ARF6 promotes hepatocellular carcinoma proliferation through activating STAT3 signaling

From January 2006 to December 2012, we collected 169 paired samples of tumor tissues and adjacent normal tissues from HCC patients who experienced tumor resection surgery at Tongji Hospital, Huazhong University of Science and Technology. The pathological examination was used to confirm all HCC tissues. The patients had not experienced systemic or local radiation and chemotherapy before surgery. The patients had undergone no antitumor treatment after surgery. Informed consent forms were signed by HCC patients and the Ethical Committee of Tongji Hospital approved each step. We made HCC staging in accordance with the seventh edition of AJCC (American Joint Committee on Cancer) TNM classification. We made a tissue microarray including 169 HCC cases at Shanghai Biochip Co., Ltd. Shanghai, China. The immunohistochemistry assay was done as previously reported [11]. 3 different pathologists scored the images without knowing about patients’ clinical pathological characteristics. We counted the total score of all images by multiplying staining area percentage score by intensity score, which was previously reported [11]. The cutoff for the definition of low or high expression group was the median value.

STAT3 inhibitor Stattic was bought from MedChemExpress, NJ, USA. Polybrene, opti-MEM medium, puromycin, and trypsin-EDTA were acquired as previous mentioned [12]. We mention Lipofectamine 3000 Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA). We make clear all antibodies used in the study in Supplementary Table S3.

We obtained cell lines (HL-7702, Alex, HLF, SK-Hep1, HLE, Hep3B, Huh7, MHCC-97 H, MHCC-LM3 and Bel7402) from the Hepatic Surgery Center, Tongji Hospital, Huazhong University of Science and Technology. We bought 293T cells lines from the American Type Culture Collection. Before the study, we examined all cell lines for their authenticity. At 37 °C in 5% CO2 and 95% air condition, we incubated above cell lines in Dulbecco’s Modified Eagle’s Medium with 4.5 g/L glucose (DMEM, Hyclone, Logan, UT, USA), which included 10% fetal bovine serum (FBS, Gibco, North America).

We obtained pBABE-puro (Plasmid #1764), gag/pol (Plasmid #14,887), pMD2.G(Plasmid #12,259), pLKO.1-TRC cloning vector (Plasmid # 10,878), psPAX2(Plasmid #12,260) from the Hepatic Surgery Center, Tongji Hospital, Huazhong University of Science and Technology. We cloned the human ARF6 cDNA into the BamHI/EcoRI site of the pBABE-puro vector to construct pBABE-Flag-ARF6 plasmid, and identified it by sequencing (TSINGKE, Wuhan, China). We annealed the target double-stranded oligonucleotides (shRNA) sequences and one non-targeting sequence (scramble) and cloned them into the AgeI/EcoRI site of pLKO.1-puro vector, in order to establish pLKO.1-scramble and pLKO.1-shARF6 plasmids. We listed all sequences of target shRNA oligo pairs and siRNA used in the study in Supplementary Table S4. Viral production, infection and establishment of stable cell clones were performed as previously described [70]. We established pBABE-Flag-ARF6Q67L plasmid in accordance with ClonExpress II One Step Cloning Kit and Mut Express II Fast Mutagenesis Kit V2 (Vazyme, Nanjing, China) protocol and confirmed it with sequencing (TSINGKE, Wuhan, China).

We conducted immunofluorescence assay as previously described [70]. Briefly, cells grew on coverslips in a 24-well culture plate for 12 h, and we fixed cells with 4% paraformaldehyde for 15 min at room temperature. Then cells were permeabilized using 0.5% Triton X-100 for 20 min. After blocking with 5% bovine serum albumin for 1 h, we cultivated cells with indicated primary antibody overnight at 4 °C. Afterwards, we washed the cells three times and cultured them with indicated secondary antibody for 4 h at room temperature. At the end, the slides were incubated with 40, 60-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 5 min and visualized under phase-contrast and confocal laser-scanning microscopy.

We performed western blot assay as shown previously [70]. In brief, cells or tissues were lysed on ice with RIPA lysis buffer, including 1% protease (Roche) and 1% phosphatase inhibitor cocktail (Sigma). We quantified protein samples using BCA assay (Sigma), and separated proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred proteins onto polyvinylidene fuoride (PVDF) membranes (Millipore). The membranes were blocked with 5% milk, and incubated with indicated primary antibodies, then probed with horseradish peroxidase (HRP)-linked secondary antibodies (Jackson ImmunoResearch, PA, USA). We used ECL for signal detection and western blot images were acquired with Bio-Rad GelDoc system.

Cells were lysed with TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) for total cell RNA extracting. We performed reverse transcription using the QuantScript RT Kit (TIANGEN, Beijing, China) as shown previously [70]. We carried out real-time fluorescence quantitative PCR using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with SuperReal PreMix Plus (SYBR Green) kit (TIANGEN, Beijing, China) as shown previously [70]. All genes expression were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same specimen. All specimens were done independently in triplicate. We used the specific primer pairs to quantify the expression of the genes that encode the proteins. We listed the primers in Supplementary Table S5.

We seeded indicated HCC cells in 96-well microplates with the appropriate density per well. After incubated for 0, 1, 2, 3, and 4 d, the cells were treated using Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology) according to.

manufacturers’ introductions. At the end, the optical density was read at 450 nm using an enzyme-linked immunosorbent assay plate reader (Bio-Tek Elx 800, USA).

We seeded indicated HCC cells on a 6-well plate with the appropriate density per wells. We incubated the cells for about 14 days, and we fixed the colonies using 4% paraformaldehyde and stained them using 1% crystal violet. At the end, we take photos of the plates, and counted the separate adherent colonies larger than 100 μm in diameter.

We conducted animal assays according to Wuhan Medical Experimental Animal Care Guidelines. Male BALB/c (nu/nu) mice (6 weeks old, male, HUAFUKANG BIOSCIENCE CO. INC. Beijing, China) were bred under specific pathogen-free.

(SPF) conditions. We divided the mice into two or more groups at random, and subcutaneously injected indicated HCC cells into the flank of the mice. 14 days later, we began measuring the tumor size every 4 days with digital vernier calipers, and calculated the tumor volume according to the following formula: volume = 1/2× (width2 × length). After tumors grew to 3–5 mm in diameter, we peritoneally injected the mice with Stattic (50 mg/kg, three times per week). We sacrificed the mice at appropriate time, and the tumors were visually examined and collected for further analysis.

We conducted data analysis with Prism 5.0 (GraphPad Software, La Jolla, CA, USA) software, and SPSS software (version 21.0, IBM Corp, Armonk, NY, USA). We showed the values as the mean ± SEM from at least done independently in triplicate. We analyzed the difference between two groups by two-tailed Student’s t-test, ANOVA test, a nonparametric test, or a parametric test. We used χ2 test or Fisher’s exact test to analyze categorical data. We analyzed the survival curve between subgroups by Kaplan-Meier and log-rank analysis. We did at least independently in triplicate to guarantee repeatability. We considered a value of P