Downregulation of miR-181a binding and targeting to cMET in CD24+ ovarian cancer stem cells is significantly associated with poor prognosis

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate posttranscriptional gene expression, and play an important role in diverse biological processes including carcinogenesis. Recently, it was documented that the subset of CD24-positive (CD24+) cells in ovarian carcinoma has cancer stem cell properties that are related to recurrence as well as tumor initiation. Although dysregulation of miRNAs in ovarian cancer has been observed, the role of miRNA in relation to CD24+ ovarian cancer stem cells has not been studied. Differential expression of miRNAs according to CD24 phenotype was investigated and the expression of the selected miRNA and its putative target were validated to determine the CD24-related miRNA in CD24+ ovarian cancer stem cells. In addition, the clinical significance of immunohistochemical expression of the targets was investigated.Differential miRNA expressions between CD24+ and CD24- CaOV3 cells, and between C4 (CD24high) and C14.2 (CD24low) cells were examined by miRNA microarray analysis. Among differentially expressed miRNAs in both comparison groups, one miRNA downregulated in both CD24+ and C4 cells was selected and its one putative target was chosen from TargetScan, microRNA.org, and PicTar databases, focusing on known oncogenes. The mRNA levels of the selected miRNA and the target were measured by real-time reverse transcription polymerase chain reaction (real time RT-PCR) in CD24+ and CD24- CaOV3 cells. To investigate whether the selected putative target is functional, a luciferase assay was performed. For in vivo validation, immunohistochemical staining of CD24 and the target protein was carried out on formalin-fixed, paraffin-embedded tissue from 74 patients with serous papillary carcinoma. Overall and disease-free survival of patients according to CD24 and cMET expression were evaluated.miRNA microarray analysis revealed 7

downregulated and 10 upregulated miRNAs in both CD24+ and C4 cells subject to greater than 2-fold change. Of them, miR-181a and its putative target, cMET were selected. The mRNA level of miR-181a was downregulated in CD24+ CaOV3 cells compared with CD24- CaOV3 cells, and the mRNA level of cMET inversely correlated with that of miR-181a. A luciferase assay revealed that miR-181a directly targets both CD24 and cMET. On immunohistochemical staining, CD24 expression positively correlated with cMET expression, and cases with immunopositivity for both CD24 and cMET (CD24(+)cMET(+)) showed a shorter disease-free survival than cases with immunonegativity for both CD24 and cMET.In conclusion, our results suggest that miR-181a may play an important role in regulating CD24+ ovarian cancer stem cells by directly targeting both cMET and CD24, and miR-181a could be a potential therapeutic target related to ovarian cancer stem cells. In addition, CD24(+)cMET(+) immunohistsochemistry can be used as a prognostic marker for poor disease-free survival.