Clinical samples
The HCC and adjacent tissues were collected from 58 diagnosed HCC patients who had not received chemotherapy, rediation therapy or other anticancer treatment before undergoing surgical resection at the First pepole’s Hospital of Huaihua from June 2019 to December 2019. The project was approved by the Medical Research Ethics Committee of Hunan University of Medicine and adhered to the principles in the Declaration of Helsinki. All patients signed informed consent at the time of surgery.
Cell culture
All cells were purchased from ATCC and cultured at 37 ℃ in 5% CO2 incubator with saturate humidity. The SMMC7721 cell line was cultured in RPMI-1640 (Gibco, USA) and other HCC cells were cultured in DMEM (Gibco, USA). All medium were supplemented with 10% FBS (Gibco, USA) and 1% pencillin-streptomycin (Sigma, USA).
Cell transfection
MiRNA-485-5p minic, control minic (Ctrol minic), inhibitors, siRNA cotrol (si-Ctrol) and siRNA of MUC1 (si-MUC1) were synthesized by GenePharma (Shanghai, China). Small molecule RNA (minics, inhibitors, si-Ctrol and si-MUC1) were transfected into SMMC7721 cells using LipofectamineR3000 reagent in accordance with manufacturer’s protocol.
qPCR
Total RNA was isolated from cells by the TransZol Up Plus RNA Kit (TRANSGEN, China). Accordance with the manufacturer’s protocol, cDNA was synthesized by the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland). The expression of miRNA-485-5p and MUC1 were analyzed using the BsetarR SybrGreen qPCR Mastermix (DBI, GER) in according to the manufacturer’s protocol by StepOnePlusTM real-time PCR system (AB, USA). 18S, U6 were selected as the internal reference of MUC1 and miRNA-485-5p, respectively. All primers were synthesized by Sangon (Shanghai, China) and shown in Table 1.
Western blot
Total proteins from patients with HCC or cells were extracted using RIPA lysis buffer (P0013B, Beyotime, China) and quantified by BCA protein assay kit (PA101-01, Biomed, China) in according to the manual’s protocol. Total proteins (15 μg/well) were separated by 8% SDS-PAGE, tansferred onto a PVDF membrane (IPVH00010, Millipore, Germany), blocked with 5% skim milk (D8340, Solarbio, China) at room temperature (RT) for 1-2 h. The membranes were incubated with anti-MUC1 (ab181133, abcam, USA) or anti-β-actin (ab179467, abcam, USA) at 4 ℃ overnight, and then incubated with the anti-rabbit IgG (ab6721, abcam, USA) at RT for 1 h. Finally, the MUC1 and β-actin proteins were visualized by chemiluminescence.
Immunohistochemistry
Sections from paraffin embedded blocks of HCC tissue were cut at 1-2 μm thickness and dewaxed. Then, heat-induced antigen retrieval of sections was performed in an autoclave at 120 ℃ for 10 min. Nonspecific antigen was blocked by 2% bovine serum albumin. The sections were incubated with an anti-MUC1 at 4 ℃ overnight, followed by incubation with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG at RT for 30 min. The sections were incubated with 3,3N-Diaminobenzidine tertrahydrochloride for a few seconds and counterstained with hematoxylin. Finally, the sections were viewed under a microscope (Carl Zeiss, GER).
Proliferation assay
The SMMC7721 and Huh7 cells were seeded into 96-well paltes at the density of 1 x 104 cells/well and transfected small molecule RNA. It was considered as 0 h when cells were attached. The cell viability of HCC cells was measured with the cell count kit-8 (CCK-8, Dojindo, Japan) according to the instruction protocol at indicated time respectively.
Apoptosis assay
After 8 h of transfection, apoptosis was detected in according to the manufacturer’s protocol by flow cytometry (BD FACSCalibur, USA) using Annexin V-FITC kit (Dojindo, Japan). Briefly, the cells were suspended in 1x Annexin V binding solution at the concentration of 1x106 cells/ml. Then, the 100 μl of cells suspension were incubated with Annexin V and PI for 15 minutes in the dark. After dilution with 400μl 1x Annexin V binding solution, the apoptosis of cells was analyzed by flow cytometry.
Dual luciferase assay
The MUC1 3’ UTR wild-type (WT) or mutant (MT) binding to miRNA-485-5p were synthesized by Songon (Shanghai, China) and inserted into the pGL3 luciferase reporter vectors (Promega, USA) to generated pGL3-MUC1-WT and pGL3-MUC1-MT plamids. The pGL3-MUC1 vector (pGL3-MUC1-WT or pGL3-MUC1-MT) was co-transfected with miRNA-485-5p or control minic (Ctrol minic) into SMMC7721 cells using LipofectamineR3000 reagent in accordance with manufacturer’s protocol. After 48 h of transfection, luciferase activity was analyzed by SpectraMax® M5 Multi-Mode microplate reader (MD, USA).
Statistical analysis
All data from three independent experiments were presnted as the mean ± standard deviation and processed by SPSS23.0 and GraphPad Prism 7.0. The student’s t test was used for analyzing difference between two groups. The difference of among groups was analyzed with two-way ANOVA. The Spearman correlation analysis was performed to analyze the correlation between expression of miRNA-485-5p and expression of MUC1 in the patients with HCC. The p value <0.05 was considered as statistically significant.