Supplementary MaterialsSupporting Data Supplementary_Data. present research indicated that miR-299-3p functions as a tumor suppressor by directly targeting HPSE, highlighting its potential as a target for the treatment of GC. (12) reported a role for miR-299-3p in the chemoresistance of lung cancer, as miR-299-3p promoted sensitivity to doxorubicin by directly targeting ATP-binding cassette E1. Recently, tumor-suppressive roles and prognostic values of miR-299-3p were further reported in hepatocellular carcinoma and thyroid cancer (13,14); however, the role of miR-299-3p in the progression of GC remains largely unknown. In the present study, the potential role of miR-299-3p in the invasion of human GC cells was explored. miR-299-3p expression analysis revealed that miR-299-3p was downregulated in GC tissues significantly. The LHW090-A7 inhibition of miR-299-3p advertised the invasion of HGC-27 cells luciferase plasmid (Promega Company) and 50 nM miR-299-3p or control mimics had been co-transfected into HGC-27 cells when the cell reach 80C90% confluence using Lipofectamine 2000 reagent, following a manufacturer’s guidelines. Luciferase activity was assessed 48 h pursuing transfection utilizing a Dual Luciferase Assay (Promega Company), based on the manufacturer’s protocols. Comparative luciferase activity was established as the percentage of firefly to luciferase activity. LHW090-A7 All transfection assays had been carried out in triplicate. Traditional western blot assay Entire cell lysates had been ready using RIPA buffer (Bio-Rad Laboratories, Inc.) containing the protease inhibitor PMSF (Invitrogen; Thermo Fisher Scientific, Inc.). The protein concentrations were determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). A total of 20 g protein per lane was loaded and resolved by 10% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% (w/v) non-fat milk for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4C. Following washing, the membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature. Immunoreactive proteins were detected using enhanced chemiluminescence (Bio-Rad Laboratories, Inc.). The relative band intensity was quantified using ImageJ software LAMC1 antibody (National Institutes of Health). The following antibodies were used: Anti-HPSE (1:500; cat. no. ab85543; Abcam), anti-GAPDH (1:2,000; cat. no. 2118; Cell Signaling Technology, Inc.) and goat anti-rabbit immunoglobulin G (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.). Statistical analysis All data are presented as the mean standard deviation. Statistical analysis was performed by unpaired Student’s t-test LHW090-A7 or one-way ANOVA followed by post hoc Tukey’s multiple comparison tests as indicated using GraphPad Prism software 6.0 (GraphPad Software, Inc.). Survival analysis was performed using the Kaplan-Meier method with a Log-rank statistical test. P 0.05 was considered to indicate a statistically significant difference. Results miR-299-3p is downregulated in GC tissues To investigate the potential involvement of miR-299-3p in GC, its expression was evaluated in human GC tissues via RT-qPCR analysis. The expression levels of miR-299-3p were significantly downregulated in GC than in non-tumor tissues (P 0.01; Fig. 1A). In addition, the expression of miR-299-3p was analyzed by an ISH assay in GC and adjacent non-tumor tissues. A moderate miR-299-3p signal was evident in the adjacent control tissues, and a normal structure of the stomach mucosa was observed. Conversely, a large number of malignant cells with low miR-299-3p expression were observed in GC tissues (Fig. 1B). Of note, reduced ISH staining of miR-299-3p was more evident in diffusive GC (Fig. 1B). This observation was further LHW090-A7 revealed by miR-299-3p expression analysis according to histological subtype, with the most notably downregulated expression in the diffuse subtype (Fig. 1C). The Cancer Genome Atlas database was subsequently explored, and it was revealed that patients with GC with reduced miR-299-3p expression exhibited significantly shorter overall survival (Fig. 1D). Collectively, these data suggested that miR-299-3p may serve a tumor-suppressive role in the development of GC. Open in a separate window Physique 1. Downregulation of miR-299-3p in GC tissues. (A) Relative expression of miR-299-3p in 30 primary GC compared with 25 adjacent NT tissues. The expression of miR-299-3p was normalized to U6. **P 0.01. (B) Representative hybridization of miR-299-3p in non-tumor tissue and different histological subtypes of GC specimens. Scale bar, 50 m. (C) Relative expression of miR-299-3p in different subtypes of GC classified according to the Lauren.