MicroRNA-125b down-regulation mediates endometrial cancer invasion by targeting ERBB2

Abstract

Background: MicroRNAs (miRNAs) are small non-coding nucleotides that regulate mRNA stability and protein expression by imperfect base pairing with the 3'-untranslated region (3'UTR) of target mRNAs. Many miRNAs have been documented to be aberrantly expressed in human cancers, but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. The objective of this study was to investigate the effect of miR-125b on EEC development and to explore its molecular mechanism in EEC carcinogenesis.

Material/methods: Real-time quantitative PCR was applied to evaluate the expression level of miRNA-125b in EEC and normal endometrium (NE) samples. The invasion ability of miR-125b in EEC HEC1B cells was analyzed by Transwell assay after pre-miR-125b or anti-miR-125b transfection. For the invasion mechanism analysis of miR-125b on HEC1B cells, miRBase, TargetScan, miRanda and PicTar were used to predict the possible target gene of miR-125b. Luciferase activities assay, cotransfection and Western blot were used to reveal that the predicted target genes of miR-125b were direct and specific. RNA interference technology was used to confirm that the invasion inhibition of miR-125b was directly induced by ERBB2.

Results: Our study showed that miR-125b was down-regulated in human EEC specimens compared to that in NC specimens. Over-expression of miR-125b in HEC1B cells inhibited EEC invasion and this inhibitory effect on HEC1B cells could be restored by miR-125b knock down. Mechanism analysis revealed that ERBB2 was a direct and specific target of miR-125b. The inhibitory effect on EEC cell invasion was mediated by miR-125b inhibition of the translation of a proto-oncogene, ERBB2.

Conclusions: Aberrantly expressed miR-125b contributes to HEC1B cells invasion partly through directly down-regulating ERBB2 protein expression in EEC. This miRNA signature offers a novel potential therapeutic strategy for EEC.

Figure 1

The expression of miR-125b was decreased in EEC samples. Quantitative analysis of the expression levels of miR-125b normalized to those of 18s rRNA by qRT-PCR. Data were shown as RQ ±SD in NE (n=30) and EEC (n=50) samples. * p<0.05 vs. NE.

Figure 2

Effects of miR-125b on HEC1B cell invasion. (A–D) represent photographs of invasive cells on the membrane by transfection vehicle, control miR, pre-miR-125b and anti-miR-125b respectively, magnification, 400×. The membrane was formaldehyde fixed and hematoxylin-stained. Randomly 5 fields were selected to calculate the trans-membrane cells, which represented the invasion ability of HEC1B cells.

Figure 3

ERBB2 is a validated target of miR-125b. (A) 3′UTR of ERBB2 is a target of miR-125b predicted by miRBase, TargetScan, miRanda and PicTar. (B) The reporter assay result with each bar representing values from three independent experiments. The transfection efficiency was normalized by co-transfected renilla luciferase and the light units were calculated by relative luciferase activity of firefly to renilla. * P<0.05. (C) Representative image of the protein level of ERBB2. GAPDH was used as a reference control. (D) quantitative analysis of the relative protein levels of ERBB2 normalized to those of GAPDH was shown. Data were mean ±SD of three independent experiments. * P<0.05.

Figure 4

miR-125b inhibits HEC1B cell invasion by down-regulating ERBB2. (A) Western blot showing ERBB2 knockdown by siERBB2 in HEC1B cells. GAPDH was used as a reference control. (B) quantitative analysis of the relative protein levels of ERBB2 normalized to those of GAPDH was shown. Data were mean ±SD of three independent experiments. * P<0.05. (C) Transwell assay was performed on HEC1B transfected with siERBB2 and controls.