The L02/HBx cells were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in Gibco™ Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS) and 250 µg/ml G418 in a 37°C incubator with 5% CO₂.

The short hairpin RNAs specially targeting Notch1 (Notch1-shRNAs) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of Notch1-shRNAs were as follows: shRNA1, 5′-CCGGGATGCCAAATGCCTGCCAGAACTCGAGTTCTGGCAGGCATTTGGCATCTTTTT-3′; shRNA2, 5′-CCGGCAAAGACATGACCAGTGGCTACTCGAGTAGCCACTGGTCATGTCTTTGTTTTT-3′; shRNA3, 5′-CCGGCTTTGTTTCAGGTTCAGTATTCTCGAGAATACTGAACCTGAAACAAAGTTTTT-3′; and shNC, 5′-CCGGGCCGAACCAATACAACCCTCTCTCGAGAGAGGGTTGTATTGGTTCGGCTTTTT-3′. For the in vitro group, L02/HBx cells were divided into 5 groups by transfection with Notch1-shRNAs, Notch2-shRNAs, Notch3-shRNAs, sh-NC or without any treatment using Invitrogen™ Oligofectamine (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The shRNA, which induced the lowest relative mRNA of Notch1, was used for subsequent experiments.

When the cell reached 80–90% confluence, cell were harvested and total RNA was extract by TRIzol RNA kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The concentration and purity of total RNA were assessed by a ultraviolet spectrophotometer. Reverse transcription into cDNA was then conducted by a reverse cDNA transcription Kit (Takara Bio Inc., Otsu, Japan). Quantitative SYBR ExScript qRT-PCR Kit (Takara Bio Inc.) was used to detect the expression of target genes and the primers used in this study are showed in Table I. The reactions were performed at 95°C for 10 min followed by 40 cycles of 95°C, 15 sec and 60°C for 1 min. Finally, the expression levels of target genes were normalized to GAPDH and then calculated using the 2^−ΔΔCq method (24).

Total protein was extracted using radioimmunoprecipitation assay (RIPA) (Sangon Biotech, Shanghai, China). The protein samples were run by 10% SDS-PAGE gel electrophoresis, transferred to the polyvinylidene fluoride (PVDF) membranes for 2 h, blocked using skimmed milk powder antigen for 30 min, and incubated with primary rat antibodies against Notch1 (cat. no. ab44986), Hes1 (cat. no. ab108937), NICD (cat. no. ab8925), Ki-67 (cat. no. ab15580), proliferating cell nuclear antigen (PCNA) (cat. no. ab18197), Bcl-2 (cat. no. ab182858), cyclin D1 (cat. no. ab16663), CDK4 (cat. no. ab199728), E2F1 (cat. no. ab4070), p21 (cat. no. ab109199), retinoblastoma gene (Rb) (cat. no. ab181616), cyclin E1 (cat. no. ab133266), caspase-3 (cat. no. ab13585), caspase-9 (cat. no. ab32539), caspase-8 (cat. no. ab25901) and actin (cat. no. ab8226) (Abcam, Cambridge, MA, USA; 1:1,000 dilution) overnight at 4°C, respectively. Actin was used as the internal control. Then the samples were incubated with secondary rabbit anti-rat antibody (cat. no. sc-516132; Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:5,000 dilution) at room temperature for 2 h, followed by luminescence development using an enhanced chemiluminescence (ECL) analysis system (Santa Cruz Biotechnology).

Cells were seeded in 96-well plates at a density of 5×10⁴ cells/well. After transfection for 24, 48, 72 and 96 h, the cells in the corresponding well were replaced with primary culture medium (no red phenol), and WST-8 solution was added to each well at 37°C for 4 h. The absorbance of each well at 450 nm was detected by a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

Cells were digested by 0.25% trypsin and filtered using a 40-µm nylon mesh. Then, cells were seeded in 24-well plates and maintained in a 37°C incubator with 5% CO₂ for 14 days. Thereafter, the cells were fixed with 4% methanol for 15 min, followed by incubation with Giemasa (Kaiji Biotechnology, Shanghai, China) for 30 min. The colonies containing >30 cells were counted under a light microscope (Olympus IX83; Olympus Corp., Tokyo, Japan).

Cells were digested by 0.25% trypsin. Cells (1×10⁶ cells/ml) were collected and then fixed with 70% volume ethanol overnight at 4°C. After discarding the supernatant by centrifugation (in 5 min × 1,500 g), 50 µl RNase A was added to the cell precipitation for 30 min at 37°C and then 200 µl propidium iodide (PI) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added for staining. Cell cycle was then analyzed by flow cytometry (Sysmex Partec, Munster, Germany).

Cells were digested by 0.25% trypsin. Cells (1×10⁶ cells/ml) were collected and then suspended in 1X binding buffer. Thereafter, the cells were stained with Annexin V-PE and 7-amino-actinomycin (7-AAD) at room temperature for 15 min. Cell apoptosis was then detected by flow cytometry within 1 h.

A total of 12 male nude mice (4–6 weeks old, 18–20 g in weight; Cyagen, Shanghai, China) were housed for one week and then divided into 2 groups: Notch1-shRNA and sh-NC groups (n=6 each group). The mice were housed in temperature-controlled cages with a 12-h light/dark cycle and given free access to water and normal chows. The maximum allowable tumor size was diameter of 2.0 cm and volume of 4.2 cm³. Then Notch1-shRNA and sh-NC transfected cells at logarithmic growth phase were digested and suspended in DMEM respectively, at a cell density of 2×10⁶. Next, 0.2 ml cells were injected subcutaneously into the neck of each nude mouse, respectively. The nude mice were sacrificed by ether anesthesia after 25 days. The tumors were removed, and the tumor volume was calculated according to the formula: V = (LxW²)/2 (V, volume, L, length and W, width). Then, the tissues were fixed in 10% formalin for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) straining. Non-retrospective ethical approval was obtained for the animal experiments conducted in the study and tumor burden did not exceed the recommended dimensions. The animal experiments in this study were approved by the Animal Care and Research Committee of Chengdu Military General Hospital. All experiments were performed in compliance with relevant laws and guidelines. In addition, all experiments were conducted following the institutional guidelines of Chengdu Military General Hospital (Chengdu, Sichuan, China).

To assess the pathological changes, the fixed samples of the lungs in 10% formaldehyde were embedded in paraffin. Tissue sections cut from paraffin-embedded samples were then stained with H&E to detect inflammatory cell infiltration in the lung tissue.

Paraffin sections (3–5 µm in thickness) on slides with suitable tissue adhesive were processed for deparaffinization and hydration. Endogenous peroxidase was inactivated by incubation with 3% hydrogen peroxidase for 15–20 min. Antigen retrieval was conducted with a microwave oven heating for 30 min with citrate buffer (0.01 M, pH 6.0). After blocked with 5% bovine serum albumin for 1 h to reduce nonspecific reactions, the sections were incubated with the appropriate monoclonal antibodies, including Notch1 (cat. no. ab44986), Hes1 (cat. no. ab108937), NICD (cat. no. ab8925), Ki-67 (cat. no. ab15580), cyclin D1 (cat. no. ab16663), retinoblastoma gene (Rb) (cat. no. ab181616), caspase-3 (cat. no. ab13585) and actin (cat. no. ab8226) (Abcam, Cambridge, MA, USA; 1:1,000 dilution) overnight at 4°C, followed by incubation with the secondary rabbit anti-rat antibody (cat. no. sc-516132; Santa Cruz Biotechnology) (1:5,000 dilution). The reaction was visualized using DAB (3,3′-diaminobenzidine), and nuclei were counterstained with hematoxylin.

Data were derived from at least three independent experiments. The data were analyzed using SPSS Software v10.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation and experiments were repeated at least three times. Group statistical comparisons were assessed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. A value of P<0.05 indicated a statistically significant difference.

Three pairs of chemically synthesized shRNAs (shRNA1, 2, 3) targeting Notch1 and negative controls (sh-NC group) were transfected into L02/HBx cells, respectively. The results of qRT-PCR showed that compared with the blank control, the expression of Notch1 was obviously inhibited by all siRNAs (P<0.01), and the inhibition efficiencies were 64.2% (shRNA1), 72.9% (shRNA2) and 22.4% (shRNA3), respectively (Fig. 1A). The L02/HBx cells were transfected with shRNA2 for subsequent experiments. Moreover, the effects of recombinant plasmid Notch1-shRNA on Notch1 signaling pathway were detected. The results showed that the mRNA and protein expression levels of Notch1 signaling pathway-related molecules, including Notch1, Hes1 and NICD, were significantly decreased in the Notch1-shRNA transfected cells compared with those in the blank control (P<0.01) (Fig. 1B and C), indicating that Notch1-shRNA could inhibit the activation of the Notch1 signaling pathway.

The effect of Notch1 on the proliferation of L02/HBx cells was detected by CCK-8 and colony formation assays. Compared with the blank control, the cell viability of the Notch1-shRNA group was significantly decreased after 48 h of transfection (P<0.05) (Fig. 2A). Consistent with the results of the CCK-8 assay, the number of clones formed by Notch1-shRNA transfected cells was significantly reduced in comparison with the blank control or sh-NC groups (P<0.05) (Fig. 2B and C). Furthermore, the protein expression levels of Ki-67, PCNA and Bcl-2 were detected. The results showed that the protein expression levels of Ki-67 and PCNA were significantly downregulated in the Notch1-shRNA transfected cells relative to those in blank control (P<0.01) (Fig. 2D).

The effect of Notch1 on L02/HBx cell cycle was detected by flow cytometry. The result showed that the proportion of cells at the G₀/G₁ phase in the Notch1-shRNA group was increased significantly compared with the blank control group (P<0.01), and the proportion of cells at S phase was decreased significantly (P<0.01); no obvious change was detected in the proportion of cells at G2/M (Fig. 3A and B). These data indicated that inhibition of Notch1 significantly arrested the cell cycle at the G₀/G₁ phase. Moreover, we determined the expression changes in cell cycle-related molecules. As shown in Fig. 3C and D, the mRNA and protein expression levels of Cyclin D1, CDK4 and E2F1 were significantly decreased in the Notch1-shRNA group (P<0.01), while the mRNA and protein levels of P21 and Rb were significantly increased compared with the blank control group, (P<0.01). These results indicate that knockdown of Notch1 led to L02/HBx cell cycle arrest at G₀/G₁ phase.

The effect of Notch1 on L02/HBx cell apoptosis was also assessed by flow cytometry. As shown in Fig. 4A and B, the percentage of apoptotic cells in the Notch1-shRNA transfected group was significantly increased compared with the blank control or sh-NC groups (P<0.05). Furthermore, the mRNA and protein expression levels of caspase-3 and caspase-9 were significantly increased in the Notch1-shRNA group compared with the blank control (P<0.01), but the expression of caspase-8 exhibited no significant difference (Fig. 4C and D).

The in vivo tumor xenograft model was established to further explore the effects of Notch1. The results showed that the tumor volume was significantly inhibited in the Notch1-shRNA group compared with that noted in the L02/HBx group (P<0.05) (Fig. 5A and B). A statistically difference was detected at 10 days after inoculation (Fig. 5A). In addition, the results of H&E staining revealed that the tumor tissues were hepatocellular carcinoma, characterized by a cord like arrangement, with round nuclei, large nuclear shelves and frequent nuclear division (Fig. 5C). The results of IHC staining showed that the expression levels of Notch1, Ki-67 and cyclin D1 in the Notch1-shRNA group were significantly decreased compared with these levels in the L02/HBx group, while the expression levels of Rb and caspase-3 were significantly increased (P<0.05) (Fig. 5D and E). Moreover, the results of western blot analysis showed that the expression levels of Notch1 pathway-related proteins, including Notch1, Hes1 and NICD were all significantly decreased in the Notch1-shRNA tumor group in comparison with the L02/HBx group (P<0.01) (Fig. 5F). There was no significant difference in the above indices between the NC and blank groups.