Between January 2011 and January 2016 data from 177 HCC patients were obtained from Zhejiang University School of Medicine Sir Run Run Shaw Hospital (Hangzhou, Zhejiang, China) for the study.
This research was conducted with the approval of the ethics committee from Zhejiang University School of Medicine Sir Run Run Shaw Hospital. All participants signed a written acknowledgment of informed consent. The clinical characteristics of patients were retrieved from medical records. None of the subjects in this study received any pre-treatment with chemotherapy or radiotherapy prior to surgical resection. HCC tissue and the adjacent normal tissues were surgically resected from each patient. After collection, all specimens were promptly frozen with liquid nitrogen and stored at -78 to -80 degrees Celsius.
The human HCC cell lines (Sk-hep1, Huh7, L02, Hep-G2, LM3, HA22T, JHH7) were provided by the American Tissue Culture Collection (Manassas, Virginia, USA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) and supplemented with 10% fetal bovine serum (FBS) (Gibco), 10 U/mL penicillin and 10 mg/mL streptomycin. Cells were grown in a humidified atmosphere incubator at 37°C in 5% carbon dioxide. All cells were cultured according to the manufacturer’s guidelines.
Plasmids were used in this experiment. All transfections were done by utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA). Sk-hep1 and Huh7 cells were seeded into 24-well plates (6 × 104 cells per well) 48 hours after successful transfection. They were then transfected with 50nm plasmids purchased from (Tsingke Biological Technology, Beijing, China). The plasmids served as negative controls to allow for high expression or down-regulation of the cell lines.
Real-time Quantitative PCR (RT-qPCR)
Total RNA was isolated by using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc.). The quantity of total RNA was measured by using NanoDrop equipment (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions.
Complementary DNA (cDNA) was synthesized using a cDNA synthesis kit (Yeasen, Shanghai, China). qPCR was performed using the SYBR Green PCR Master Mix (Yeasen, Shanghai, China). All target genes were normalized to the endogenous reference gene GAPDH by using an optimized comparative Ct (2-ΔΔCt) value method. qPCR assay was performed on the 7500 Fast qPCR system using a One-Step SYBR PrimeScript RT-PCR kit (Applied Biosystems, Foster City, California, USA). The specific primer sequences of LOXL1-AS1 and GAPDH were as follows:
LOXL1-AS1 forward primer: GGTGCCACGGCTTACCAA
LOXL1-AS1 reverse primer: TCCTATCCCTGCCATTCCCA
GAPDH forward primer: GAAGGTGAAGGTCGGAGTC
GAPDH reverse primer: GAAGATGGTGATGGGATTTC
The relative gene expression was quantified using the 2-ΔΔCtmethod.
Fluorescence In Situ Hybridization (FISH)
The expression of LOXL1-AS1 was measured in paraffin-embedded tissue microarrays using an in situ hybridization detection probe. Digoxigenin-labeled sense and antisense LOXL1-AS1 probes were designed and made by Wuhan Servicebio Technology Co., Ltd. (China) for this purpose. The probe signals were detected using an optimization FISH kit (Wuhan Servicebio Technology Co., Ltd., China). We then followed the steps below.
The slides were deparaffinized and rehydrated before incubation with Proteinase K 15 μg/ml at 37°C for 40 minutes at room temperature.Than,The slides were then washed three times with phosphate-buffered saline (PBSor 15 minutes. Next, the slides were incubated with 5x saline-sodium citrate (SSC) solution at room temperature for 15 minutes. LOXL1-AS1 probes were added to the hybridization buffer for 1 hour at 50°C and then overnight at 4°C. The next day, the slides were washed with graded-diluted solutions at 50°C for 20 minutes and then placed in a blocking solution for 1 hour at room temperature. Finally, the slides were placed in a blocking solution containing alkaline phosphatase conjugated with anti-DIG Fab fragment overnight at 4°C.
The hybridization signals were visualized using NBT/BCIP (Thermo Fisher Scientific) according to the manufacturer's instructions. These slides were scored according to the staining intensity and number of positive cells. All images were acquired and scanned with an Eclipse Ci positive fluorescence microscope (Nikon Corporation). The laser-microscope was used to examine the results. The samples were divided into two groups, low expression and high expression.
Follow-up data were obtained via telephone contact. The end point was overall survival. Survival time was defined according to the dates between surgical resection and patient’s death or last follow-up.
Cell Proliferation Assay (CCK-8 assay)
Using the cell counting kit-8 (CCK-8) assay, transfected cells were incubated at a density of
2 x 103 cells/well into 24-well plates and then cultivated for 0, 24, 48 or 72 hours. After incubation 20 μL of CCK-8 reagent (Yeasen, Shanghai) was added to each well and cultured for another hour at 37°C. The absorbance at 450 nm was recorded with a standard microplate reader (Multiskan MK3, Thermo Scientific). The absorbance on days 1 to 3 was normalized to the absorbance on day 0, which was used as a control (100%). Each experiment was performed independently three times
Colony Formation Assay
HCC cells with concentrations of 1 x 103/mL were seeded in a 6-well plate. The culture medium was discarded after 2 weeks and the colonies were carefully washed with PBS two times. The colonies were fixed with 10% paraformaldehyde for 10 minutes and stained with 0.5% crystal violet for 20 minutes. After that, the formed colonies were counted and recorded.
Cell Migration Assay In Vitro
Assay cells were incubated in a 24-well plate equipped with a transwell chamber using an 8-μm pore size polycarbonate membrane (Corning). After transfection, cells were resuspended in a serum-free medium and plated into the upper chamber. The bottom chamber was filled with DMEM containing 10% FBS. The cells were incubated at 37oC and 24 hours after incubation the cells on the lower surface of the chamber were gently wiped clean with a cotton swab. The lower chamber was fixed using 95% ethanol for 20 minutes and then stained by 0.5% crystal violet for 10 minutes. The cells were then counted in five random fields under a microscope. Each experiment was independently performed three times.
Flow Cytometry and Apoptosis Analysis
HCC cell cycle and cell apoptosis analyses were performed via flow cytometry using a FACScan (BD Biosciences, USA). For the cell cycle analysis, cells were seeded into 6-well plates, then harvested and fixed with 70% ethanol at 4°C overnight, after which the cells were collected and resuspended in a binding buffer. The cells were stained with propidium iodide (50 μg/ml) containing 100 μg/ml of RNase A for 15 minutes.
Cells were harvested for the apoptosis analysis then washed with PBS and incubated with Annexin V-FITC and propidium iodide (Beyotime, China), according to the manufacturer’s instructions. The cells were then analyzed using a BD LSRFortessa cell analyzer (BD Biosciences, USA).
Western Blot Analysis
Proteins from analyzed cells were extracted using the RIPA lysis buffer (Beyotime, Shanghai, China) with a protease inhibitor. Protein concentration was determined using a Bio-Rad protein assay system (Bio-Rad, Hercules, California, USA). All of the extracted protein was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked in Tris-buffered saline (TBS) containing 5% non-fat milk at room temperature for 1 hour, followed by an overnight incubation at 4°C with the appropriate primary antibody. After incubating overnight, the membranes were washed by TBS with Tween 20 (TBST) three times and then the second corresponding antibody (Beyotime, Shanghai, China) was applied.
The following primary antibodies were used: CDK2 (1:1000, Cell Signaling Technology, Massachusetts, USA), CDK4 (1:2000, Abcam, Cell Signaling Technology, Massachusetts, USA) and Tubulin (1:20000, Abcam). Incubation with the secondary antibodies was done overnight at 4°C, then for another 2 hours at room temperature with a horseradish peroxidase-conjugated anti-rabbit antibody . After that, the proteins were visualized using the ECL Western blotting kit (Hangzou Fude Biological Co., Ltd., China) to check for color reaction. The densities of the bands were detected using BioImaging Systems (Bio-Rad, Hercules, California, USA). All experiments were performed three times.
All statistical analyses were implemented using SPSS 20.0 software (SPSS, Inc., Chicago, Illinois) and GraphPad Prism 6.0 (Graph-Pad Software, Inc., USA). The experimental data were presented as mean ± SD. The survival curves were calculated using the Kaplan-Meier method and the differences were assessed by a log-rank test. Statistical significance was tested using the Student’s t-test or a Chi-square test. The Pearson’s test was used to analyze the relationship between LOXL1-AS1 expression and the clinicopathologic features of HCC. The Student’s t-test was used to detect significant differences in data obtained from qPCR experiments and colony formation assays. A multi-way classification analysis of variance tests was performed to assess data obtained from the CCK-8 assays, tumor growth and correlations among LOXL1-AS1 expression. All data were presented as mean ± SD. A p value less than 0.05 indicated statistical significance.