To construct the recombinant adenovirus vector Adv-LASS2-GFP, the LASS2-GFP (human LASS2 coding sequence, NM_022075; 627 ng/µl) and the pShuttle-CMV recombinant shuttle vector (BAC Biological Technology Co., Ltd., Beijing, China) were digested with HindIII and NotI (New England Biolabs, Inc., Ipswich, MA, USA). The digested vector and insert segments were ligated with T4 DNA ligase. Transformation, plasmid mini-extraction and sequencing were performed to obtain a verified pShuttle-LASS2-GFP recombinant shuttle plasmid. Following this, the pAdxsi vector (BAC Biological Technology Co., Ltd.) and pShuttle-LASS2-GFP were separately digested with I-CeuI and I-SceI, followed by ligation and transformation, to obtain a pAdxsi-LASS2-GFP viral plasmid. Extraction of the viral plasmids was performed, linearized pAdxsi-LASS2-GFP viral plasmid was transfected into 293 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), which was followed by collection and amplification to finally obtain a recombinant Adv-LASS2-GFP with a titer of 1.2×10¹⁰ plaque-forming units/milliliter.

Human HB HepG2 cells (19) were kindly provided and authenticated using short tandem repeat markers by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). It is notable that the HepG2 cells are typically misidentified as a HCC cell line, which is frequently erroneously used in HCC research (20,21).

Cells were cultured in the recommended medium, Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Cells in the exponential phase of growth were counted, seeded in 6-well plates and transfected with the recombinant adenoviruses (Adv-LASS2-GFP or Adv-GFP control) at 50–70% confluence following 24-h incubation. All assays were performed in triplicate.

Adv-GFP or Adv-LASS2-GFP vectors were transfected into HepG2 cells that were subsequently incubated for 48 h. Cells were placed on slides, washed three times with phosphate-buffered saline (PBS) and fixed by incubation in 4% paraformaldehyde for 30 min at room temperature. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in the dark for 5 min at room temperature and washed three times. A confocal microscope (LSM710; Carl Zeiss AG, Oberkochen, Germany) was used to analyze the expression of the GFP-LASS2 protein, which was detected with the GFP filter at 525 nm, following excitation at 488 nm. The cell nuclei were identified with laser excitation at 340 nm and emission at 488 nm. Images were acquired at ×200 magnification.

Cell viability and cell number were assessed using a CCK-8 assay (Beyotime Institute of Biotechnology, Shanghai, China). HepG2 cells were seeded into 96-well plates at 10⁴ cells/well. pAdv-GFP or pAdv-LASS2-GFP vectors were transfected into the cells, which were subsequently incubated for 0, 24, 48, 72 or 96 h. The cells were incubated with 10 µl CCK-8 solution for 1 h. The optical density (OD) was measured at 450 nm. The cell proliferation rate (%) = (OD value of the test well - OD value of the background control well) / (OD value of the control well - OD value of the background control) × 100%.

The transfected cells were collected and seeded in 6-cm sterile petri dishes at a density of 10³ cells/well. At 14 days from the initial appearance of colonies, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature and stained with crystal violet for 20 min at room temperature. Subsequently, the number of colonies (≥50 cells) was determined.

HepG2 cells were transfected with Adv-GFP or Adv-LASS2-GFP for 48 h. Subsequently, the cells were harvested, washed with PBS and prepared into single-cell suspension (10⁶ cells/ml, 1 ml/group). The precipitate was removed by centrifugation at 800 × g for 5 min at room temperature and the cells fixed in 500 µl 70% ice-cold ethanol for 2 h, or overnight. Following this, cells were incubated with 100 µl RNAase at 37°C for 30 min and stained with 400 µl propidium iodide (Nanjing KeyGen Biotech, Nanjing, China) for 30 min at 4°C while protected from light. The stained cells were subjected to flow cytometry using a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) and the proportion of cells phases in G₀/G₁, S and G₂/M were analyzed using ModFit software (version 3.3; Verity Software House, Inc., Topsham, ME, USA).

Transfected HepG2 cells were harvested with 0.25% trypsin without EDTA, washed twice with cold PBS and resuspended in 500 µl binding buffer. Following this, 5 µl Annexin V-APC and 5 µl 7-AAD (Nanjing KeyGen Biotech) were added in sequence according to the manufacturer's protocol. Cells were incubated for 15 min at room temperature while protected from light, then immediately analyzed using flow cytometry as described above and CellQuest software 6.0 (BD Biosciences).

Apoptosis-associated nuclear DNA fragmentation was detected using a commercial biotin dUTP-labeling TUNEL kit (cat. no. KGA702; KeyGen Biotech) according to the manufacturer's instructions. Briefly, HepG2 cells were seeded onto slides in 6-well plates. Following 48 h of incubation, the slides were dried and washed with PBS three times. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 1% Triton X-100. Subsequently, cells were incubated in 3% H₂O₂ in methanol for 10 min at room temperature to block endogenous peroxidase activity, washed in PBS and incubated with a mixture of TdT and fluorescein isothiocyanate dUTP solutions in a humidified chamber at 37°C for 60 min. This was followed by washing in PBS and incubated with streptavidin-conjugated horseradish peroxidase (dilution, 1:100; from the TUNEL kit) in a humidified chamber at 37°C for 30 min. When cells were washed with PBS, DAPI solution was applied, followed by counterstaining with hematoxylin. The dehydrated sections were cleared in xylene, mounted with neutral balsam and enclosed with coverslips. Over 200 cells with brown granules in the nuclei were considered to be TUNEL-positive. Cells were assessed in at least three high-powered fields of view (magnification, ×200) under an optical microscope (Olympus Corporation, Tokyo, Japan) to determine the mean percentage of positive cells.

Intracellular ROS were monitored using dihydroethidium (DHE; Nanjing KeyGen Biotech) as the probe. HepG2 cells were trypsinized, washed with PBS and resuspended in 1 ml serum-free medium supplemented with 10 µM DHE at density of 1×10⁶/ml following transfection. Cells were incubated at 37°C for 20 min in the dark and inverted every 3–5 min to maximize the contact between the probe and cells. Following this, cells were washed three times with serum-free medium to remove excess DHE. Cellular ROS were subsequently measured using flow cytometry, and fluorescence was read using a fluorescence spectrometer with a 488 nm excitation and 605 nm emission wavelength.

ΔΨm was measured using the JC-1 apoptosis detection kit (Nanjing KeyGen Biotech). Briefly, HepG2 cells transfected with the Adv-GFP or Adv-LASS2-GFP vectors were incubated for 48 h. Cells were collected, washed with PBS, resuspended in 500 µl JC-1 working solution and incubated at 37°C with 5% CO₂ for 20 min. Cells were collected by centrifugation (800 × g for 5 min at room temperature), washed twice with 1X incubation buffer and resuspended in 500 µl 1X incubation buffer. The concentration of retained JC-1 dye was determined by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

ATPases decompose ATP to produce ADP and inorganic phosphate (22). Therefore, Ca²⁺-ATPase activity was determined using a colorimetric method to detect the inorganic phosphate content. Transfected cells were digested and centrifuged as described above. A total of 500 µl cell suspension was added to each tube to a density of 10⁶ cells/ml. The cells were disrupted by ultrasonic homogenization. The detection of inorganic phosphate was performed with the Ultra Trace Ca²⁺-ATPase assay kit (Nanjing KeyGen Biotech) according to the manufacturer's instructions.

Transfected cells were washed twice with PBS and a single-cell suspension was prepared by centrifugation of 10⁶cells/ml at 800 g for 5 min. Calbryte 630 AM (AAT Bioquest, Inc., Sunnyvale, CA, USA) was dissolved in dimethyl sulfoxide to prepare a 2 mM stock solution; a working solution of 20 µM Calbryte 630 AM in Hank's balanced salt solution was also prepared. A total of 500 µl Calbryte 630 AM working solution was added into each well, and the dye-loaded plate was incubated for 60 min, followed by incubation at room temperature in the dark for a further 15 min. Cells were collected by centrifugation at 800 × g for 5 min at room temperature, and washed twice with 1X Hank's buffer with HEPES (HHBS) to remove excess dye. Cells were resuspended in 500 µl 1X HHBS and analyzed using flow cytometry for Calbryte 630 AM content, with an excitation wavelength of 610 nm and an emission wavelength of 640 nm, to determine the [Ca²⁺]_i.

Total RNA was isolated from HepG2 cells using the RNAiso Plus reagent (Takara Bio Inc., Otsu, Japan). cDNA was synthesized using the PrimeScript RT Master Mix kit (Takara Bio Inc.). qPCR was performed with the SYBR Premix Ex Taq II kit (Takara Bio Inc.) and the ABI 7500 Real-Time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The thermocycling conditions used with the ABI7500 detection system were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. Relative quantification was performed using the comparative Cq (2^−ΔΔCq) method (23). All data were normalized to the internal control, GAPDH. The specific primers for human LASS2 and GAPDH used in the present study were as follows: GAPDH, sense 5′-GGAGCGAGATCCCTCCAAAAT-3′ and antisense 5′-GGCTGTTGTCATACTTCTCATGG-3′ (197 bp product); and LASS2, sense 5′-ATCGTCTTCGCCATTGTT-3′ and antisense 5′-CGGTCACTGCGTTCATCT-3′ (233 bp product).

Cells were cultured on glass coverslips and transfected for 48 h, washed with PBS for 5 min, fixed with 4% pre-cooled paraformaldehyde for 20 min, washed in PBS three times for 5 min each time and incubated with PBS containing 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) on ice for 10 min. Two drops of 3% H₂O₂-methanol solution were added onto each coverslip for 10 min at room temperature to inactivate endogenous peroxidase, followed by washing three times with PBS again. Cells were blocked with ready-to-use normal goat serum kit (cat. no. AR0009; Boster Biological Technology, Pleasanton, CA, USA) for 20 min at 37°C and incubated with 50 µl rabbit anti-human NF-κB p-p65 (Ser536; dilution, 1:100; cat. no. 3033; Cell Signaling Technology, Inc., Danvers, MA, USA) on each coverslip overnight at 4°C. Following this, cells were incubated with tetramethylrhodamine-conjugated goat anti-rabbit IgG secondary antibodies (dilution, 1:100; cat. no. KGAA99; Nanjing KeyGen Biotech) at 37°C in the dark for 1 h. Cells were counterstained with DAPI for 5 min at room temperature and the coverslips were mounted in fluoroshield with DAPI histology mounting medium (cat. no. F657; Sigma-Aldrich; Merck KGaA) and the slides were scanned under a confocal microscope (LSM710; Carl Zeiss AG).

The total protein was extracted from HepG2 cells using a protein extraction kit (Nanjing KeyGen Biotech); protein concentrations were measured using a bicinchoninic acid assay kit (Nanjing KeyGen Biotech) according to the manufacturer's protocols. An equal amount of protein (10 µg per lane) for each group was separated using SDS-PAGE (10% gels) and electrotransferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk for 2 h at room temperature and incubated overnight at 4°C with the primary antibodies, including GAPDH (dilution, 1:5,000; cat. no. EM1101; Hangzhou HuaAn Biotechnology Co., Ltd., Hangzhou, China), anti-LASS2 (dilution, 1:300; cat. no. ab85567; Abcam, Cambridge, UK), cyclin D1 (dilution, 1:5,000; cat. no. ab137875; Abcam), CDK4 (dilution, 1:2,000; cat. no. ab137675; Abcam), NF-κB p65 (dilution, 1:1,000; cat. no. 8242), NF-κB p-p65 (Ser536; dilution, 1:1,000; cat. no. 3033) and p27^kip1 (dilution, 1:1,000; cat. no. 2552; all from Cell Signaling Technology, Inc.). The membranes were washed and incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; dilution, 1:4,000; cat. no. 5220-0283; KPL, Milford, MA, USA) at room temperature for 1–2 h. The membranes were washed, developed using enhanced chemiluminescent kit (cat. no. SQ101; EpiZyme, Shanghai, China) and analyzed by densitometry relative to the control gene using Gel-Pro analyzer 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

All statistical analyses were performed with SPSS (version 22.0; IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 7.0; GraphPad Software, Inc., La Jolla, CA, USA) software. Data are expressed as the mean ± standard deviation of at least three independent experiments. One-way analysis of variance with a least-significant-difference test were used for group comparisons. P<0.05 was considered to indicate a statistically significant difference.

Adenovirus vectors Adv-GFP (vector control) and Adv-LASS2-GFP were generated and successfully transiently transfected into HepG2 cells. The subcellular localization of the LASS2-GFP fusion protein was identified using confocal laser scanning microscope at 48 h post-transfection. The LASS2-GFP fusion protein was localized to the nuclear membrane and cytosol (Fig. 1A).

Furthermore, using RT-qPCR, it was identified that the Adv-LASS2-GFP-transfected cells exhibited a significantly greater LASS2 mRNA expression level compared with those transfected with Adv-GFP or negative control (NC) cells (51.62-fold difference; Fig. 1B). The LASS2 protein expression levels were also analyzed by western blot analysis. The increased expression of LASS2 protein was evident in the Adv-LASS2-GFP transfected cells when compared with the Adv-GFP or NC cells (10.55-fold; Fig. 1C).

To determine the effect of LASS2 overexpression on cell proliferation, CCK-8 and colony formation assays were performed. Cell proliferation was significantly decreased at 48 h post-transfection with Adv-LASS2-GFP when compared with cells transfected with Adv-GFP (Fig. 1D). The overexpression of LASS2 significantly suppressed HepG2 colony formation (Fig. 1E). These results indicate that LASS2 overexpression inhibited the proliferation of HepG2 HB cells.

To investigate the role of LASS2 in the regulation of the cell cycle in HepG2 cells, the effect of its overexpression on cell cycle progression was assessed. The cell cycle distribution was analyzed using flow cytometry with propidium iodide. The results indicated in Fig. 2A revealed that the overexpression of LASS2 inhibited the cell cycle progression of HepG2 cells in the G₀/G₁ phase. In the cells transfected with Adv-LASS2-GFP for 48 h compared with NC or Adv-GFP groups, the percentage of cells in the G₀/G₁ phase was significantly increased to 65.13±3.07%; the proportion of cells in the S and G₂/M phases was reduced, with the cells in S phase significantly decreasing from 46.11±1.77 to 33.54±2.30% and the cells in the G2/M phase decreasing from 11.58±1.39 to 2.00±0.84%.

To explore the potential mechanisms for the G₀/G₁ cell cycle arrest induced by LASS2 in HepG2 cells, the expression of members of the NF-κB p65/p27^kip1/cyclin D1/CDK4 signaling pathway, associated with the cell cycle and proliferation in cancer cells, were examined using western blot analysis and immunofluorescence (Fig. 2B-E).

NF-κB is an inducible transcription factor critical for the expression of a variety of genes that affect inflammation, immunity, apoptosis, cell proliferation (24,25) and the cell cycle (26,27). The present results demonstrated that NF-κB p65 activity was suppressed following Adv-LASS2-GFP transfection (Fig. 2B and C). Immunofluorescence analysis revealed that NF-κB p-p65 was widely expressed in the cytoplasm, with negative staining in the nuclei (Fig. 2B). Western blot analysis indicated that the relative expression of NF-κB p-p65 was significantly decreased compared with NC or Adv-GFP groups (Fig. 2C). Although the relative expression level of inactive NF-κB p65 was also downregulated, the difference was not statistically significant (Fig. 2C).

CDKs are a family of kinases initially identified for their role in the regulation of the cell cycle (28). CDK4 is a key partner for cyclin D1 in the regulation of cell cycle progression from the G₁ phase (28). The western blot analysis results indicated that the relative expression of cyclin D1 and CDK4 were significantly reduced by 40.89±5.91 and 46.95±6.43%, respectively, in the cells transfected with Adv-LASS2-GFP compared with the control group (Fig. 2D). To investigate the mechanism for the effect of LASS2 on cyclin D1 expression, western blot analysis was performed to detect the changes in the expression of the CDK inhibitor p27. p27 is a negative regulator of cell cycle progression from G₁ to S phase and is considered the most characteristic of CDK regulators. Furthermore, p27 inhibits cyclin-CDK complexes such as cyclin D1-CDK4 (29,30). The present results revealed a significantly upregulated expression of p27 protein (1.46-fold) in the cells transfected with Adv-LASS2-GFP compared with the NC group (Fig. 2E).

To confirm whether LASS2 overexpression induced apoptosis in HepG2 cells, the phosphatidylserine on the surface of apoptotic cells and nuclear DNA fragmentation during apoptosis was detected using Annexin V-APC/7-AAD and TUNEL assays, respectively. The proportion of Annexin V-APC and/or 7AAD-positive cells (Fig. 3A) and TUNEL-positive cells (Fig. 3B) was significantly higher in cells transfected with Adv-LASS2-GFP compared with cells transfected with Adv-GFP or NC cells.

To further investigate the mechanisms of LASS2-induced apoptosis, changes in intracellular ROS, ∆Ψ_m and [Ca²⁺]_i were considered. The intracellular ROS level was determined by flow cytometry analysis with DHE staining. It was revealed that the overexpression of LASS2 significantly elicited ROS generation at 48 h post-transfection (Fig. 4A). To study whether LASS2 induced ∆Ψ_m collapse in HepG2 cells, ∆Ψ_m was measured using flow cytometry analysis with JC-1 dye. The LASS2-overexpressing HepG2 cells demonstrated a clear reduction of ∆Ψ_m (Fig. 4B). Furthermore, the effect of LASS2 on [Ca²⁺]_i was determined by flow cytometry using Calbryte 630 AM fluorescence intensity. LASS2 overexpression promoted a significant increase of [Ca²⁺]_i in HepG2 cells (Fig. 4C), leading to the disruption of intracellular calcium homeostasis. Ca²⁺-ATPase is the major active calcium transport protein in the maintenance of normal [Ca²⁺]_i in a variety of cell types (31). Ca²⁺-ATPase activity was determined using a colorimetric method by the inorganic phosphate content. A significantly reduced ATPase activity was detected in cells transfected with Adv-LASS2-GFP HepG2 cells compared with the Adv-GFP or NC cells (Fig. 4D).