O-GlcNAc and O-GlcNAc transferase (OGT) levels were significantly lower in chemoresistant ovarian cancer tissues than in chemosensitive tissues, whereas O-GlcNAcase (OGA) levels did not differ. The down-regulation of OGT increased cisplatin resistance in ovarian cancer cells but had no effect on the efficacy of paclitaxel. The down-regulation of OGT improved tumor resistance to cisplatin in a mouse xenograft tumor model. OGT knockdown enhanced cisplatin-induced autophagy, which reduced apoptotic cell death induced by cisplatin, and promoted autolysosome formation. A reduction in O-GlcNAcylated SNAP-29 levels caused by the down-regulation of OGT promoted the formation of the SNARE complex and autophagic flux. Conclusion: Our findings suggest that down-regulation of OGT enhances cisplatin-induced autophagy via SNAP-29, resulting in cisplatin-resistant ovarian cancer. OGT may represent a novel target for overcoming cisplatin resistance in ovarian cancer.
Project description:Ovarian cancer is the leading cause of death in women worldwide. Cisplatin is the core of first-line chemotherapy for patients with advanced ovarian cancer. Many patients eventually become resistant to cisplatin, diminishing its therapeutic effect. MicroRNAs (miRNAs) have critical functions in diverse biological processes. Using miRNA profiling and polymerase chain reaction validation, we identified a panel of differentially expressed miRNAs and their potential targets in cisplatin-resistant SKOV3/DDP ovarian cancer cells relative to cisplatin-sensitive SKOV3 parental cells. More specifically, our results revealed significant changes in the expression of 13 of 663 miRNAs analyzed, including 11 that were up-regulated and 2 that were down-regulated in SKOV3/DDP cells with or without cisplatin treatment compared with SKOV3 cells with or without cisplatin treatment. miRNA array and mRNA array data were further analyzed using Ingenuity Pathway Analysis software. Bioinformatics analysis suggests that the genes ANKRD17, SMC1A, SUMO1, GTF2H1, and TP73, which are involved in DNA damage signaling pathways, are potential targets of miRNAs in promoting cisplatin resistance. This study highlights candidate miRNA-mRNA interactions that may contribute to cisplatin resistance in ovarian cancer.
Project description:Platinum-based chemotherapy remains one of the major choices for treatment of ovarian cancer (OC). However, primary or acquired drug resistance severely impairs their efficiency, thereby causing chemotherapy failure and poor prognosis. SH3 domain containing ring finger 2 (SH3RF2) has been linked to the development of cancer. Here we find higher levels of SH3RF2 in the tumor tissues from cisplatin-resistant OC patients when compared to those from cisplatin-sensitive patients. Similarly, cisplatin-resistant OC cells also express higher levels of SH3RF2 than normal OC cells. Through in vitro and in vivo loss-of-function experiments, SH3RF2 is identified as a driver of cisplatin resistance, as evidenced by increases in cisplatin-induced cell apoptosis and DNA damage and decreases in cell proliferation induced by SH3RF2 depletion. Mechanistically, SH3RF2 can directly bind to the RNA-binding protein mRNA processing factor (RBPMS). RBPMS has been reported as an inhibitor of cisplatin resistance in OC. As a E3 ligase, SH3RF2 promotes the K48-linked ubiquitination of RBPMS to increase its proteasomal degradation and activator protein 1 (AP-1) transactivation. Impairments in RBPMS function reverse the inhibitory effect of SH3RF2 depletion on cisplatin resistance. Collectively, the SH3RF2-RBPMS-AP-1 axis is an important regulator in cisplatin resistance and inhibition of SH3RF2 may be a potential target in preventing cisplatin resistance.
Project description:Previously, we have demonstrated that NRF2 plays a key role in mediating cisplatin resistance in ovarian cancer. To further explore the mechanism underlying NRF2-dependent cisplatin resistance, we stably overexpressed or knocked down NRF2 in parental and cisplatin-resistant human ovarian cancer cells, respectively. These two pairs of stable cell lines were then subjected to microarray analysis, where we identified 18 putative NRF2 target genes. Among these genes, ABCF2, a cytosolic member of the ABC superfamily of transporters, has previously been reported to contribute to chemoresistance in clear cell ovarian cancer. A detailed analysis on ABCF2 revealed a functional antioxidant response element (ARE) in its promoter region, establishing ABCF2 as an NRF2 target gene. Next, we investigated the contribution of ABCF2 in NRF2-mediated cisplatin resistance using our stable ovarian cancer cell lines. The NRF2-overexpressing cell line, containing high levels of ABCF2, was more resistant to cisplatin-induced apoptosis compared to its control cell line; whereas the NRF2 knockdown cell line with low levels of ABCF2, was more sensitive to cisplatin treatment than its control cell line. Furthermore, transient overexpression of ABCF2 in the parental cells decreased apoptosis and increased cell viability following cisplatin treatment. Conversely, knockdown of ABCF2 using specific siRNA notably increased apoptosis and decreased cell viability in cisplatin-resistant cells treated with cisplatin. This data indicate that the novel NRF2 target gene, ABCF2, plays a critical role in cisplatin resistance in ovarian cancer, and that targeting ABCF2 may be a new strategy to improve chemotherapeutic efficiency.
Project description:Ubiquitin carboxyl terminal hydrolase 1 (UCHL1) catalyzes the hydrolysis of COOH-terminal ubiquityl esters and amides. It has been reported as either an oncogene or a tumor suppressor in cancers. However, UCHL1's role in ovarian cancer is still unclear. Therefore, we conducted an analysis to understand the role of UCHL1 in ovarian cancer. Firstly, we detected UCHL1 promoter methylation status in 7 ovarian cancer cell lines. 4 of them with UCHL1 silencing showed heavy promoter methylation while the other 3 with relative high UCHL1 expression showed little promoter methylation. Then we reduced UCHL1 expression in ovarian cancer cell line A2780 and IGROV1 and found that inhibition of UCHL1 promoted cell proliferation by increasing cells in S phases of cell cycle. Knockdown of UCHL1 also reduced cell apoptosis and contributed to cisplatin resistance. Furthermore, the expression level of UCHL1 in several ovarian cancer cell lines correlated negatively with their cisplatin resistance levels. Microarray data revealed that UCHL1 related genes are enriched in apoptosis and cell death gene ontology (GO) terms. Several apoptosis related genes were increased after UCHL1 knockdown, including apoptosis regulator BCL2, BCL11A, AEN and XIAP. Furthermore, we identified up-regulation of Bcl-2 and pAKT as well as down-regulation of Bax in UCHL1 knockdown cells, while no significant alteration of p53 and AKT1 was found. This study provides a new and promising strategy to overcome cisplatin resistance in ovarian cancer via UCHL1 mediated pathways.
Project description:Cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance are believed to be responsible for treatment failure and tumor relapse in ovarian cancer patients. However, it is still unclear how CSCs survive DNA-damaging agent treatment. Here, we report an elevated expression of DNA polymerase η (Pol η) in ovarian CSCs isolated from both ovarian cancer cell lines and primary tumors, indicating that CSCs may have intrinsically enhanced translesion DNA synthesis (TLS). Down-regulation of Pol η blocked cisplatin-induced CSC enrichment both in vitro and in vivo through the enhancement of cisplatin-induced apoptosis in CSCs, indicating that Pol η-mediated TLS contributes to the survival of CSCs upon cisplatin treatment. Furthermore, our data demonstrated a depletion of miR-93 in ovarian CSCs. Enforced expression of miR-93 in ovarian CSCs reduced Pol η expression and increased their sensitivity to cisplatin. Taken together, our data suggest that ovarian CSCs have intrinsically enhanced Pol η-mediated TLS, allowing CSCs to survive cisplatin treatment, leading to tumor relapse. Targeting Pol η, probably through enhancement of miR-93 expression, might be exploited as a strategy to increase the efficacy of cisplatin treatment.
Project description:Enhanced chemoresistance is, among other factors, believed to be responsible for treatment failure and tumor relapse in patients with epithelial ovarian cancer (EOC). Here, we exposed EOC cells to interleukin-6 (IL-6) to activate oncogenic STAT3, which directly repressed miR-204 via a conserved STAT3-binding site near the TRPM3 promoter region upstream of miR-204. Repression of miR-204 was required for IL-6-induced cisplatin (cDDP) resistance. Furthermore, we identified the IL-6 receptor (IL-6R), which mediates IL-6-dependent STAT3 activation, as a direct miR-204 target. Importantly, the resulting IL-6R/STAT3/miR-204 feedback loop was identified in patients with EOC, and its activity correlated with chemosensitivity. Moreover, exogenous miR-204 blocked this circuit and enhanced cDDP sensitivity both in vitro and in vivo by inactivating IL-6R/STAT3 signaling and subsequently decreasing the expression of anti-apoptotic proteins. Our findings illustrate the function of this feedback loop in cDDP-based therapy and may offer a broadly useful approach to improve EOC therapy.
Project description:BNIP3 is a proapoptotic protein that mediates apoptosis, necrosis and autophagy. However, the involvement of BNIP3 in cisplatin-induced apoptosis in ovarian cancer is not clear. In this study, we examined the role of BNIP3 in ovarian cancer during cisplatin treatment and its correlation with clinical outcomes. We first measured cisplatin cytotoxicity and BNIP3 levels before and after cisplatin exposure for ovarian cancer cell lines A2780, SKOV3, OVCAR4, OV2008, ES2 and HO8910. BNIP3 was observed to be differentially expressed in these cell lines, and cisplatin induced a significant increase in BNIP3 levels in A2780 and OVCAR4. BNIP3 knockdown with siRNA in A2780 and OVCAR4 significantly reduced cisplatin cytotoxicity in these two cell lines and alleviated cisplatin-induced apoptosis. We searched the online databases Gene Expression Omnibus and The Cancer Genome Atlas to analyze the correlation between BNIP3 level and overall survival and progression-free survival in patients with ovarian cancer. Pooled analyses showed that higher BNIP3 level was correlated with poorer overall survival (95% confidence intervals; hazard ratio = 1.18, 1.04-1.34; P = 0.013) and progression-free survival (95% confidence intervals; hazard ratio = 1.26, 1.10-1.43; P = 0.00049). However, the results of individual datasets and stratification analyses of histology, FIGO (Federation Internationale de Gynecolgie et d'Obstetrique) stage, chemotherapy regimen and P53 mutation status varied. These findings indicate that cisplatin-induced apoptosis is dependent on BNIP3 level in ovarian cancer cell lines. Targeting BNIP3 may therefore be a potential way of restoring cisplatin sensitivity.