Comparative Analysis of Differentially Expressed miRNAs and their Downstream mRNAs in Ovarian Cancer and its Associated Endometriosis

Abstract

Objective: There is an increased risk of developing ovarian cancer (OC) in patients with endometriosis. Hence, development of new biomarkers may provide a positive clinical outcome for early detection. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in biological and pathological process and are currently used as diagnostic and prognostic markers in various cancers. In the current study, we assessed the differential expression of miRNAs from 19 paired ovarian cancer and its associated endometriosis tissue samples. In addition we also analyzed the downstream targets of those miRNAs.

Methods: Nineteen paired cases of ovarian cancer and endometriosis foci were identified by a gynecologic pathologist and macro-dissected. The total RNAs were extracted and subjected to comprehensive miRNA profiling from the pooled samples of these two different entities using microarray analysis. Later, the abnormal expressions of few selected miRNAs were validated in individual cases by quantitative real-time PCR (qRT-PCR). Ingenuity pathway analysis revealed target mRNAs which were validated by qRT-PCR.

Results: The miRNA profiling identified deregulation of greater than 1156 miRNAs in OC, of which the top seven were further validated by qRT-PCR. The expression of miR-1, miR-133a, and miR-451 were reduced significantly (p<0.0001) in the OC patients compared to its associated endometriosis. In contrast, the expression of miR-141, miR-200a, miR-200c, and miR-3613 were elevated significantly (p<0.05) in most of the OC patients. Furthermore, among the downstream mRNAs of these miRNAs, the level of PTEN expression was significantly (p<0.05) reduced in OC compared to endometriosis while no significant difference was observed in NF-κB expression.

Conclusion: The expression of miRNAs and mRNAs in OC were significantly different compared to its concurrent endometriosis. These differential expressed miRNAs may serve as potential diagnostic and prognostic biomarkers for OC associated with endometriosis.

Keywords: Endometriosis; FFPE; NF-κB; Ovarian cancer; PTEN; miRNA.

Figure 1

Representative foci of endometriosis and ovarian cancers (OC) for miRNA array assay. Endometriosis (A, B, C), Endometrioid carcinoma (D), Clear cell carcinoma (E), Serous carcinoma (F).

Figure 2

Heatmap of clustering miRNA microarray data with columns arranged according to hierarchical clustering method. miRNA groups that are increased in endometriosis compared to ovarian cancer (A). miRNA groups that are reduced in endometriosis compared to ovarian cancer (B).

Figure 3

Comparative expression analysis of miR-1, miR-133a and miR-451 in 19 paired samples of FFPE blocks of endometriosis (Endo) and ovarian cancer (OC) tissue samples individually using qRT-PCR. There was a significant down regulation of all three miRNAs tested in OC compared to respective Endo. *** represent comparison between Endo and OC using t test and the p value was found to be < 0.0001. RNU48 was used as loading control.

Figure 4

Comparative expression analysis of miR-141, miR-3613, miR-200a and miR-200c in 19 paired samples of FFPE blocks of endometriosis (Endo) and ovarian cancer (OC) tissue samples individually using qRT-PCR. There was a significant up regulation of all four miRNAs tested in OC (ovarian cancer) compared to respective Endo (endometriosis) as assessed by qRT-PCR. * represent comparison between Endo and OC using t test and the p value was found to be < 0.05. RNU48 was used as loading control.

Figure 5

Ingenuity pathway analysis showing up (green) and down-regulation (red) of miRNAs involved in ovarian cancer tumor samples when compared to endometriosis samples. Target genes are also represented, such as Insulin, NF-κB, and MAP2K 1/2 pathways.

Figure 6

Comparative expression analysis of PTEN in 19 paired samples of FFPE blocks of endometriosis and ovarian cancer tissue samples individually using qRT-PCR. PTEN mRNA was significantly reduced in ovarian cancer compared to endometriosis as assessed by qRT-PCR. * represent comparison between Endometriosis and ovarian cancer using t test and the p value was found to be < 0.05. GAPDH was used as loading control.