Deregulation of miR-100, miR-99a and miR-199b in tissues and plasma coexists with increased expression of mTOR kinase in endometrioid endometrial carcinoma

Abstract

Background: Alterations of mTOR gene expression have been implicated in the pathogenesis of endometrioid endometrial cancer however only few studies explored the cause of increased mTOR activation in this malignancy. miRNAs are small, noncoding RNAs, which were proven to regulated gene expression at the posttranscriptional level. The study aimed to explore deregulation of miRNAs targeting mTOR kinase (miR-99a, miR-100 and miR-199b) as a possible cause of its altered expression in EEC tissues. In addition expression of the three miRNAs was investigated in plasma of EEC patients and was assessed in terms of diagnostic and prognostic utility.

Methods: We investigated expression of mTOR kinase transcripts in 46 fresh tissue samples. Expression of miR-99a, miR-100 and miR-199b was investigated in the same group of fresh samples, and in additional 58 FFPE sections as well as in 48 plasma samples using qPCR. Relative quantification was performed using experimentally validated endogenous controls.

Results: mTOR kinase expression was increased in EEC tissues and was accompanied by decreased expression of all three miRNAs. Down-regulation of the investigated miRNAs was discovered in plasma of EEC patients and miRNA signatures classified EEC tissues (miR-99a/miR-100/miR-199b) and plasma (miR-99a/miR-199b) samples with higher accuracy in comparison to single miRNAs. We also revealed that miR-100 was an independent prognostic marker of overall survival.

Conclusions: We conclude that increased expression of mTOR kinase coexists with down-regulation of its targeting miRNAs, which could suggest a new mechanism of mTOR pathway alterations in EEC. In addition, our findings implicate that miRNA signatures can be considered promising biomarkers for early detection and prognosis of endometrioid endometrial carcinoma.

Figure 1

mTOR gene expression in endometrioid endometrial cancer samples (n = 31) and in the control group (n = 15). Values are presented in the log scale; * p = 0.033

Figure 2

Expression of miR-99a, miR-100 and miR-199b in endometrioid endometrial cancer (EEC n = 73) tissue samples in comparison to control group (NE n = 31). Values are presented in the log scale; * p = 0.007 (miR-99a), * p = 0.02 (miR-100), * p = 0.002 (miR-199b)

Figure 3

Expression of miR-99a, miR-100 and miR-199b in endometrioid endometrial cancer (EEC n = 34) plasma samples in comparison to control group (NE n = 14). Values are presented in the log scale; * p < 0.0001 (miR-99a), * p = 0.001 (miR-100), * p = 0.0007 (miR-199b)

Figure 4

Expression of miR-99a, miR-100 and miR-199b in endometrioid endometrial cancer (EEC) plasma samples in groups distinguished based on FIGO stage. FIGOIA (n = 17), FIGO > IA (n = 17), NE, normal endometrium (n = 14). Values are presented in the log scale; * p = 0.039, # p < 0.05

Figure 5

Receiver operating characteristics curve (ROC) analysis using tissue miRNAs expression for discriminating endometrioid endometrial cancer (EEC) samples (EEC (n = 73) vs. NE (n = 31). A miRNA signature consisting of three miRNAs yielded area under curve (AUC), which was higher in comparison to single miRNAs (0.774, 95% CI 0.678–0.853, p < 0.0001)

Figure 6

Receiver operating characteristics curve (ROC) analysis using plasma miRNAs expression for discriminating endometrioid endometrial cancer (EEC) samples. A miRNA signature consisting of two miRNAs yielded area under curve (AUC), which was higher in comparison to single miRNAs (0.903, 95% CI 0.780–0.970, p < 0.0001, EEC (n = 34) vs. NE (n = 14)

Figure 7

Kaplan-Meier overall survival curve for patients with endometrioid endometrial cancer (EEC) based on miR-100 expression. Red line – values above median, blue line – values below median; HR 0.43, 95% CI 0.222–0.830, p = 0.02