Patient recruitment and tissue collection
We recruited 8 HBV-related and pathologically confirmed primary HCC patients, who received radical hepatic resection at the Zhongshan hospital, Fudan university between October and December in 2011. None of the patients had received any auxiliary therapy before surgery. Primary HCC and adjacent normal tissues were obtained from patientsThe study protocol was approved by the Ethics Committee of Zhongshan Hospital, and informed consent was obtained from all subjects prior to inclusion in the study.
Expression profiles of LncRNAs from 8 human HCC and matched normal tissues were analyzed using ArrayStar Human LncRNA Microarray (8×60 k, ArrayStar, Rockville, MD, version 2.0). This dataset comprises a total of 33,045 lncRNAs collected from several databases, such as RefSeq, the UCSC Known genes, and Ensembl. Total RNA was extracted, amplified, and transcribed into fluorescent complementary RNA (cRNA) using the Quick Amp Labeling Kit, One-Color (Palo Alto, CA, USA) according to the manufacturer’s instructions. Labeled cRNAs were hybridized onto the human lncRNA arrays, and after washing steps, the arrays were scanned using the Agilent Microarray Scanner (G2565BA) and analyzed with the Feature Extraction software (version 126.96.36.199). Quantile normalization and data processing were performed using the GeneSpring GX software (version 11.5.1, Agilent Technologies). The threshold for screening of differential lncRNAs comprised a fold change >2.0 and P value <0.05.
Subsequent processing of DNA methylation microarray data was conducted using ArrayStar Human 2.1M LncRNA Promoter Microarray (Rockville, MD, USA). Briefly, total DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA), sonicated, denatured and immunoprecipitated with anti-5- methylcytosine antibodies. The DNA-antibody complex was isolated and purified by immunomagnetic beads (Millipore, Bedford, MA, USA), followed by methylated DNA Immunoprecipitation (MeDIP) as previously described (18). After quality control, MeDIP and input DNA fragments were amplified using the Sigma WGA kit, and purified using the QIAquick PCR purification kit (QIAGEN, Valencia, CA, USA). Next, the fluorescent-labeled MeDIP (Cy5) was mixed with input DNA (Cy3), denatured and hybridized onto the DNA methylation microarray. The mixture was washed, and the arrays scanned and analyzed. To accurately quantify methylation levels of CpGs, we applied a novel analytical methodology, known as modeling experimental data with MeDIP enrichment (MEDME), which utilizes the absolute methylation score (AMS) as an indicator of DNA methylation to identify differentially methylated regions (DMRs) of lncRNA promoters. Next, we merged differentially methylated probes, based on AMS, into candidate DMRs, then recalculated and re-tested the average AMS of candidate DMRs via t-test. Finally, we selected DMRs with average AMS that were significantly different between two groups, and defined them as AMS DMRs.
RNA isolation, reverse transcription and quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from tissues or cells using the TRIzol reagent (Invitrogen), then 2 µg of the RNA reverse transcribed to complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription kit (Ambion Inc., Austin, USA) according to the manufacturer’s instructions. qRT-PCR was performed using the Power SYBR Green PCR Master Kit (Applied Biosystems, Foster City, CA) on the ABI 7900HT Fast Real-Time PCR System, targeting specific genes whose primers are listed in Supplementary Table 1. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as an endogenous control, while relative expression of LncRNAs was evaluated using the 2 −ΔΔCt method. All amplifications were performed in triplicates.
Bisulphite modification combining sequencing PCR (BSP) and Methylation specific PCR (MSP)
Total genomic DNA was isolated from the tissues using the QIAamp DNA Mini Kit (QIAGEN) and 500 ng bisulfate-modified using the Methyl Code™ Bisulfite Conversion Kit (Invitrogen, Carlsbad, CA, USA). Bisulfate-treated DNA was amplified and cloned into the pMD18-T vector. Methylation level for each site is indicated as the mean percentage of the total methylation on the sequencing data obtained from 10 clones. MSP was conducted as previously described(19), targeting genes whose primers are listed in Supplementary Table 2
Human hepatocellular carcinoma cell lines (HepG2, SMMC-7721, MHCC-97L, MHCC-97H, HCC-LM3) and the normal hepatocytes cell line (L02) were obtained from the Liver Cancer Institute, Fudan University (Shanghai, China), while human HCC cell line (Huh-7) was purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Mycoplasma detections revealed negative results in all cell lines. All cells were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco-BRL, Grand Island, New York, USA), supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, Grand Island, New York, USA), and maintained in a humidified incubator with 5% CO2 and a temperature of 37℃.
Synthesis of lnc-SCARF1, plasmids and lentivirus construction and transfections
Full-length lnc-SCARF1(NR_028076) was cloned and synthesized by Sangon (Shanghai, China), and transfected into pCDH vectors (CD513B-1, bylensystembio science). The resultant pCDH expression constructs were then cloned into Pseudoviral Particles. Thereafter, MHCC-97H or HepG2 cells were co-cultured with lentiviral vectors, with non-transfected cells eliminated using puromycin.
Cell proliferation assay
Proliferation of MHCC-97H and HepG2 cells overexpressing lnc-SCARF1 (Lv-lnc-SCARF1) and controls (Lv-GFP) was evaluated using the CCK-8 assay kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer’s instructions. Briefly, cell lines were seeded into 96-well plates (Corning Costar Corp., Kennebunk, ME, USA), at a density of 3 × 103 per well, and incubated overnight with 200 μl of cell culture medium at 37℃. The rate of cell proliferation was determined at 24, 48, and 72 h after seeding.
Cell invasion assay
The invasive ability of MHCC97H-Lv-lnc-SCARF1 and HepG2-Lv-lnc-SCARF1 cells were also tested using 6.5-mm Transwells with 8.0-μm pore polycarbonate membrane in 24-well plates (Corning Costar Corp., Kennebunk, ME, USA). Briefly, the transwell chambers were first coated with 100 μl Matrigel (0.8 mg/ml) and incubated for 2 h at 37℃ . Next, 3 × 105 cells were inoculated into 100 μl of serum-free medium, and seeded onto the upper compartment of the chamber, while the lower chamber was loaded with 600 μl DMEM supplemented with 10% FBS, followed by a 24-h incubation at 37℃. The cells and Matrigel on the upper surface were then removed, and invasive cells located on the lower surface fixed and stained with Giemsa. The invasive cells were numbered in five microscopic fields (×200 magnification). All individual experiments were performed in triplicate.
Determination of cell apoptosis
Cell apoptosis was performed using the Annexin V-PE Apoptosis Detection Kit (Becton Dickinson, San Jose, CA, USA) according to the manufacturer’s instructions, with apoptotic cells detected via flow cytometry (Becton Dickinson, San Jose, CA, USA).
Western blot assay
Total proteins were extracted from cell lysates using 100 μL pre-cooled lysis buffer (1 ml RIPA buffer + 25 μL PMSF + 110 μL Phosphatase inhibitor cocktail), then quantified using the BCA protein quantification kit. Next, 30 μg of denatured proteins were separated on a 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with TBST containing 10% nonfat milk powder for 2 h, and incubated overnight with primary antibodies of CUL9 at 4℃. Next, the membranes were washed with TBST for 30 mins and incubated with horseradish peroxidase (HRP)–labeled secondary antibodies (Jackson ImmunoResearch). The membranes were then exposed on the Pierce ECL Plus (Thermo Fisher Scientific, MA, USA) in the ChemiDocTM XRS+ system (Bio-Rad, CA, USA), and images acquired and analyzed using the Image Lab Software.
Chromatin isolation by RNA purification (ChIRP) and mass spectrometry (MS)
MHCC-97H and HepG2 cells were used for the ChIRP-MS experiment (100 million – 500 million cells depending on the cell type). Summarily, cell harvesting, lysis, disruption, and ChIRP procedures were performed as previously described(20). Thereafter, protein samples were size-separated on bis-tris SDS-PAGE gels (Invitrogen) then subjected to western blot assay and mass spectrometry.
Establishment of a xenograft tumor mouse model
MHCC97H-Lv-lnc-SCARF1, and HepG2-Lv-lnc-SCARF1 cells were subcutaneously inoculated into the left armpits of 6 male BALB/c nude mice (4-6 weeks old), while control cell lines were inoculated into the right armpit. The sizes of subcutaneous tumors were measured at day 0, 7, 14, 21, and 28 after injection, And on the 28th day, all animals were euthanized, active tumors removed and cut into small pieces (2×2×2 mm3). Orthotopic implantation was conducted on 24 male athymic BALB/c nude mice (4-6 weeks old). Briefly, each mouse was orthotopically implanted with a tumor mass, into the left lobe of their liver (12 mice per group). 6 mice were implanted with lnc-SCARF1 overexpression tumor mass while the other six were implanted with controls. All mice were euthanized by intraperitoneal injection of pentobarbital, after 4 weeks, then tumor sizes, location, and frequency of both intrahepatic and extrahepatic tumors detected and measured. Athymic BALB/c nude mice were housed in laminar-flow cabinets, under specific pathogen-free conditions, and handled according to the recommendations of the ARRIVE guidelines for the care and use of laboratory animals. All related experimental protocols were approved by the Shanghai Medical Experimental Animal Care Committee.
All statistical analyses and illustration were performed using SPSS version 23.0 for Windows (SPSS Inc., Chicago, IL, USA) and Prism.v8.0 for Mac (GraphPad Software, La Jolla, CA, USA). Continuous data were first subjected to normality tests, then expressed as means ± standard errors of the mean (SE). Differences in tumor recurrence, between control and treatment groups, were determined using the Chi-square test, while cumulative survival and recurrence rates were estimated using the Kaplan-Meier method with a log-rank test. Independent prognostic factors were calculated using the Cox proportional hazards regression model. Data followed by P < 0.05 were considered statistically significant.