Datasets
Clinical information and RNA sequencing data of HCC and paired normal tissues were downloaded from TCGA data up to February 7, 2019. HCC RNA sequencing and microarray datasets GSE55191 and GSE67260 downloaded from GEO datasets was analyzed using Affymetrix Human Genome U133 Plus 2.0 Array. The differently expressed lncRNAs (DElncRNAs) between HCC tumor and adjacent normal tissues were screened using edgeR package of the R platform, with adjusted P< 0.05 and the thresholds of |log2FC| >2.0.
Survival analysis and Clinical significance of DElncRNAs
The association of each lncRNA expression level and overall survival (OS) was calculated by univariate Cox model. Then, the contribution of lncRNA as independent prognosis factors of OS was evaluated via multivariate Cox analysis. Kaplan-Meier curves of DElncRNA was plotted using the “survival” package in R software with P< 0.05.
The association of LncRNA with clinicopathological characteristics of HCC patients, including age (over or under 60 years), gender (male or female), clinical stage (I, II or III, IV), histologic grade (G1, G2, G3 or G4), Pathologic(T,N,M), and risk factors (alcohol consumption, hepatitis B, hepatitis C, no history of primary risk factors and non-alcoholic fatty liver disease) were analyzed with P<0.05.
Tissue collection
Fifteen paired HCC tumors and adjacent normal tissues were collected from HCC patients without any local or systemic anticancer treatment before surgical resection at the Second Affiliated Hospital of Nanjing Medical University. The specimens were collected during surgery, and immediately kept in RNA Later stabilization solution (Invitrogen, USA) and froze. Ethics approval was obtained from the Ethics Committee of the Second Affiliated Hospital of Nanjing Medical University and written informed consent was obtained from all participants.
Cell culture and transfection
Human normal liver cell line L02 and HCC cell lines HepG2, Hep3B, and Huh7 used in this study were stored in our laboratory. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, USA), supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator with 5% CO2 atmosphere at 37℃. The siRNAs sequences were chemically synthesized by Gene Pharma Company (China) and listed as follows: SREBF2-AS1, si-1: 5′-CCAUCAUACAUGCCUGCAATT-3′; si-2:5′-CGUCCGGUCAUCAUCUUAAT-3′; si-3:5′-GCAGCUGGUCGUGUUGUAA TT-3′. si-SREBF2: 5′-GCUGGUAAAUGGUGUGAUUTT-3′. Negative control (NC): 5′-UUCUCCGAACGUGUCACGUT T-3′. For tumor assay in nude mouse model, siRNAs were modified by 2OMe + 5Chol (Ribobio, China). Full-length SREBF2-AS1 cDNA was synthesized and subcloned into pcDNA3.1 vector (Genebay, China). Cells in the logarithmic growth phase at 70% confluence were transfected with siRNAs or vectors by lipofectamine 3000 reagent (Invitrogen, USA).
Fluorescence in situ immunohybridization (FISH)
The probe of SREBF2-AS1 was designed and synthesized by Invitrogen (Shanghai, China), and its sequences were 5′-ACGCACCGCTTCGCTCGCCCATTG G-3′. Cells were fixed in 4% formaldehyde, washed, and treated with pepsin K for 3 min. Next the cells were air-dried and incubated with FISH probe diluted in hybridization buffer. After the hybridization, the cells were washed, dehydrated, and visualized under a fluorescence microscope (DMI4000B, Leica, German).
RNA isolation and detection of lncRNA and mRNA
The total RNA was isolated from cell lines or tissue samples using TRIzol® reagent (Invitrogen, USA) and reversely transcribed to cDNA using HiScript® II Q RT SuperMix kit (Vazyme, China). Then, qRT-PCR analyses were performed using the SYBR Green PCR Master Mix (ThermoFisher, USA). The primers were as follows: GAPDH (forward: 5′-TGT GGGCATCAATGGATTTGG-3′, reverse: 5′-ACACCATG TATTCCGGGTCAAT-3′), SREBF2-AS1 (forward: 5′-GTCATCCAATCCCGCTTC T-3′, reverse:5′-GTTCCGA GGTGCCAGAGATT-3′), SREBF2 (forward: 5′-CAGAC ATCATCTGTCGGTGG T-3′ and reverse: 5′-CGCAA TGGCAGAAGGAACTC-3′). The relative gene expression was analyzed by 2△△ct method in triplicate
Western blot analysis
Proteins were extracted from cells or tissue samples using RIPA buffer containing Phenyle methane sulfonyl fluoride (Beyotime, China). Proteins were separated by SDS‐PAGE and transferred onto PVDF membranes, which were then blocked with 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 (TBST) and incubated overnight with following primary antibodies: rabbit polyclonal antibody SREBF2(Abcam, USA) and mouse monoclonal antibody GAPDH (Abmart, China). The membranes were washed with TBST and then incubated with horseradish peroxidase conjugated secondary antibodies: goat anti-rabbit IgG (Biosharp, China) or goat anti-mouse IgG (Abmart). Finally, the immunoreactive protein bands on the membrane were visualized using an ECL Kit (Beyotime, China).
Cell proliferation assay
Cells proliferation was evaluated using Cell Counting Kit-8 assays (CCK8) (Dojindo Laboratories, Japan). Huh7 and HepG2 cells transfected by siRNAs after 24h were seeded into the 96-well plates at a density of 5,000 cells/well and then incubated for 0 h, 24 h, 48 h and 72 h. Subsequently, 10 µl CCK8 solution was added to each well and incubated for 2 h at 37˚C. The optical density (OD) of 450 nm was measured by a microplate reader.
Colony formation assay
Huh7 and HepG2 cells were transfected by siRNAs and after 24 h were seeded into 6-well plates with 500 cells per well. After culture for two weeks, most of the colonies contained at least 50 cells, the colonies were fixed in methanol and stained with 0.1% crystal violet.
Flow-cytometric analysis for apoptosis and cell cycle
For apoptosis assay, Huh7 and HepG2 cells were harvested after transfection for 24h. Firstly, 5×105 cells were suspended in binding buffer and stained by Annexin V-FITC and Propidium Iodide (PI) (BD Biosciences, USA). Secondly, cells were vortexed and then incubated for 15-20 min after mixing, and immediately subjected to flow cytometry within an hour. For cell cycle assay, cells were collected and fixed using 70% ethanol at -20 ˚C for 18 h, and stained with 400 ul of propidium iodide (PI) for 30 min. The results were analyzed with FlowJo software (FlowJo LLC, USA). The Calculation of apoptosis rate was: (Q2+Q3)/(Q1+Q2+Q3+Q4). Q2: early apoptosis; Q3: later apoptosis.
Xenograft model
Nude mice (male, five-weeks old) were subcutaneously injected with 1 × 107 HepG2 cells randomly and divided into two groups (n=5). When tumors grew to 50 mm3, siRNAs modified by 2OMe + 5Chol were injected into tumors in si-SREBF2-AS1 group every two days, while NC group were injected with control siRNAs. Tumor diameter was measured every two days by digital calipers. After three weeks, the mice were sacrificed and the tumor size and weight were measured. The protocol and procedures employed were ethically reviewed and approved by Animal Ethical and Welfare Committee of NJMU.
Immunohistochemistry
The tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into sections (4 µm thin). After antigen retrieval, the sections were blocked with bovine serum albumin, and incubated with primary antibody at 4°C overnight. Next, the sections were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h, and staining signal was detected with Tissue Staining HRP-DAB Kit (DAKO).
Statistical analysis
Data were analyzed using GraphPad Prism 7 (GraphPad, USA). The comparison of two groups was performed using Student’s t-test, and analysis between multiple groups was conducted by one-way analysis of variance (ANOVA) with the Bonferroni correction. P value< 0.05 indicated significant difference.