Role Of MiR-130b In Drug-resistant Ovarian Cancer

Ovarian cancer is the leading cause of death in gynecologic cancers, the incidence is the sixth in all of malignancies among women. The large mortality is mainly due to drug-resistance and its late clinical appearance. The ovarian cancer cells are inclined to resist to chemo-therapeutic drugs, such as cisplatin and paclitaxel, which could lead to treatment failure. The development of drug-resistant in ovarian cancer influences the prognosis of it. Therefore, the study of the mechanisms in the development of drug-resistant ovarian cancer is very necessary. The effect of chemotherapy could be improved through reverse the chemo-resistance.MicroRNAs (miRNAs) are a class of endogenous, non-coding RNA (～22nt) that negatively regulate gene expression via base pairing with target mRNA. miRNAs are involved in the post-transcriptional regulation of genes either by mRNA cleavage and degradation or by repressing the translation of mRNA into proteins. Recent studies suggested miRNAs are contributing to the development of cancers. Further studies indicated that miRNAs also play important roles in the development of drug-resistant ovarian cancer. We focused on the role of miR-130b in the development of drug-resistant ovarian cancer. PartⅠmiR-130b expression in ovarian tissuesObjectiveTo examine the difference of miR-130b expression among malignant, benign ovarian tumors and normal ovarian tissues.MethodsIsolation the RNA of 47 malignant,7 benign and 16 normal ovarian tissue samples. The expression levels of miR-130b in ovarian tissues were measured with stem-loop RT-PCR.ResultsFrom normal ovarian tissues to benign ovarian tumors then to ovarian cancers, a decreasing tendency of miR-130b level was observed. The miR-130b expression of FIGOⅠ～Ⅱwas significantly higher than that of FIGOⅢ～Ⅳ(P=0.027). The expression of miR-130b was not significantly related with ages and histological types (P>0.05). The relapse rate of ovarian cancer patients with low miR-130b expression was significantly higher that of the patients with high miR-130b level (P=0.014).ConclusionThe down-regulation of miR-130b was correlated with the relapse of ovarian cancer. It suggested that miR-130b may be involved in the development of drug-resistant ovarian cancer.PartⅡRole of miR-130b in drug-resistant ovarian cancer cellsObjectiveTo determine the role of miR-130b in drug-resistant ovarian cancer cells MethodsThe expression level of miR-130b in drug-resistant ovarian cancer cell lines (A2780/TAX, A2780/DDP and SKOV3/TAX) was measured by using stem-loop qRT-PCR. After transfect miR-130b mimics/inhibitors into the cells, assessment of chemo-resistance to cisplatin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The viability of cells was determined by cell number counting.ResultsThe expression levels of miR-130b in drug-resistant ovarian cancer cells (A2780/TAX, A2780/DDP and SKOV3/TAX) were lower than in parental cell lines (A2780, SKOV3, P <0.001). After transfected drug-resistant ovarian cancer cells with miR-130b mimics, the drug-resistance was significantly reduced. On the other hand, the drug-resistance of the parental cells was significantly increased after being transfected with miR-130b inhibitors.ConclusionmiR-130b was down-regulated in drug-resistant ovarian cancer cells, and the chemo-resistance was reversed after the expression of miR-130b was increased.Part III miR-130b is regulated by cytosine methylationObjectiveTo investigate whether miR-130b is regulated by methylation, and the effect of demethylation in drug-resistance.MethodsIsolation the DNA of 26 malignant,5 benign ovarian tissue samples. The methylation level was measured by using qMSP. The methylation level of drug-resistant ovarian cancer cells and parental cells were measured by using qMSP. After cells were treated with 5-aza-CdR, the methylation level of miR-130b was determined by using qMSP, the expression level of miR-130b was measured by using stem-loop qRT-PCR, and the drug-resistance was measured by using MTT.ResultsThe methylation level of miR-130b in ovarian cancer was significantly higher than in benign ovarian tissues (P=0.028), and a significant inverse correlation between methylation level and expression level of miR-130b was found (P=0.024). The methylation level of miR-130b in drug-resistant ovarian cancer cells was significantly higer than in parental cells. After demethylation, the methylation level of miR-130b was down-regulated, and the expression level of miR-130b was increased. Furthermore, the chemoresistance was partly reversed.ConclusionmiR-130b is regulated by cytosine methylation, demethylation could partly reverse the chemo-resistance.PartⅣCSF-1 is a direct target of miR-130bObjectiveTo determine CSF-1 is a direct target of miR-130bMethodsThe CSF-1 mRNA expression of 29 malignant,7 benign and 10 normal ovarian tissue samples were measured by qRT-PCR. The CSF-1 expression was measured by immunohistochemistry. The CSF-1 mRNA and protein expression of drug-resistant ovarian cancer cells transfected with miR-130b mimics/inhibitors was measured by ELISA. A luciferase targeting assay was performed to determine that CSF-1 is a direct target of miR-130b. Resultsl.The expression level of CSF-1 mRNA in malignant tissues was higher than benign and normal tissues (P=0.009)2. Positive CSF-1 expression was more frequent in ovarian cancer compared with benign tumors (P=0.003)3. A significant inverse correlation between CSF-1 mRNA and miR-130b expression levels in ovarian tumor tissues was detected (P=0.034).4. The mimics transfection dramatically inhibited CSF-1 mRNA and protein expression in drug-resistant ovarian cancer cells. Reversely, a significant increase in CSF-1 protein expression of parental cells in response to miR-130b inhibitors transfection.5. Luciferase targeting assay suggested miR-130b negatively regulates CSF-1 expression by direct binding to 3'-UTR of CSF-1 mRNA.ConclusionCSF-1 is a direct target of miR-130bPart V miR-130b modulates drug-resistance by targeting CSF-1 Experiment 1 Role of CSF-1 in drug-resistant ovarian cancerObjectiveDown-regulate the expression of CSF-1 by shRNA to study the role of CSF-1 in drug-resistant ovarian cancer.Methods1. The expression level of CSF-1 in drug-resistant ovarian cancer cells was measured by ELISA.2. ShRNA sequences targeting CSF-1 gene were synthesized and inserted into supersilencing shRNA plasmid expression vector pGPU6/GFP/Neo.3. Transfect the CSF-1 shRNA plasmid into drug-resistant ovarian cancer cells, then the chemoresistance was measured with MTT.4. Transfect the CSF-1 shRNA plasmid into drug-resistant ovarian cancer cells, then the proliferation was measured using EdU assay.Results1. The expression level of CSF-1 in drug-resistant ovarian cancer cells was significantly higher than parental cells.2. After downregulation the expression of CSF-1 by shRNA, the drug sensitivity of ovarian cancer cells was significantly increased.3. After downregulation the expression of CSF-1 by shRNA, the proliferation of cells was significantly decreased.ConclusionDown-regulation of CSF-1 could partly reverse chemo-resistance in drug-resistant ovarian cancer cells.Experiment 2 miR-130b modulates drug-resistance by targeting CSF-1ObjectiveTo confirm miR-130b modulates drug-resistance by targeting CSF-1.MethodsTransfect miR-130b inhibitors and CSF-1 shRNA into A2780 and SKOV3 simutaneously, then the change of chemo-resistance was determined by MTT. The viability of cells was assessed by cell number counting after treatment. ResultsThe drug sensitivity of cells transfected with miR-130b inhibitors and CSF-1 shRNA was significantly lower than the cells only transfected with miR-130b inhibitors. The viability assay confirmed the result.ConclusionmiR-130b modulates drug-resistance by targeting CSF-1.