Verification of microRNA expression in human endometrial adenocarcinoma

Abstract

Background: MicroRNAs are small non-coding RNAs that have been implicated in tumor initiation and progression. In a previous study we identified 138 miRNAs as differentially expressed in endometrial adenocarcinoma compared to normal tissues. One of these miRNAs was miRNA-34a, which regulates several genes involved in the Notch pathway, which is frequently altered in endometrial cancer. The aims of this study were to verify the differential expression of a subset of miRNAs and to scrutinize the regulatory role of mir-34a on the target genes NOTCH1 and DLL1.

Methods: Twenty-five miRNAs that were previously identified as differentially expressed were subjected to further analysis using qPCR. To investigate the regulation of NOTCH1 and DLL1 by mir-34a, we designed gain- and loss-of-function experiments in Ishikawa and HEK293 cell lines by transfection with a synthetic mir-34a mimic and a mir-34a inhibitor.

Results: Of the 25 validated miRNAs, seven were down-regulated and 18 were up-regulated compared to normal endometrium, which was fully consistent with our previous findings. In addition, the up-regulation of mir-34a led to a significant decrease in mRNA levels of NOTCH1 and DLL1, while down-regulation led to a significant increase in mRNA levels of these two genes.

Conclusions: We verified both up-regulated and down-regulated miRNAs in the tumor samples, indicating various roles of microRNAs during tumor development. Mir-34a functions as a regulator by decreasing the expression of NOTCH1 and DLL1. Our study is the first to identify a correlation between mir-34a and its target genes NOTCH1 and DLL1 in endometrial adenocarcinoma.

Keywords: Endometrial adenocarcinoma; Target genes; microRNA; mir-34a.

Fig. 1

Detection of NOTCH1 and DLL1 expression in transfected Ishikawa and HEK293 cell lines. BLOCK-iT Alexa Fluor red fluorescent and mir-34a inhibitor transfection in (a) Ishikawa cells and (b) HEK293 cells. The photos were taken 24 h after transfection corresponding to morphology of cells (left) and BLOCK-iT Alexa Fluor red fluorescent (right). c The level of NOTCH1 and DLL1 expression in Ishikawa cells 48 h after transfection with mir-34a inhibitor compared with the negative control. d The level of NOTCH1 and DLL1 expression in HEK293 cells 48 h after transfection with mir-34a inhibitor compared with the negative control

Fig. 2

Detection of NOTCH1 and DLL1 expression in transfected Ishikawa and HEK293 cell lines. BLOCK-iT Alexa Fluor red fluorescent and mir-34a mimic transfection in (a) Ishikawa cells and (b) HEK293 cells. The photos were taken 24 h after transfection corresponding to morphology of cells (left) and BLOCK-iT Alexa Fluor red fluorescent (right). c The level of NOTCH1 and DLL1 expression in Ishikawa cells 48 h after transfection with mir-34a mimic compared with the negative control. d The level of NOTCH1 and DLL1 expression in HEK293 cells 48 h after transfection with mir-34a mimic compared with the negative control