Reagents and antibodies

Primary antibodies applied in this study include antibodies against ORMDL3 (Abcam, UK, ab211522), Bax, Bcl-2, PARP, Caspase-3, Caspase-9, P62, Beclin1 (Proteintech-Group, Wuhan, China, 50599-2, 12789-1, 1337-1, 19677-1, 10380-1, 66184-1, 11306-1), LC3B (Novus-Biologicals, USA, NB100-2220), ATF4 (Cell Signaling Technology, D4B8, Danvers, USA), PERK, Phospho-PERK (Bioworld Technology, Shanghai, China, BS2156, BS66100), Goat anti-Rabbit IgG-HRP H&L, Goat anti-Mouse IgG-HRP H&L (Bioworld Technology, Shanghai, China, BS13279, BS12478), Alexa Fluor®488 Goat Anti-Rabbit IgG H&L, Alexa Fluor®488 Goat Anti Mouse IgG H&L(Abcam, UK, ab150077, ab150113). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), BCA Protein Assay Kit, BeyoECL Plus, DAPI Staining Solution, Apoptosis and necrosis Assay Kit, Annexin V-FITC/PI Apoptosis Detection Kit, Reactive Oxygen Species Assay Kit, and ATP detection Kit were purchased from Biyuntian Institute of Biotechnology (Nanjing, China). MitoSOX Red Mitochondrial Superoxide Indicator was purchased from ThermoFisher Scientific (Shanghai, China).

Cell culture and transfection

HepG2, SMMC-7721 hepatoma cell lines, and normal human HL-7702 liver cell lines were purchased from the Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China). The SMMC-7721 cell line was used in most of the experiments, and short tandem repeat (STR) analysis was performed on the cell line to determine its origin from HCC. Cells were grown in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum (FBS, Gibco, MD, USA), 100 U/mL penicillin G, and 100 μg/mL streptomycin, and incubated at 37 °C in a humidified incubator containing 5% CO2.

siRNA and plasmid transfections

The lentiviruses with scrambled shRNA against ORMDL3 (Lv-shORMDL3) and the shRNA control (Lv-shCon) were constructed in GV248 (Genechem, Shanghai, China). The ORMDL3 overexpressed plasmid was constructed in GV362, with pcDNA3.1-CMV-GFP plasmid as the backbone (Genechem, Shanghai, China) [23]. SMMC-7721 cells were plated and grown in 6-well plates before transfection at approximately 60% confluence. According to the manufacturer's protocol, the cells were transfected with siRNA targeting ORMDL3, and the overexpressed cells were transfected with a plasmid encapsulated with Lipofectamine® 2000. After culturing for 48 h, the transfected cells were screened with puromycin (4 μg/mL) for 2 weeks to form a stable transduction reagent pool. The transfected cells were used for subsequent experiments.

Cell viability assay

SMMC-7721 cells were treated with trypsin, counted, and then plated in a 96-well plate at 10,000 cells/well. After 24 h of treatment with sorafenib at 4 μM, 20 μL of MTT was added to each sample, and the plate was placed in the incubator for another 4 h. Next, the supernatant was discarded, 120 μL of DMSO was added to each well, and the plate was shaken for 10 min in the dark to sufficiently dissolve the formazan crystals. Then, the absorbance at 490 nm was measured with SpectraMax 190 (Molecular Devices, USA). Three independent experiments were performed and the data were statistically analyzed.

Total RNA isolation and quantitative real-time PCR

Total RNA was isolated using Trizol RNA extraction reagent (Life Technologies, Grand Island, NY) and reverse transcribed into cDNA using Superscript III first chain synthesis system (Invitrogen, China). SYBR Green PCR Master Mix (Vazyme, China) was used to prepare the PCR reaction mixture. The primers were used for qRT-PCR as follows:

Forward, 5'-AACACGCGGGTGA TGAACAG-3',

Reverse, 5'-AGGGACACTCACAAACGGGA-3' for human ORMDL3 gene;

Forward, 5'-GGAGCGAGA TCCCTCCAAAA T-3',

Reverse, 5'-GGCTGTTGTCA TACTTCTCA TGG-3' for human GAPDH gene.

The QuantStudio-Q3 Detection System (ABI, Singapore) performed a thermal cycle at 42 °C for 2 min, then at 95 °C for 15 s, 60 °C for 60 s, 95 °C for 1 s with 40 cycles of amplification. Relative expression was evaluated using the competitive critical threshold (ΔΔCt) method using GAPDH as the reference gene. Three independent experiments were performed.

Western blot

SMMC-7721 hepatoma cells were lysed on ice for 15 min and then collected into a centrifuge tube and centrifuged (12,000 rpm, 4 °C) for 15 min. The protein content of the supernatant was determined by BCA. Purified protein samples were incubated with 4× loading buffer and heated at 95 °C for 10 min. Protein samples from each group were isolated by SDS-PAGE and transferred to PVDF membranes under constant electric current (Amersham hybond-P, GE Healthcare, Buckinghamshire, UK). The membrane was sealed with 5% skim milk for 90 min and then incubated overnight with the primary antibody at 4 °C, followed by incubation with secondary antibodies for 1 h and visualized with an ECL Plus Western Blotting Detection System (GE Healthcare). Films were scanned or photographed and molecular weights and net light density of target bands were analyzed using a gel image processing system (Image lab). Each experiment was repeated independently more than three times, and then quantitative analysis was performed.

Colony formation assay

SMMC-7721 cells were treated with trypsin, counted, and then plated in a 6-well plate at 1000 cells/well. After 24 h of treatment with sorafenib at 4 μM, the SMMC-7721 cells were cultured for 12 days and fixed with 4% paraformaldehyde for 30 min. After drying, 1 mL of crystal violet staining solution (Biyuntian Institute of Biotechnology, Nanjing, China) was added, shaken evenly, dyed for 5 min, air dried naturally, and then photos were taken. The colonies were counted and the clone formation rate of each group was compared. The experiment was repeated three times for statistical analysis.

Apoptosis assay

SMMC-7721 hepatoma cells were seeded into a 6-well plate with 200,000 cells per well. Apoptosis levels were detected using the PI/Hoechst double-staining kit. 1 mL of the prepared staining solution (PI staining solution:Hoechst staining solution:cell staining buffer = 5 μL:5 μL:1 mL) was added to each well, shaken well, wrapped with aluminum foil to avoid light, and incubated for 30 min at 4 °C. Then, the fluorescence was observed with an inverted fluorescence microscope (Nikon, Japan) and the image was collected. Each experiment was independently repeated three times.

Annexin V-FITC/propidium iodide (PI) apoptosis assay

Annexin V-FITC and PI were detected by flow cytometry using two channels to determine the proportion of apoptotic cells. The drug-treated suspension and adherent cells were collected into a flow tube and incubated with 5 μL Annexin V-FITC and 10 μL PI in a dark room at room temperature for 15 min. Data were obtained using Beckman Cytoflex LX flow cytometry (Beckman Coulter, USA) and processed using CytExpert software (Beckman Coulter, USA). Each experiment was repeated three times independently, and the data were statistically analyzed.

Measurement of intracellular reactive oxygen species

SMMC-7721 cells were seeded into a 6-well plate with 200,000 cells per well. The fluorescent probe DCFH-DA (dichlorodihydrofluorescein acetoacetate) was used to detect reactive oxygen species. The DCFH-DA was diluted in serum-free medium at 1:1000 and incubated at a final concentration of 10 mM/L, 37 °C, and 5% CO2 for 20 min. After treatment, data were obtained using Beckman Cytoflex LX flow cytometry (Beckman Coulter, USA) and processed using CytExpert software (Beckman Coulter, USA). Each experiment was performed in triplicate.

Immunofluorescence

SMMC-7721 cells were uniformly seeded on sterile cover slips in 6-well culture plates for adherent growth. After the indicated treatment for a specified time, the cells were sequentially fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100, and then, the coverslips were immersed in 5% BSA at room temperature for 1 h. Subsequently, the pretreated samples were incubated with primary antibodies (dilution 1:100) overnight at 4 °C and then with goat anti-mouse/rabbit secondary antibody (dilution 1:100) in the dark for 2 h. Finally, following incubation with DAPI for nuclear staining. Confocal microscopy (Nikon, Japan) was used to observe cell fluorescence and image collection was carried out. Image J software was used to analyze cell fluorescence intensity. Five regions were randomly selected from each sample for evaluation and each experiment was repeated independently for three times for statistical analysis.

Immunohistochemical analysis

The tissue sections were dewaxed, hydrated, and heated in 0.01 mol/L sodium citrate buffer for antigen extraction, and then incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. The tissue microarray was then incubated with the specific primary antibody for 1.5 h at room temperature or 4 °C overnight, followed by the corresponding secondary antibody and streptomycin anti-biotin protein. Subsequently, DAB was used for color development, DAPI and hematoxylin were used for dyeing, alcohol was used for hydrating, and xylene was used for transparency. The plates were sealed with neutral resin. The stained tissue sections were observed with a forward microscope (Nikon, Japan), and the images were collected. Image Pro Plus 6 software was used to calculate the mean optical density (AOD) of the five regions of each sample for quantitative analysis.

In vivo experiment

Animal researches were carried out in compliance with the National Institutes of Health's Laboratory Animal Care and Use Guidelines, and approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University (license no. wydw 2021–0619).

Male BALB/C-nu nude mice (6 weeks) (WeitongLihua Laboratory, Zhejiang, China) were used in the experiment. The mice were divided into the shControl group, shORMDL3 group, shControl + Sora group, and shORMDL3 + Sora group. At least, 5 nude mice were randomly allocated to each group. Prepared SMMC-7721 cells of shControl or shORMDL3 (1 × 106) were injected into the right axilla fossa of each mouse, respectively. Sorafenib (30 mg/kg) treatment or saline as control was initiated when tumor volumes reached 60–100 mm3 and delivered intraperitoneally every 2 days for 28 days [24]. Tumor size and body weight were measured every 2 days, and tumor volume was calculated according to the equation: volume (cm3) = L × W2/2 with L and W representing the largest and smallest diameters, respectively. On the 28th day, mice were euthanized by cervical dislocation, and then tumors were isolated from each mouse and prepared for subsequent experiments.

Statistical analysis

The statistical analyses were performed using GraphPad Prism 7 software. All results are expressed as the mean ± SD of at least three independent trials. Differences between the two groups were analyzed using Student’s t test. One-way ANOVA was used for multiple groups to assess significant differences between study groups. P < 0.05 was considered statistically significant.