HCC tissues and cell lines
HCC samples and adjacent tissues were obtained from Shandong Provincial Hospital Affiliated to Shandong University. The clinicopathological features of patients with HCC were summarized in Table 1. Written consent was obtained from all participants who were involved in the study. All tissues were frozen in liquid nitrogen after surgery and stored at -80 °C. This study was approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University. The HCC cell lines (HepG2, SNU449, SMMC-7721, Huh7 and PLC/PRF/5), HEK-293T and normal liver cell lines (LO2) were purchased from the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in DMEM containing with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/ml penicillin/streptomycin in a humid incubator at 37 °C.
pcDNA3.1 vector, pcDNA3.1 vector carrying ASPM (ASPM), miR-26b-5p mimic, negative control oligonucleotides (miR-NC), miR-26b-5p inhibitor (anti-miR-26b-5p), negative control oligonucleotide (anti-miR-NC), small interfering RNA of ASPM (si-ASPM) and scramble siRNA (si-Con) were bought from RiboBio (Guangzhou, China). Cells were transfected utilizing lipofectamine 3000 (Thermo Fisher Scientific) following to manufacturer’s protocol. sh-NC and shRNA targeting ASPM (sh-ASPM) were purchased from RiboBio (Guangzhou, China) and transfected into SMMC-7721 cell. To stably overexpress miR-26b-5p in SMMC-7721 cell, the lentiviral packaging kit was used. Lentivirus containing miR-26b-5p was packaged following the manufacturer’s manual. Lentivirus was packaged in HEK-293T cell and secreted into culture medium. SMMC-7721 cell was infected by lentivirus carrying miR-26b-5p with the presence of polybrene (Sigma) and selected by puromycin (Sigma) for two weeks. To overexpress ASPM in SMMC-7721 cells, lentivrius carrying ASPM cDNA (GeneCopoeia) were packaged and used to infect cell following to manufacturer’s protocol.
Total RNAs were extracted using Trizol reagent (Thermo Fisher Scientific). miRNAs were extracted using RNAsimple Total RNA kit (Tiangen Biotech Co., Ltd., Beijing, China). cDNA was synthesized using a TransScript First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China). qPCR reactions were performed using PowerUp SYBR Green kit (ABI, Foster City, CA, United States) and QuantStudio 6 System (ABI). For miR-26b-5p, total RNA (2 µg) was transcribed into cDNA using a microRNA First-Strand cDNA Synthesis Kit (Sangong Biotech, Shanghai, China) according to the manufacturer's protocol. Data was analyzed using the comparative Ct method (2−ΔΔCt). U6 or GAPDH was served as the internal control. Primers used were listed below: ASPM, forward primer (5′-GGCCCTAGACAACCCTAACGA-3′) and reverse primer (5′-AGCTTGGTGTTTCAGAACATCA-3′); GAPDH, forward primer (5′-TGGATTTGGACGCATTGGTC-3′) and reverse primer (5′-TTTGCACTGGTACGTGTTGAT-3′); miR-26b-5p, forward primer (5′-TTCAAGTAATTCAGGATAGGT-3′) and reverse primer (5′-CTTCGCAGCATACT-3′); U6, forward primer (5′-CTCGCTTCGGCAGCACA-3′) and reverse primer (5′-AAAATATGCAACGCTTCACG-3′).
ASPM 3′-untranslated region (3′-UTR) carrying the binding sites for miR-26b-5p was cloned into psiCHECK-2 vector (Promega, Madison, WI, USA) to construct wild-type (wt) reporter vector (wt-ASPM) or mutant (mut) reporter vector (mut-ASPM). The ASPM 3′-UTR-mut was constructed by mutating the binding sites. Luciferase vector plasmid and miR-26b-5p mimics were cotransfected into SMMC-7721 and PLC/PRF/5 cells. After 48 h, the luciferase activity was detected using Luciferase Assay Kit (Promega).
Cell Proliferation And Colony Formation Assays
Cell growth was detected using CCK-8 Kit (Beyotime, Nanjing, China) according to the protocol. In colony formation assay, cells were cultured into six-well plates and incubated for two weeks. After that, cells were stained and photographed.
Migration And Invasion Assays
Cell migration assay was performed using transwell chamber (BD, Franklin Lakes, NJ, United States). 5 × 104 SMMC-7721 and PLC/PRF/5 cells were seeded into the upper chambers and the lower chambers were filled with medium with 20% FBS. Cell migration was assessed by counting migrative cells under an inverted microscope. Cell invasion assay was conducted using transwell chamber coated with Matrigel (BD). 5 × 104 SMMC-7721 and PLC/PRF/5 cells were seeded into the upper chambers and the lower chamber was filled with DMEM with 20% FBS. The number of invasion cell was counted under microscope.
miR-NC or miR-26b-5p transfected SMMC-7721 cells were subcutaneously implanted into nude mice (Beijing Vital River Laboratory, Beijing, China). Tumor volume = 0.5 x length x width2. Tumor volume was examined every week. Mice were euthanized at 5 weeks post implantation. The tumor tissues were extracted for further immunohistochemical staining (IHC). All procedures involving experimental animals were performed in accordance with protocols that were approved by the Committee for Animal Research of Shandong Provincial Hospital Affiliated to Shandong University and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86 − 23, revised 1985).
Proteins were extracted from cells using RIPA buffer. 20 µg protein lysis were loaded onto 10% SDS-PAGE. After separated, the proteins were transferred to PVDF membrane (Millipore, Braunschweig, Germany). The membrane was incubated with anti-GAPDH (ab8245, Abcam, Cambridge, UK) or ASPM (26223-1-AP, Proteintech Group, Rosemont, IL, USA) at 4 °C overnight. After washed with TBST, the membrane was incubated with secondary antibody for 2 h and the bands were detected using Enhanced chemiluminescence (ECL) reagent (Pierce, Rockford, IL, USA).
GraphPad Prism software was used for statistical analyses. Data were presented as Mean ± SD. Differences were calculated using Student’s t-test or Mann-Whitney U test. The correlation analysis was performed by Spearman test. P < 0.05 was statistically significant.