Clinical Sample and Tissue Specimen Acquisition
Hepatocellular carcinoma specimens and adjacent noncancerous tissues were obtained from Harbin Medical University Cancer Hospital (HMUCH), and patients with a histological diagnosis of hepatocellular carcinoma who had received neither chemotherapy nor radiotherapy before surgical resection were recruited for the present study between 2009 and 2019. This study conformed to clinical research guidelines and was approved by the research ethics committee of Harbin Medical University Cancer Hospital. We obtained written informed consent from all patients.
The human immortalized normal hepatocyte cell line LO2 and HCC cell lines HepG2, Hep3B, Huh7 and SMMC-7721 were obtained from the Chinese Academy of Sciences Cell Bank and Cellbio (China) and were cultured according to the suppliers’ instructions. All cell lines were cultured according to instructions.
Plasmids and Transfection
LINC00645 and sh-LINC00645 and controls were constructed by Genechem (Shanghai, China). Concentrated viruses were used to infect 5 × 105 cells in a 6-well plate with 4–6 µg/ml Polybrene. The infected cells were then subjected to selection with 1 µg/ml puromycin (Cat#540411, Calbiochem, USA) for two weeks. Stable over-expression cell lines or knockdown cell lines were identified using qRT-PCR or western blotting. The miR-141-3p mimic and inhibitor were purchased from Ribobio (Guangzhou, China). Oligonucleotide or plasmid transfection into HCC cells was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were collected for further experiments at 48 hours after transfection. The shRNA sequences are listed in Table S1.
RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was prepared with a Transcriptor First Strand cDNA Synthesis Kit (Cat# 04897030001, Roche, USA). Real-time PCR was performed using FastStart Universal SYBR Green Master (ROX) (Cat#04913914001, Roche) on a 7500 Fast Real-Time PCR system (ABI, USA). For the quantification of gene expression, we used the 2−ΔΔCt method. GAPDH or U1 expression was used for normalization. The primer sequences were synthesized by Genepharma (Shanghai, China). All the primer sequences are available in Table S2.
Nuclear/cytoplasmic fractionation was performed by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Cat#78835, Thermo Fisher) according to the manufacturer’s protocols. U1 was used as a nuclear control, while GAPDH was used as a cytoplasmic control.
Cell Proliferation and Colony Formation Assay
Cell proliferation was measured by the CCK-8 method and colony formation method. Briefly, in the CCK-8 experiment, 1 × 103 cells were cultured in 96-well plates at 37˚C. 96 well plates were incubated with 10 µl of CCK-8 solution per well for 1 hour. Cell proliferation curves were drawn by measuring the absorbance at 450 nm at each indicated time point. The cell proliferation curves were plotted by measuring the 450 nm absorbance at each indicated time point. Experiments were performed in triplicate. For the colony formation assay, cells were exposed to the indicated treatments, were seeded in 6-well plates and were cultured for 2 weeks. Cell colonies were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and imaged using an optical microscope.
Ethynyl Deoxyuridine (EdU) Incorporation Assay
A Cell-Light™ EdU Apollo567 In Vitro Kit (Catalogue Number C10310–1, RiboBio, China) was used to perform the EdU proliferation assay according to the manufacturer’s instructions as previously described.
Cell Migration and Invasion Assay
Cell migration and invasion assays were performed as described previously. In a wound healing assay, cells were seeded in 6-well plates to form a confluent monolayer. Then, a scratch wound was induced by a pipette tip. Photographs of cells migrating to the scratched area were taken and the data were shown as a percentage of the control group. In the transwell invasion and migration assay, cells (5 × 104 cells per well) were seeded in the upper chambers of the transwell plate and placed in FBS-free medium with or without matrix gel. After 24 hours of incubation, the cells that invaded/migrated to the lower surface of the membrane were fixed, stained with crystal violet and observed using an inverted microscope.
Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed using a commercially available kit (Beyotime) according to the manufacturer’s protocol. Briefly, cells were cross-linked with 1% formaldehyde and were sonicated on ice to create 200–500 bp fragments. Stained chromatin was cultured overnight with an anti-MAZ antibody (Novus Biologicals, NB100-86984, 1:50) or IgG (Cell Signaling Technology, Cat#3900)as an isotype control. The precipitated chromatin DNA was recovered and analyzed by qRT-PCR. The primer sequences are shown in Table S2
RNA Immunoprecipitation (RIP)
RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation (RIP) Kit (EMD Millipore). HepG2 cells lysis solution (Sigma Aldrich Chemical Company, USA) was added to 3 mg of cells and left to incubate at 4 ˚C for 1 h. Cells were centrifuged at 12,000 g at 4 ˚C for 10 min in order to collect the supernatant, which was employed for RIP experiments using an anti-AGO2 antibody (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.. The RNA fraction isolated by RIP was subjected to qRT-PCR analysis to identify the direct binding between linc00645 and miR-141-3p. These experiments were repeated three times.
Western Blot Analysis
Cells were lysed with RIPA extraction reagent (Beyotime) supplemented with a protease inhibitor cocktail (Roche). Proteins were separated by 6%-15% SDS-PAGE, transferred to 0.22 mm polyvinylidene fluoride membranes (Millipore), and then incubated with antibodies. The bands on the blots were captured by using an Odyssey Infrared Imaging System (LI-COR Biosciences) and were quantified with Odyssey v1.2 software (LI-COR Biosciences). GAPDH was used as the internal controls. Antibodies against the following proteins were used. MAZ (Abcam, ab85725,1:1000), GP73 (Santa Cruz, sc-365817,1:500), E-cadherin(Cell Signaling Technology, Cat#3195,1:1000), N-cadherin(Cell Signaling Technology ,Cat#4061,1:1000), Cyclin D1 (Cell Signaling Technology ,Cat#2978,1:1000), GAPDH (Cell Signaling Technology Cat#5174,1:1000). Alexa Fluor® 800 goat anti-mouse (LI-COR Biosciences, Cat#926-32210, 1:10000) or anti-rabbit (LI-COR Biosciences, Cat#926-32211, 1:10000) was used as a secondary antibody.
Immunohistochemical (IHC) Analysis
The paraffin-embedded sections were dewaxed in xylene and rehydrated in alcohol. Endogenous peroxidase was blocked by 3% H2O2, and microwave heating was performed for antigen retrieval. After blocking nonspecific antigen binding with 5% BSA at 37 °C for 1 h, the sections were incubated with a specific primary antibody against Ki67 (Abcam, ab15580, 1:1000), at 4 °C overnight. After incubating with the corresponding secondary antibodies ( Abcam, ab205718) at 37 °C for 1 h, the sections were stained with diaminobenzidine and counterstained with hematoxylin. Representative images were taken using an Olympus light microscope
Luciferase Reporter Assays
HepG2 cells were seeded at 5 × 104 cells/well in 24-well plates and were cultured overnight. On the next day, the cells were cotransfected with pmirGLO-LINC00645-WT, pmirGLO-LINC00645-MUT, pmirGLO-GP73-3’UTRWT, pmirGLO-GP73-3’UTR-MUT reporter plasmids (Genechem, Shanghai), NC mimics or miR-141-3p mimics. Twenty-four hours posttransfection, cells were lysed using passive lysis buffer (Promega), and the luciferase activity was measured by a GloMax20/20 Luminometer (Promega) using the Dual-Luciferase Reporter Assay System (Promega) and was normalized to the Renilla luciferase activity.
Tumor Xenograft Model
The animal study protocol was approved by the Institutional Animal Care and Use Committee of Harbin Medical University Cancer Hospital and was performed in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council). Mouse xenograft models were established using 4-week-old BALB/c nude female mice. HepG2 cells (5 × 105 per injection) that were transfected with sh-LINC00645 and sh-control, respectively, were implanted into the mice via subcutaneous injection. Tumor volumes were measured every 3 days after being apparently observed and calculated with the following formula: Volume = (length × width2)/2. After 4 weeks, all mice were sacrificed under anesthesia.
The Cancer Genome Atlas (TCGA) (https://tcga-data. nci.nih.gov) was used to compare the expression of LINC00645 in HCC tissues and normal by using R software. The LncBook (https://bigd.big.ac.cn/lncbook), starBase V3.0 (http://starbase.sysu.edu.cn/) and TargetScan (http://www.targetscan.org/) databases were used to examine putative miRNA interactions between LINC00645 and GP73. The MAZ binding motif in the promoter region of LINC00645 was identified by TRAN SFAC (http://gene-regulation.com/) and JASPAR (http://jaspar.genereg.net/).
Data are expressed as mean ± SEM. And all data represent at least three independent experiments. Statistical analysis was performed using unpaired Student t-test or 1-way ANOVA followed by Tukey post hoc analysis. P < 0.05 was considered statistically different and indicated by *P < 0.05, ** P < 0.01, ***P < 0.001.