| BackgroundOvarian cancer is one of the most common malignant tumors in women, which is ranking the first among the leading gynecologic cancer. About 70% percent of patients have local or distant metastases before they seek medical attention due to lack of typical symptom and effective tests to make a diagnosis. As a result, the five-year survival rate of patients with an advanced disease is still dissatisfied. Relapse and metastasis always result in poor clinical outcome. Recent studies indicate that there is a small subpopulation of cancer cells with stem cell-like characteristics, namely tumor stem cells(TSCs), which have the ability to self-renew and to differentiate into the heterogeneous non-tumorigenic cancer cell type and to form tumor in the distance, leading to the generation, progression, relapse and metastasis of the tumor. Strategies targeting to restrain the metastasis of TSCs would develop if the the molecular mechanism of TSCs is clear.miRNAs are non-coding, single-stranded RNAs about 18-25 nucleotides long and involved in post-transcriptional gene repression via degradation of target mRNA or inhibition of translation. They are found in both plants and animals and confirmed to be involved in diverse biological response in human being due to one miRNA could regulate more than hundreds of gene. Recent studies indicate that miRNA mutations or mis-expression correlate with various human cancers and suggest that miRNAs can function as tumour suppressors or oncogene via activate or inactivate the expression of TSCs-related genes. Further investigation into the expression and function of miRNAs in TSCs play an important role for the study of mechanism of tumor generation and metastasis as well as prove useful approaches for the diagnosis and treatment of cancer.Hu et al. reported that miR-200a was frequently low-expressed in advanced ovarian cancer. Patients with low expression of miR-200a were found to have a significantly poor survival rate. The mechanisms of miR-200a predict the poor prognosis of ovarian cancer patients have not been clarified. Human miR-200a located in Chromosome 1p36, which had been demonstrated could regulate the transcription of ZEB2 in breast cancer, colon cancer and gastric cancer cells, leading to indirectly regulate the expression of E-cadherin, result in the inhibition of tumor cells invasion and migration via reversion of epithelial to mesenchymal transition (EMT).But the pathway in ovarian tumor stem cell has not been verified. E-cadherin is a member of cadherin family which play an important role in the formation and maintenance of epithelium, the function deficiency of E-cadherin is associated with tumor invasion and metastasis.ObjectivemiRNAs mis-expression in CD133/1+ and CD133/1- subpopulation were identified using microarray in our previous study. The results showed that miR-200a was low-expressed in CD133/1+ cells compared with CD133/1- cells. The aim of the study is to further validate the expression of mature miR-200a in CD133/1+ cells, and investigate the effect of miR-200a on invasion and migration of CD133/1+ cells and their molecular mechanism, so as to provide experimental evidence for the development of TSCs targeting therapeutic strategies for ovarian cancer. Methods1. CD133/1+ cells sorting: Human ovarian cancer cell line OVCAR3 was grown in DMEM, cells in logarithmic phase were trypsinized and resuspended in PBS, then incubated with Mouse anti-human CD133/1-PE monoclonal antibodies following the manufacturer's instruction. After finishing staining, BD FACSAria was use to sort CD133/1+ cells and CD133/1- cells.2. The expression level of mature miR-200a in CD133/1+ and CD133/1- cells: Total RNA from CD133/1+ and CD133/1- cells was extracted with TriZol Reagent, First strand cDNA synthesis and amplification was performed using a stem-loop primers, SYBR Green quantitative PCR amplifications were performed in a 7500 Sequence Detection System, U6 snRNA as an endogenous control.3. Transwell and wound healing assay: Transwell and wound healing assays were used to investigate the ability of CD133/1+ cells transfected with miR-200a mimic or negative control.4. We utilized the following three bioinformatics algorithms to predict the target sites between miR-200a and the 3'-UTR of ZEB2 mRNA(TargetScan,miRanda and PicTar).5. A fragment of the 3'-UTR of ZEB2 mRNA was extracted from OVCAR3 cells genomic DNA. Construct three mutants containing one binding site mutation and a mutant containing three binding sites mutation simultaneously, then the mutants were cloned into the psi-CHECK2 vector, after cotransfected with miR-200a mimic or negative control, Luciferase assays were performed using the Dual Luciferase Reporter Assay System.6. Total RNA from CD133/1+ cells transfected with miR-200a mimic or negative control was extracted with TriZol Reagent, SYBR Green quantitative RT-PCR were used to detect the mRNA expression of ZEB2 and E-cadherin, 18srRNA as an endogenous control.7. Western blot was used to detect the ZEB2 and E-cadherin protein expression in CD133/1+ cells transfected with miR-200a mimic or negative control.Results1. The proportion of CD133/1 positive in OVCAR3 cell line was 0.3%.2. The expression level of mature miR-200a in CD133/1+ cells was much less than CD133/1- cells(1.00±0.00vs2.17±0.25,P=0.015).3. Effects of miR-200a on invasion and migration of CD133/1+ cells: In transwell experiment ,the cell number that infiltrated transwell membrane in miR-200a group(81.7±12.1) was significantly less than that in negative control group (162±20.7) and blank group(164±23.3).The invasion of CD133/1+ cells treated with miR-200a was weakened than the other two groups(P<0.01). In wound healing assay, 24hr after wounded, the migration distance in miR-200a group (632μm±53μm) was less than that in negative control group(912μm±75μm) and blank group (935μm±35μm). The migration of CD133/1+ cells treated with miR-200a was weakened than the other two groups(P<0.01).4. All of three bioinformatics algorithms predicted the same miR-200a binding site. There were two conserved binding sites among three binding sites.5. The Dual Luciferase Reporter Assay result showed that overexpression of miR-200a in CD133/1+ cells resulted in decreased of Renilla luciferase compared with negative control and blank group (P<0.05).6. Compared with negative control and blank group, ZEB2 mRNA in miR-200a group decreased 44%, the difference was significant(P<0.05). E-cadherin mRNA in miR-200a group was 2.35-fold Compared with negative control and blank group,the difference was significant(P<0.05).7. Western blot result showed that the expression of ZEB2 protein in miR-200a group decreased 42%, Compared with negative control and blank group, the difference was significant(P<0.05). The expression of E-cadherin protein in miR-200a group was upregulation, 1.4-fold Compared with negative control and blank group,the difference was significant(P<0.05). Conclusions1. There was a small subset of CD133/1+ TSCs in ovarian cancer OVCAR3 cell line, mature miR-200a was low-expressed in CD133/1+ TSCs.2. Over-expression of miR-200a in CD133/1+ TSCs inhibited its invasion and migration.3. miR-200a inhibited CD133/1+ TSCs invasion and migration by targeting E-cadherin repressor ZEB2. |
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