Prognostic implications of microRNA-100 and its functional roles in human epithelial ovarian cancer

Abstract

Dysregulation of microRNAs (miRNAs) has been found to be associated with a variety of diseases, including epithelial ovarian cancer (EOC). Recently, miR-100 was reported to be downregulated in human ovarian carcinoma, however, the clinical significance and functional roles of miR-100 expression in human EOC are unclear. TaqMan real-time quantitative RT-PCR assay was performed to detect the expression of miR-100 in 98 EOC tissues and 15 adjacent normal epithelial tissues. The relationship between miR-100 expression and clinicopathological factors in 98 EOC patients was statistically analyzed. The effect of miR-100 expression on patient survival was determined. Finally, the role of miR-100 in EOC cell growth and its possible mechanisms were analyzed with miR-100 precursor or inhibitor-transfected cells. We showed that the level of miR-100 was significantly lower in EOC tissues compared to adjacent normal tissues. Low miR-100 expression was found to be closely correlated with advanced FIGO stage, higher serum CA125 expression level and lymph node involvement. Also, low miR-100 expression was correlated with shorter overall survival of EOC patients, and multivariate analysis showed that the status of miR-100 expression was an independent predictor of overall survival in EOC. Additionally, miR-100 could affect the growth of EOC cells by post-transcriptionally regulating polo-like kinase 1 (PLK1) expression. Together, these results suggest that low miR-100 expression may be an independent poor prognostic factor and miR-100 can function as a tumor suppressor by targeting PLK1 in human EOCs.

Figure 1

TaqMan real-time RT-PCR analysis of relative miR-100 expression in tissue samples. (A) Levels of miR-100 was significantly lower in EOC tissues (n=15) than in a adjacent normal tissue (n=15) (P<0.001). (B) Levels of miR-100 were confirmed to be lower in patients with FIGO stage III/IV (n=43) as compared to stage I/II (n=55). Statistical significance comparing the expression of miR-100 between EOC and adjacent normal tissue was calculated using the Wilcoxon signed-rank test.

Figure 2

Survival curves of EOC patients according to the status of miR-100 expression. Patients with low miR-100 expression (n=50) showed significantly poorer prognosis than those with high miR-100 expression (n=48) (P=0.021; log-rank test).

Figure 3

Effect of miR-100 expression on the growth of the EOC cell line (SKOV-3). (A) TaqMan real-time RT-PCR analysis of relative miR-100 expression in the miR-100 precusor (pre-miR-100) or inhibitor (anti-miR-100)-tranfected SKOV-3 cells. Pre-miR-NC or anti-miR-NC was used as a corresponding control. (B) MTT analysis of growth in SKOV-3 cells transfected with pre-miR-100 (pre-miR-NC) or anti-miR-100 (anti-miR-NC). Data are presented as the mean ± SD of three experiments.

Figure 4

PLK1 is a target of miR-100. (A) Western blot analysis of PLK1 protein expression in SKOV-3 cells transfected with pre-miR-100 (pre-miR-NC) or anti-miR-100 (anti-miR-NC). (B) PLK1 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in pGL3-Basic vector and validated by DNA sequencing. The sequences of the predicted miR-100 binding sites within the PLK1 3′-UTR, including wild-type UTR or UTR segments containing mutant binding site are shown. (C) Detection of luciferase activity. For luciferase reporter assays in 6-well plates, 1-mg luciferase reporter plasmid containing either wild-type (wt) or mutant (mut) PLK1 3′-UTR, and 200 pmol of pre-miR-100, anti-miR-100, pre-miR-NC or anti-miR-100 were transfected. The parental luciferase plasmid was also transfected as a control. At 24 h after transfection, cells were assayed using the Luciferase Gene Reporter Assay kit. The data are presented as the mean ± SD of three experiments. ^*P<0.05 or ^**P<0.01.

Figure 5

Functional analysis of PLK1 gene in SKOV-3 cells. (A) Western blot analysis of PLK1 protein expression in mock or siRNA/PLK1 (siRNA/control)-transfected SKOV-3 cells. (B) MTT analysis of cell growth in mock SKOV-3 or SKOV-3 cells transfected with siRNA/PLK1 or siRNA/control. (C) Western blot analysis of PLK1 protein expression in pre-miR-100 (or pre-miR-NC)-transfected SKOV-3 cells or co-transfected with pcDNA/PLK1 (or pcDNA/control). (D) MTT analysis of growth in pre-miR-100 (or pre-miR-NC)-transfected SKOV-3 cells or co-transfected with pcDNA/PLK1 (or pcDNA/control). The data are presented as the mean ± SD of three experiments. ^*P<0.05.