Scientific Papers

TMEM147 aggravates the progression of HCC by modulating cholesterol homeostasis, suppressing ferroptosis, and promoting the M2 polarization of tumor-associated macrophages | Journal of Experimental & Clinical Cancer Research

HCC specimens

Only patients with pathological diagnosis of HCC were included in this study. We collected paired HCC and adjacent non-cancerous liver tissue samples from patients who underwent liver resection at the First Affiliated Hospital of Harbin Medical University between January 2012 and August 2017. Senior pathologists were invited to perform pathological diagnoses on the paraffin sections.

The study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Research Ethics Committee of the First Affiliated Hospital of Harbin Medical University. All patients provided written informed consent.

Cell lines and cell culture

Human HCC cell lines SK-Hep-1, Huh7, HepG2, HCCLM3, and BEL-7402 were obtained from the Chinese Academy of Science (Shanghai, China). The normal liver cell line WRL-68 was obtained from AcceGen (Fairfield, USA). The cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. Human monocytic leukemia cells, THP-1, were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/mL penicillin, and 100 µg/mL streptomycin. THP-1 cells were treated with 100 nM phorbol myristate acetate (PMA) (Sigma-Aldrich, USA) for 48 h to induce differentiation into macrophages. All cells were incubated at 37 °C in a humidified atmosphere (5% CO2).

Lentivirus infection

Lentiviral vectors for human TMEM147 overexpression (lenti-TMEM147) and downregulation (lenti-TMEM147) and DHCR7 and GPX4 downregulation (lenti-sh DHCR7 and lenti-sh GPX4, respectively) were obtained from Hanbio Biotechnology Co. Ltd. (Shanghai). Corresponding empty vectors (lenti-Con and lenti-shcon) were used as negative controls. (Hanbio, Shanghai, China).

The transfection was performed with a multiplicity of infection (MOI) of 10–30 in the presence of polybrene (5 µg/mL). Following lentiviral infection, single-cell clones were selected with 3.5 µg/mL puromycin (Sigma-Aldrich) after 2 weeks of incubation. Stably transfected clones were isolated and used for in vitro and in vivo experiments.

Immunohistochemical staining

The tissue sections were deparaffinized, rehydrated, blocked with 10% normal goat serum, and incubated with the anti-TMEM147 (Abcam, ab97624; 1:200 dilution), anti-4HNE (Abcam, ab46544; 1:250 dilution), and anti-ARG1 (Abcam, abab96183; 1:250 dilution) primary antibodies overnight at 4 °C. The slides were then incubated sequentially, first with a secondary antibody (Vector lab, Burlingame, CA, USA) for 1 h and then with Vectastain Elite ABC reagent (Vector Lab) for 30 min. The tissue sections were stained with diaminobenzidine (DAB kit; Vector Laboratories) and counterstained with hematoxylin (Sigma-Aldrich). The percentage score was defined as follows: 0: 0–5%; 1: 5–25%; 2:, 26–50%; 3: 51–75%; 4: 76–100%; and the staining intensity was defined as 1: weak; 2: moderate; and 3: strong and provided a Multiple index (MI) score (MI = intensity × percentage) as follows: MI = 0, scored as 1; MI = 1–4, scored as 2; MI = 5–8, scored as 3; MI = 9 or 12, scored as 4. Samples with scores ≥ 3 were considered to exhibit high expression, whereas those with scores ≤ 2 were classified as exhibiting low expression.

Western blotting

Protein samples extracted from tissues or cells were separated by gel electrophoresis, and transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, USA). Subsequently, the membrane was blocked with 5% skim milk and incubated with primary antibodies overnight at 4 ℃. Finally, the membrane was incubated with IRDye 800CW secondary antibody (LI-COR, USA) (1:10,000 dilution) at room temperature for 1 h, and the Odyssey® Imaging System (LI-COR, USA) was used to visualize and analyze the proteins. Details of the primary antibodies against the target proteins are listed in Additional file 1: Table S3.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Total RNA was isolated from freshly frozen tissue and logarithmically growing cells using the Total RNA Miniprep Kit (Axygen Scientific, Inc., USA) according to the manufacturer’s instructions and reverse-transcribed into cDNA using a qPCR RT Kit (TOYOBO, Shanghai, China) after RNA quantification. qRT-PCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Shanghai, China) on an ABI PRISM 7500HT instrument (Applied Biosystems). The expression level of the target mRNA was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level and was determined according to the 2−ΔΔCT method. The primers used are listed in Additional file 1: Table S2.

Cell counting Kit-8 (CCK-8) and colony formation assays

1000–1500 stably transfected cells were seeded onto a 96-well plate and cultured for 4 h for attachment. Subsequently, the culture solution was replaced with a solution containing CCK-8 reagent and the optical density (OD) value was determined. Colony formation was assayed by plating 1000 cells in petri dishes with 6 cm diameter after 14 days of culture. The medium was then discarded, and the colonies were fixed with 4% paraformaldehyde (PFA) and stained with 0.5% crystal violet.

Wound-healing assay

Stably transfected cells were seeded in a 6-well plate at 30 × 104 cells/well and cultured until cell fusion occurred. A 10-µL pipette tip was used to scratch a straight cut at the bottom of the plate. The floating cells were washed away with phosphate buffered saline (PBS) and cultured in serum-free medium, and wound closure was photographed at 0 and 24 h.

Transwell migration and invasion assay

Matrigel-coated (BD Biosciences, Franklin Lakes, NJ, USA) or non-Matrigel-coated Transwell plates were used to examine the invasion and migration abilities of the cells. Cells were inoculated into the upper chamber of the transwell and serum-free medium was added. Normal media was injected into the plate wells. After a 48 h incubation period, cells from the upper layer filter were discarded, and the cells in the bottom layer were fixed, stained, and counted.

Immunofluorescence (IF) assay

Cells were seeded on polylysine-coated coverslips, cultured for 24 h, and fixed with 4% paraformaldehyde, followed by 0.1% Triton-X-100 permeabilization. Next, cells were incubated with primary antibodies, secondary antibodies (Invitrogen) and DAPI (Vector Laboratories) in sequence. The images were captured with a camera attached to a microscope.

Co-immunoprecipitation (IP) assay

Cells were harvested and then lysed in 500 µL co-IP buffer containing a protease inhibitor cocktail (Sigma-Aldrich). After centrifugation, cell lysates were collected and pre-cleared by incubating with 20 µL immobilized protein A/G beads for 1 h at 4 °C. The beads were then discarded using a magnetic frame, and the lysates incubated with primary antibody or control immunoglobulin (Ig)G on a rotator at 4 °C overnight. On the following day, 20 µL of immobilized protein A/G beads were added to precipitate the protein complex at 4 °C for 4 h. Subsequently, samples were washed five times, the beads boiled in loading buffer, and the proteins were prepared for Western blot as described above.

Mass spectrometric analysis

Similar to those in the IP assay, cellular protein extracts from HCC cells were incubated with anti-Flag-TMEM147, followed by incubation with protein A/G agarose beads. The recovered proteins bound with Flag-TMEM147 or IgG were resolved via gel electrophoresis. The bands specifically bound to Flag-TMEM147 were excised, and proteomics screening was performed via mass spectrometric analysis on a MALDI-TOF-MS instrument (Bruker Daltonics).

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed using a ChIP Kit (Beyotime, Shanghai, China). Briefly, cells were treated with 1% formalin solution for 10 min and quenched with glycine for 5 min at room temperature to generate DNA–protein cross-links. Cell lysates were sonicated to produce chromatin fragments of 200–1000 bp and immunoprecipitated with anti-DHCR7, anti-STAT2, or IgG antibodies. The immunoprecipitated DNAs was analyzed using qRT-PCR. Information on the primers and antibodies used is provided in Additional File 1: Table S2.

Dual‑luciferase reporter assay

The purpose of the dual-luciferase reporter gene assay was to analyze the transcriptional regulation of transcription factors on target genes. The full-length promoter of DHCR7 carrying mutant or wild-type sequences was cloned into pGLO4.10 vectors (Promega, Madison, WI, USA) and co-transfected with a STAT2 overexpression vector or mock vector into Huh7 cells, using Lipofectamine TM 2000 (Invitrogen, CA, USA). After 48 h of culture, firefly and Renilla luciferase activities were measured using a dual-luciferase reporter gene assay system (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocols.

Enzyme-linked immunosorbent assay (ELISA) assay

Cholesteryl Esters’ (CEs) concentrations were measured using a Cholesterol/Cholesterol Ester Quantification Kit (ab65359, Abcam). 27HC concentrations were measured with a Human 27-Hydroxycholesterol ELISA Kit (EH4025, FineTest). Lipid content was measured using a lipid ELISA Kit (SBJ-M0736-96T, GoldenRain/sbj). ELISA was performed according to the manufacturer’s instructions. All experiments were performed in triplicate.

Glutathione (GSH) assay

A reduced glutathione (GSH) assay kit (A006-2-1, Nanjing Jiancheng Bioengineering Institute) was used to measure intracellular GSH levels. Cells (3 × 105) were seeded into a 6-well plate and treated with erastin or RSL3 for 24 h. The cells were harvested, and 0.3 mL PBS was added to the homogenization medium. A 100 µL supernatant was removed for GSH determination. Simultaneously, a standard GSH concentration curve was also generated. The plate was incubated for 5 min after mixing the sample and reagents, and absorbance was measured at 405 nm. The exact GSH concentration in the different cell lines was then calculated based on the GSH standard curve, following the manufacturer’s instructions.

Mitochondrial iron, malondialdehyde, and membrane potential assays

The mitochondria were isolated using a mitochondrial isolation kit (Thermo Fisher Scientific). Ferrous iron levels in cells or mitochondria were measured using an iron assay kit (Sigma-Aldrich). Lipid peroxidation was assessed in HCC cell lysates by measuring the concentration of malondialdehyde (MDA), an end product of lipid peroxidation, using a lipid peroxidation assay kit (Abcam, Cambridge, MA, USA). Mitochondrial membrane potential (MMP) was measured by staining the cells with 200 nM tetramethylrhodamine ethyl ester (TMRE, Thermo Fisher Scientific) for 20 min. The mean fluorescence intensity of each group was normalized to that of the control group.

Reactive oxygen species (ROS) assay

Intracellular ROS generation was detected by reactive oxygen species assay kit (Beyotime Biotechnology, Haimen, China). Before the experiment, 1 × 104 cells per well were seeded in a 96-well black plate and incubated for 24 h. Following series of indicated experimental treatments, cells were loaded with 10 µM fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in serum-free DMEM medium for 20 min at 37 ℃. The cells were then washed thrice with serum-free medium. DCF fluorescence intensity was detected using a Tecan Infinite 200 PRO microplate reader to quantify the ROS levels.

Transmission electron microscopy (TEM)

TEM was used for ultrastructural analysis of mitochondria. HCC cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4 °C for 2.5 h, washed three times with 0.1 M PBS, and post-fixed in 1% OsO4 for 2 h at 4 °C. The samples were dehydrated using an ethanol gradient and embedded in Spurr’s resin. Ultrathin sections were collected, stained with uranyl acetate or lead citrate, and examined under a JEOL 1200EX transmission electron microscope.

Flow cytometric analysis

PMA-stimulated THP-1 cells that had been co-cultured with HCC cell supernatants for 24 h were stained with PE-conjugated mouse anti-human CD86 (560,957, BD Biosciences) or CD206 (555,954, BD Biosciences) antibodies according to the manufacturer’s instructions. The cells were then fixed and permeabilized using a fixation and permeabilization solution (554,722; BD Biosciences). After washing with BD Perm/Wash buffer (554,723; BD Biosciences), the cells were stained with an FITC-conjugated anti-human CD68 antibody (562,117; BD Biosciences). After 30 min, the cells were washed thrice with PBS and resuspended in 1 mL of FACS buffer for flow cytometry analysis.

Oxygen consumption rate (OCR) determination

Cells (2 × 104 cells/well) were seeded in XFe24 seahorse cell culture microplates (Seahorse Bioscience) for 24 h, and the Xfe24 sensor cartridges were hydrated overnight. The cells were treated with a cystine-free medium for 8 h, then the cell medium was replaced with basic seahorse DMEM supplemented with glucose (10 mM), sodium pyruvate (1 mM), and glutamine (2 mM). Subsequently, the cell culture microplate was kept in the CO2-free incubator for 1 h, and OCR was measured in real-time with the sequential injection of oligomycin (1.5 µM), carbonyl cyanide chlorophenylhydrazone (CCCP) (2.0 µM), and Antimycin A/Retenone (0.5 µM) by the Seahorse Xfe 24 Bioanalyzer (Seahorse Bioscience).

Animal model

Female BALB/c athymic nude mice (4–6 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under specific pathogen-free conditions and raised following institutional guidelines for animal care.

All experimental protocols involving animals were approved by the Animal Ethics Committee of Harbin Medical University, China.

The subcutaneous xenograft model was established by injecting 2 × 106 HCC cells in 200 µL PBS into the flanks of mice. Tumor volume was calculated at the 6th week when all mice were euthanized. Subcutaneous xenograft tumors were cut into 1 mm3 cubes and transplanted into the livers (left lobes) of mice to establish an orthotopic tumor model. Tumor size was assessed weekly with NIGHTOWL LB983 system (Berthold Technologies, Wildbad, Germany).

The pulmonary metastasis model was established as follows: 4 × 106 cells suspended in 0.15 mL PBS were injected into the tail vein of each mouse. After 6 weeks, the mice were euthanized, and their lungs were extracted.

All animals were euthanized to collect tumor tissues at the 6th week, and their livers were resected for IHC staining and ELISA.

Statistical analysis

Differences between groups were calculated using Student’s t-test, the chi-square test, or the Fisher exact test. The probability of differences in survival was ascertained using the Kaplan–Meier method with a log-rank test for significance. Analyses were performed with SPSS version 23.0 and Graphpad Prism 7.0 statistical analysis software. Representative data are shown as mean ± SD. P < 0.05 was considered statistically significant.

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