Samples
Biospecimens of endometrioid adenocarcinoma and corresponding nontumorous endometriums from 30 patients diagnosed with endometrioid adenocarcinoma were used after institutional review board-approved consents. All the patients were treated at Changning Maternity and Infant Health Hospital (Shanghai, China) in 2009–2011. Inclusion criteria were all women greater than 45 years of age with histological diagnosis consistent with EEC. Among these cases, 25 were postmenopausal. The pieces of tumor tissue and nontumorous endometrium tissues were carefully selected and snap frozen in liquid nitrogen, followed by storage at −80 °C.
RNA isolation, quantitative reverse transcriptase polymerase chain reaction and miRNA expression profiling and validation
For E-cadherin, Vimentin and α-SMA mRNA expression, total RNA was isolated using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) and transcribed to cDNA using the SuperScript® Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Primer sequences used in this study are provided in Additional file 1: Table S1. Data were normalized using human GAPDH housekeeping gene and analyzed by comparing the 2−ΔCt values of the normalized data.
For miRNA expression profiling, total RNA was isolated with a mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, USA). miRNA microarray analysis, including labeling, hybridization, scanning, normalization and data analysis, was performed by CloudSeq Bio-tech, Shanghai, China, on a miRCURY LNA™ microRNA Array Kit v.19.0 (Exiqon, Vedbaek, Denmark). The microRNA profiling contains 3100 capture probes cover human, mouse and rat microRNAs.
All specific primers for miRNA expression were designed and synthesized by Guangzhou RiboBio Co Ltd, Guangzhou, China, using the mirVana™ qRT-PCR Primer Sets. The levels of an endogenous control, U6 (RiboBio, Guangzhou, China), were used to normalize the expression levels of each miRNA. All reactions were performed in triplicate and included controls without a template for each miRNA. The fold change in miRNA expression was calculated using the comparative CT method.
Cell culture
HEC-1-A cells (human endometrial carcinoma cells-1-A) are human endometrium adenocarcinoma cell line and were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in McCoy’s 5A modified medium supplemented with 10 % fetal bovine serum and 1 % penicillin/streptomycin (Sigma-Aldrich, St Louis, MO, USA). HEC-1-A cells are used as an accepted model to study endometrial cancer [15]. MiR-23a mimic, inhibitor, agomir or antagomir were purchased from RiboBio Inc (GuangZhou, China). Human recombinant TGFβ1 was obtained from R & D Systems Inc (Minneapolis, MN, USA).
MicroRNA target gene prediction
In silico programs, a computational approach was used to predict the miRNA target genes with the following three different miRNA target prediction algorithms: PicTar, miRanda, and TargetScan. The potential binding sites in the messenger RNA 3′ according to specific base-pairing rules were identified, and second, implementation of cross-species conservation requirements was performed.
Luciferase activity assay
Human SMAD3 or SMAD5 3′-UTR, containing the putative target site for miR-23a, was amplified from genomic DNA by PCR amplification and inserted into the pmiR-REPORT™ (RiboBio). The mutation from a site of perfect complementarity was also generated by the QuikChange II® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and by following the manufacturer’s instructions. The HEC-1-A cells were transiently transfected with wild-type or mutant reporter plasmid. Luciferase activity was measured 24 h after transfection, as described previously. Three independent experiments were performed in triplicate.
Immunoblotting
Samples were homogenized in 100 μL ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with a proteinase inhibitor cocktail. The blots were probed with the primary antibodies for E-cadherin (ab76055, Abcam, Cambridge, MA, USA), Vimentin (ab92547, Abcam), α-SMA (ab5694, Abcam), SMAD3 (ab40854, Abcam) and SMAD5 (ab40771, Abcam).
Statistical analyses
The results are expressed as the mean ± standard deviation (SD). For the results shown in Fig. 1, the difference was calculated using an unpaired t test. For the other results, statistical analyses were performed using a one-way analysis of variance by comparing the groups using the Student–Newman–Keuls test. The least significant difference procedure was performed with GraphPad Prism software 5.0 (GraphPad Software, Inc., San Diego, CA). A P value <0.05 was considered to be statistically significant.