The present study was conducted with the permission of the Ethics Committee of Wannan Medical College. All participants provided written informed consent. HCC tissues were surgically resected and stored in liquid nitrogen for further analysis.
Cell culture and reagents
The human HCC cell lines (Hep3B and Huh-7) and human normal hepatic cell line (LO2) were purchased from ATCC ((American Type Culture Collection, Manassas, US). The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (GIBCO, MA, USA). SiRNA-SLC16A1-AS1 and negative control miRNA were synthesized by Biossci Company (Wuhan, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, CA, USA).
Quantitative real-time PCR (qRT-PCR)
The total RNA was isolated from cells and tissues using Trizol reagent (Life Technologies, USA). First-strand cDNA was carried out using using reverse transcription Reagents (ABI, CA), QRT-PCR was performed using SYBRH Select Master Mix for CFX (Invitrogen) and using the CFX Connet TM real-time PCR system (Bio-Rad, USA). GAPDH and U6 were chosen as the internal standard.
Cell viability assay
Cell viability was measured using Cell Counting Kit-8 assay (Beyotime, China). After being inoculated into 96-well plates at a density of 2 × 103 cells/well, cells were stained with 20 µL of CCK8 reagent 48 hours after transfection. Cell viability was determined at the 450 nm absorbance.
In invasion test, 1*105 cells were inoculated into the upper chambers (8 µm pore diameter, BD, USA) with serum-free medium. Complete medium (600 µL) was added to the bottom chamber to simulate the chemoattractant. 48 h after incubation, cells on the top membrane were wiped out. Invaded cells on the bottom surface were fixed and stained. counted in five random scopes and photographed.
Wound healing assay
Cells were seeded in 6-well plates. After 12 h of incubation, wounds were generated with a 200 µL pipette tip. Cells were washed and incubated with serum-free medium for 48 h. The wounded gap was photographed at different time points. Cell migration was caculated as wound closure (%) = (wounded area (A0-A1)/wounded area A0) × 100%.
The psi-CHECK-2 psi-check Luciferase Expression Reporter (Promega) vector was used to synthesize SLC16A1-AS1 vector. Hep3B and Huh-7 cells were used for the dual-luciferase-reporter assays. Luciferase activity was measured and compared 48 h later. Luciferase assay was performed 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega, WI, USA).
Cells were washed twice with PBS, and total protein was extracted using RIPA buffer (Beyotime Biotechnology, China). Protein concentration was determined using a BCA Protein Assay Kit (Beyond Biotechnology). Protein amples were separated using 10 % SDS-polyacrylamide gel (Solarbio, Beijing) after denaturation. Then, proteins were transferred to a PVDF membrane (Millipore, USA) which were blocked with 5% skimmed milk for 1 h. The blots were incubated overnight with the first antibodies (anti MITD1 antibody, 1:5000, Santa Cruz; anti-GAPDH antibody, 1:5000, Santa Cruz.). ECL (Millipore) After incubation with secondary antibodies, an enhanced chemiluminescence reagent was applied for detection.
All statistical analyses used SPSS v22.0 (SPSS Inc., Chicago, USA). Each experiment was repeated at least three times. Data was shown as mean ± standard deviation. The significance of differences between groups were assessed by Student’s t test and ANOVA test. P-values < 0.05 were considered statistically significant.