Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. Currently, the global incidence of HCC is showing a rising trend [1, 2]. According to the “World Cancer Report 2014,” which was published by the World Health Organization on February 3, 2014, the number of new cancer cases in China ranked No. 1 in the world; among these, both the number of new HCC cases and the number of HCC-related deaths were the highest in the world [3]. The causes of HCC have not yet been clarified, and the disease still lacks specific medicines and treatment approaches. As a result, the prognosis for HCC patients is poor [4, 5]. Therefore, the discussion on the influence of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) on the proliferation, migration and invasion of HCC cells by regulating the expression of G protein-coupled receptor kinase 2 (GRK2) is very significant, which will provide clinicians with new directions of thought toward the prevention and treatment of HCC.

TNF-α and Ang II play important biological roles in human physiology, and previous studies have shown that they both participate in the formation and progression of HCC [6–8]. The biological effects of TNF-α are mainly conducted through two receptors that have different structures: tumor necrosis factor receptor 1 (TNFR1) and tumor necrosis factor receptor 2 (TNFR2) [9]. TNFR1 is expressed on the surface of almost every histiocyte, and TNFR2 is mainly expressed by cells of the immune system. Therein, the signal transduction of TNFR1 has a dual biological effect of causing inflammation and promoting apoptosis [10, 11]. The main receptors related to the biological effects of Ang II are angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) [12–14]. Ang II can promote the vasoconstriction, inflammation, fibrosis and cell proliferation in various tissues through AT1R, while the signal transduction through AT2R has the opposite biological effects [15].

G protein-coupled receptors (GPCRs) are seven-transmembrane helical proteins that function as signal transducers and mediate various signaling pathways related to the growth and metastasis of tumor cells [16, 17]. GRK2 belongs to the serine/threonine-protein kinase family and is widely distributed in various tissues [18]. It can specifically phosphorylate and desensitize activated GPCRs, thereby switching the signaling pathways mediated by GPCRs [19]. GRK2 has a variety of different physiological and pathological functions, while its activity and expression can be regulated by many factors. It is reported that GRK2 can significantly reduce the proliferation and invasion of HCC cells; therefore, GRK2 functions as a negative regulator in the formation and progression of HCC [20, 21].

Previous studies have suggested that TNF-α and Ang II may play roles in inflammation, proliferation and metastasis of histiocytes by regulating GRK2 expression [22, 23]. However, there are no reports have investigated the relationship between TNF-α, Ang II and GRK2 in HCC cells. Therefore, this study explores the effects of TNF-α and Ang II on the proliferation, migration and invasion of HepG2 cells by examining their influence on the expression of GRK2 and relevant receptors TNFR1, AT1R and AT2R.

Materials and methods

Patients and clinical samples

A total of 32 non-metastatic HCC patients and 32 benign hepatic tumor patients who were treated with hepatectomy in our hospital (the First Affiliated Hospital of Anhui Medical University, Hefei, China) from 2014 to 2015 were enrolled in this study. The HCC and benign hepatic tumors were preoperatively diagnosed using appropriate imaging characteristics and were verified via histological examination after the operation. None of the patients received presurgical chemotherapy or radiation therapy. The matched cancerous and noncancerous samples were obtained during each surgery, and clinicopathological data were available for each patient. Approval for these studies was obtained from the institutional review board of our hospital and performed in accordance with the ethical standards of the responsible committee on human experimentation as well as the Declaration of Helsinki.

Reagents and antibodies

Dulbecco’s modified Eagle’s medium (DMEM) and 0.25% trypsin were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from GIBCO Co., Ltd. (Carlsbad, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). BD Matrigel™ Basement Membrane Matrix was purchased from BD Biosciences (Bedford, MA, USA). Enhanced chemiluminescence (ECL) kit was purchased from Pierce Inc. (Rockford, IL, USA). GRK2 and scrambled small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). TNF-α and Ang II were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). TNFR1, AT1R and AT2R primary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-GRK2 polyclonal antibody was purchased from Proteintech Inc. (Chicago, IL, USA). Other reagents used in the experiments were of analytical grade and obtained from commercial sources.

Cell culture and transfection

Human HCC cell line HepG2 was purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM supplemented with 10% (v/v) FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin. HepG2 cells were maintained in a humidified incubator with a 5% CO2 atmosphere at 37 °C. GRK2 or scrambled siRNAs were transfected into HepG2 cells by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The transfected cells were grown for a further 48 h before experimentation to allow for maximal suppression of GRK2 protein expression.

Cell proliferation assay

CCK-8 assay was performed to determine the number of viable cells. HepG2 or siRNAs-transfected HepG2 cells in the logarithmic phase were digested and centrifuged to collect the cells. Then, the cells were seeded into 96-well culture plates at a density of 5000 cells per well in triplicate for each group. The cells were cultured with fresh medium containing TNF-α and Ang II alone or in combination at the specified concentrations. After treatment for 12, 24 and 48 h, 10 μl of CCK-8 reagent (Dojindo, Kumamoto, Japan) was added to each well, and the plates were incubated for another 1 h at 37 °C. The absorbance was measured at 450 nm using an Infinite M1000 PRO microplate reader (Tecan, Mannedorf, Switzerland).

Transwell migration and invasion assays

The cell migration assay was performed using Transwell co-culture chambers (24 wells, 8-µm pore size, Costar, Corning, NY, USA). Each upper chamber was seeded with 1 × 105 HepG2 or siRNAs-transfected HepG2 cells suspended in 100 μl of serum-free DMEM containing TNF-α and Ang II alone or in combination at the specified concentrations, while the lower chamber was filled with 500 μl of DMEM supplemented with 10% FBS as a chemoattractant. After incubation at 37 °C for 24 h, the cells in the upper chambers were carefully removed with swabs, and the cells that had migrated to the bottom chamber were fixed and stained with 0.1% crystal violet. Following air-drying, migrated cells in five random visual fields for each chamber (×200) were counted using a light microscope (BX53, Olympus, Tokyo, Japan). The cell invasion assay was carried out in a similar manner, except that 40 μl of 1:6 phosphate buffer saline (PBS)-diluted Matrigel (BD Biosciences) was added to each well 2 h before cells were seeded onto the membrane. These experiments were performed independently three times.

Flow cytometry (FCM) analysis

HepG2 cells (1 × 106 cells per well) were seeded into six-well plates and treated with TNF-α and Ang II alone or in combination at the specified concentrations for 48 h. After being harvested by trypsinization and washed twice with cold PBS, HepG2 cells were incubated with rabbit antihuman TNFR1, AT1R and AT2R antibodies (Santa Cruz, CA, USA) at 37 °C for 1 h, respectively. Then, FITC-labeled goat anti-rabbit IgG antibody (ZSGB-BIO, Beijing, China) was added to the cells for 30 min at room temperature in the dark. The cells were then collected, and the expression levels of TNFR1, AT1R and AT2R were examined using an FC500 flow cytometer (Beckman Coulter, Brea, CA, USA).

Western blot analysis

HepG2 cells were treated with TNF-α and Ang II alone or in combination at the specified concentrations for 12, 24 and 48 h. Then, protein was extracted from the cells in RIPA lysis buffer (Beyotime, Shanghai, China), and the protein concentration was determined via the Bradford assay [24]. Protein samples were mixed with the 5 × sample buffer (4:1) (Bio-Rad, Hercules, CA, USA) and heated in boiling water for 10 min. The proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After being blocked with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with anti-GRK2 polyclonal antibody (1:1000 dilution, Proteintech, Chicago, IL, USA) overnight at 4 °C. After washing the membranes with Tris-buffered saline containing Tween-20 (TBST) three times for 10 min each, horseradish peroxidase (HRP)-conjugated secondary antibodies were applied for 2 h at room temperature. After being washed three more times with TBST, the target protein bands were visualized using an ECL kit (Pierce, Rockford, IL, USA). Auto-radiographs were scanned using an ImageQuant LAS 4000 mini-system (GE Healthcare Biosciences AB, Uppsala, Sweden). The density of the specific bands was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry (IHC) analysis

IHC was performed to test the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues. Surgical samples were fixed in 4% paraformaldehyde and embedded in paraffin; then, the tissue samples were cut into 4 μm sections and were dewaxed at 60 °C in an incubator. The sections were rinsed in absolute xylene and rehydrated through graded alcohol solutions. Antigen retrieval was performed for 10 min at 95 °C in citrate buffer (pH 6.0), and endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 10 min. The sections were blocked with 5% goat serum for 30 min at 37 °C, and then, the samples were incubated with rabbit polyclonal antibodies against TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 (1:100 dilution, Proteintech, Chicago, IL, USA) at 4 °C overnight. On the next day, the sections were incubated with diluted HRP-conjugated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. After being washed with PBS twice for 3 min each time, the sections were stained with fresh DAB solution (ZSGB-BIO, Beijing, China), counterstained with hematoxylin and dehydrated in graded alcohol and xylene. Finally, the results were quantitatively analyzed using an Olympus BX53 video microscope (Olympus, Tokyo, Japan) and Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) in five random fields (×400) per section. The relative intensities were reflected by optical density values.

Statistical analysis

The data are expressed as the mean ± standard deviation (SD) and were analyzed by one-way ANOVA or Student’s t-test. All statistical analyses were performed using the statistical package SPSS 19.0 (SPSS, Chicago, IL, USA). The results were considered statistically significant at P < 0.05.

Results

Effects of TNF-α and Ang II on HCC cells proliferation

To determine the roles of TNF-α and Ang II in HepG2 cells proliferation, cells were incubated with various concentrations of TNF-α (2.5, 5, 10, 20, 40 ng/ml) and Ang II (1 × 10−9, 1 × 10−8, 1 × 10−7, 1 × 10−6, 1 × 10−5 mol/l) for 12, 24 and 48 h. The results showed that HepG2 cells were sensitive to 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II (Fig. 1a, b). Then treating HepG2 cells with 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination for other 12, 24 and 48 h, the CCK-8 assay confirmed that the proliferative activity of HepG2 cells in all three treatment groups was obviously increased compared with the blank control group in a time-dependent manner. Moreover, compared with the Ang II alone and combination groups, the TNF-α alone group had a more evident effect on cell proliferation, and there were no obvious differences between the Ang II alone and combination groups (Fig. 1c). Thus, TNF-α and Ang II alone or in combination had different levels of stimulatory effects on HCC cells proliferation. The concentrations of 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II were used for the subsequent experiments.

Fig. 1
figure 1

Effects of TNF-α and Ang II on the proliferation of HepG2 cells. a, b The CCK-8 absorbance values of HepG2 cells treated with various concentrations of TNF-α and Ang II for 12, 24 and 48 h. c The CCK-8 absorbance values of HepG2 cells treated with TNF-α and Ang II alone or in combination for 12, 24 and 48 h. The data are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 compared with the control group. # P < 0.05 compared with the TNF-α alone group

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Effects of TNF-α and Ang II on HCC cells migration and invasion

Since HCC cells motility is associated with metastatic potential, Transwell assays were performed to corroborate the effects of TNF-α and Ang II on HepG2 cells migration and invasion (Fig. 2a). After treating HepG2 cells with 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination for 24 h, the Transwell assays results showed that the migratory and invasive ability of HepG2 cells in all three treatment groups was significantly enhanced compared with the blank control group. Furthermore, compared with the Ang II alone and combination groups, the TNF-α alone group had a more prominent effect on cell migration and invasion, and there were no obvious differences between the Ang II alone and combination groups (Fig. 2b, c). Therefore, TNF-α and Ang II alone or in combination had different levels of stimulatory effects on HCC cells migration and invasion.

Fig. 2
figure 2

Effects of TNF-α and Ang II on the migration and invasion of HepG2 cells. a Representative images of the Transwell assays showing HepG2 cells migration and invasion (magnification ×200). b, c Quantitative analysis of the HepG2 cells migration and invasion induced by TNF-α and Ang II alone or in combination for 24 h. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared with the control group. # P < 0.05, ## P < 0.01 compared with the TNF-α alone group

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Expression of TNFR1, AT1R and AT2R on the surface of HCC cells

To determine the effects of TNF-α and Ang II on the expression of TNFR1, AT1R and AT2R, HepG2 cells were treated with 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination for 48 h, and the percentages of TNFR1, AT1R and AT2R-positive cells were identified by FCM analysis (Fig. 3a). The FCM results demonstrated that TNF-α alone group significantly enhanced the expression of TNFR1 compared with the blank control, Ang II alone and combination groups. In addition, compared with the blank control and Ang II alone groups, the combination group had a higher TNFR1 expression, and there were no obvious differences between the blank control and Ang II alone groups. As shown in Fig. 3b, the Ang II alone and combination groups significantly enhanced the expression of AT1R compared with the blank control and TNF-α alone groups, and there were no obvious differences between the blank control and TNF-α alone groups or between the Ang II alone and combination groups. Meanwhile, the combination group significantly enhanced the expression of AT2R compared with the blank control, TNF-α alone and Ang II alone groups. Moreover, compared with the blank control and TNF-α alone groups, the Ang II alone group had a higher AT2R expression, and there were no obvious differences between the blank control and TNF-α alone groups. Accordingly, TNF-α and Ang II alone or in combination had different levels of stimulatory effects on the expression of TNFR1, AT1R and AT2R in HCC cells.

Fig. 3
figure 3

Effects of TNF-α and Ang II on TNFR1, AT1R and AT2R expression levels in HepG2 cells. a Representative FCM plots showing TNFR1, AT1R and AT2R expression on the surface of HepG2 cells. b The percentage of TNFR1-, AT1R- and AT2R-positive HepG2 cells induced by TNF-α and Ang II alone or in combination for 48 h. The data are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 compared with the control group. # P < 0.05, ## P < 0.01 compared with the TNF-α alone group. △ P < 0.05, △△ P < 0.01 compared with the Ang II alone group

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TNF-α and Ang II modulate the expression of GRK2 in HCC cells

Since TNF-α and Ang II were found to be very important in HCC cells proliferation, migration and invasion, Western blot analysis was performed to investigate whether TNF-α and Ang II influence the proliferation, migration and invasion of HepG2 cells by regulating the expression of GRK2 (Fig. 4a). After treating HepG2 cells with 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination for 12 h, the Western blot results showed that there were no obvious differences in GRK2 expression between the blank control, TNF-α alone, Ang II alone and combination groups. As shown in Fig. 4b, when the testing time was prolonged to 24 h, the TNF-α alone and Ang II alone groups significantly inhibited the expression of GRK2 compared with the blank control group, and there were no obvious differences between the blank control and combination groups or between the TNF-α alone, Ang II alone and combination groups. After 48 h of treatment, the TNF-α alone, Ang II alone and combination groups significantly inhibited the expression of GRK2 compared with the blank control group. Furthermore, compared with the combination group, the TNF-α alone group had a lower GRK2 expression, and there were no obvious differences between the TNF-α alone and Ang II alone groups or between the Ang II alone and combination groups. These results indicated that TNF-α and Ang II alone or in combination had different levels of inhibitory effects on the expression of GRK2 in a time-dependent manner, which was related to HCC cells proliferation, migration and invasion.

Fig. 4
figure 4

TNF-α and Ang II modulate the expression of GRK2 in HepG2 cells. a Representative Western blot showing GRK2 expression in HepG2 cells. b Semiquantitative results of GRK2 expression in HepG2 cells induced by TNF-α and Ang II alone or in combination for 12, 24 and 48 h. The data are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 compared with the control group. # P < 0.05 compared with the TNF-α alone group

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Effects of GRK2 on HCC cells proliferation, migration and invasion

To determine the effects of GRK2 on the proliferation, migration and invasion of HepG2 cells, cells were transfected with GRK2 siRNA to reduce GRK2 protein expression level (Fig. 5a, b). GRK2 or scrambled siRNAs-transfected HepG2 cells were seeded into 96-well culture plates and incubated for 12, 24 and 48 h, and the CCK-8 assay confirmed that the proliferative activity of HepG2 cells in the GRK2 siRNA-transfected group was obviously increased compared with the scrambled siRNA-transfected group (Fig. 5c). Meanwhile, GRK2 or scrambled siRNAs-transfected HepG2 cells were seeded into Transwell chambers and incubated for 24 h (Fig. 5d). The Transwell assays results showed that the migratory and invasive ability of HepG2 cells in the GRK2 siRNA-transfected group was clearly enhanced compared with the scrambled siRNA-transfected group (Fig. 5e, f). Thus, downregulation of GRK2 expression in HCC cells by transfecting with GRK2 siRNA can significantly promote cell proliferation, migration and invasion.

Fig. 5
figure 5

Effects of GRK2 on the proliferation, migration and invasion of HepG2 cells. a, b Representative Western blot and semiquantitative evaluation showing the expression of GRK2 in siRNAs-transfected HepG2 cells. c The CCK-8 absorbance values of siRNAs-transfected HepG2 cells incubated for 12, 24 and 48 h. d Representative images of the Transwell assays showing siRNAs-transfected HepG2 cells migration and invasion (magnification ×200). e, f Quantitative analysis of the siRNAs-transfected HepG2 cells migration and invasion incubated for 24 h. The data are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 compared with the scrambled control group

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Effects of TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells

Since GRK2 was found to be very important in HCC cells proliferation, migration and invasion, GRK2 siRNA-transfected HepG2 cells were used to investigate whether TNF-α and Ang II influence the proliferation, migration and invasion of HepG2 cells depending on the expression of GRK2. When treating GRK2-knockdown HepG2 cells with 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination for 12 and 24 h, the CCK-8 assay confirmed that there were no obvious differences in the proliferative activity between the blank control, TNF-α alone, Ang II alone and combination groups. After 48 h of treatment, the TNF-α alone and Ang II alone groups had a more evident effect on cell proliferation compared with the blank control group, and there were no obvious differences between the blank control and combination groups or between the TNF-α alone, Ang II alone and combination groups (Fig. 6a). Meanwhile, GRK2-knockdown HepG2 cells were seeded into Transwell chambers and incubated with 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination for 24 h (Fig. 6b). The Transwell assays results showed that the migratory and invasive ability in the TNF-α alone group was clearly enhanced compared with the blank control group, and there were no obvious differences between the blank control, Ang II alone and combination groups or between the TNF-α alone, Ang II alone and combination groups (Fig. 6c, d). Therefore, downregulation of GRK2 expression in HCC cells by transfecting with GRK2 siRNA can significantly reduce the effects of TNF-α and Ang II alone or in combination on cell proliferation, migration and invasion.

Fig. 6
figure 6

Effects of TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HepG2 cells. a The CCK-8 absorbance values of GRK2-knockdown HepG2 cells treated with TNF-α and Ang II alone or in combination for 12, 24 and 48 h. b Representative images of the Transwell assays showing GRK2-knockdown HepG2 cells migration and invasion (magnification ×200). c, d Quantitative analysis of the GRK2-knockdown HepG2 cells migration and invasion induced by TNF-α and Ang II alone or in combination for 24 h. The data are expressed as the mean ± SD of three independent experiments. * P < 0.05 compared with the control group

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Expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues

IHC was performed to test the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in 32 matched samples (Fig. 7a). The IHC results demonstrated that the expression of TNF-α and TNFR1 proteins in tumor-adjacent tissues was obviously increased compared with those in HCC and normal liver tissues. And compared with normal liver tissues, HCC tissues had a higher expression of TNF-α and TNFR1. Meanwhile, the expression of Ang II, AT1R and AT2R proteins in HCC tissues was clearly increased compared with those in normal liver tissues. In addition, compared with normal liver tissues, tumor-adjacent tissues had a higher expression of Ang II and AT1R. Furthermore, the optical density value of GRK2 in HCC tissues was obviously decreased compared with that in tumor-adjacent and normal liver tissues, and there was no obvious difference in GRK2 protein expression between tumor-adjacent and normal liver tissues (Fig. 7b). Accordingly, HCC, tumor-adjacent and normal liver tissues showed significant differences in the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins.

Fig. 7
figure 7

Expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues. a, b Representative IHC images and positive optical density values showing the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues (magnification ×400). The data are expressed as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 compared with normal liver tissues. # P < 0.05, ## P < 0.01 compared with tumor-adjacent tissues

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Discussion

HCC is a common malignant tumor of the digestive system that can cause serious harm to the physical and mental health of HCC patients, and the prognosis of HCC patients is closely related to the proliferation, migration and invasion of HCC cells [25–27]. Currently, most researchers consider that high concentrations of TNF-α have significant effects on inhibiting the proliferation and inducing the apoptosis of HCC cells, while the effects of low concentrations of TNF-α are mainly on promoting the proliferation and invasion [28–30]. Ang II is the most crucial biological effector in the renin–angiotensin–aldosterone system, and it plays an important role in the formation and growth of tumor vessels [31]. As found in this paper, 5 ng/ml TNF-α and 1 × 10−7 mol/l Ang II alone or in combination can promote the proliferation, migration and invasion of HepG2 cells obviously, which demonstrated that TNF-α and Ang II played key roles in the formation and progression of HCC.

Previous studies have shown that TNF-α and Ang II interact in influencing the inflammation, proliferation and metastasis of synoviocytes, endotheliocytes and other cells [32, 33]. Accordingly, the main aim of the present study was to investigate the interaction of TNF-α and Ang II in the formation and progression of HCC, as well as to further determine the functions of relevant receptors in HCC cells. The results of CCK-8 and Transwell assays revealed that TNF-α and Ang II can promote the proliferation, migration and invasion of HepG2 cells but that the two reagents combined did not have synergistic effects. TNFR1, AT1R and AT2R expression levels on the surface of HepG2 cells were identified by FCM after treatment with TNF-α and Ang II alone or in combination. The results suggested that overexpression of TNFR1 and AT1R perhaps enhanced the proliferation, migration and invasion of HCC cells, while high AT2R expression had the opposite effect.

GRK2 is an important signaling molecule that inhibits the proliferation, migration and invasion of HCC cells [34]. Thus, to explore the effect of TNF-α and Ang II on GRK2 expression in HCC cells has great importance for further understanding how they influence the formation and progression of HCC. Here, we showed via Western blot analysis that treatment with TNF-α and Ang II alone or in combination can significantly suppress GRK2 expression in HepG2 cells in a time-dependent manner. Furthermore, the level of inhibition on GRK2 expression in each of the experimental groups was related to their impact on the proliferation, migration and invasion of HCC cells. Meanwhile, downregulation of GRK2 expression in HepG2 cells by transfecting with GRK2 siRNA can significantly reduce the effects of TNF-α and Ang II alone or in combination on cell proliferation, migration and invasion. Therefore, TNF-α and Ang II could promote the formation and progression of HCC by reducing GRK2 expression, while the two reagents combined did not have synergistic effects.

There are significant differences in TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins expression between HCC, tumor-adjacent and normal liver tissues, indicating that these proteins play different roles in the proliferation, migration and invasion of HCC cells [35–37]. The differential expression of TNF-α and TNFR1 in HCC, tumor-adjacent and normal liver tissues was observed by IHC analysis, and the results implied that a moderate increase in TNF-α and TNFR1 expression could promote the formation and progression of HCC, while excessive expression played an opposite role. Meanwhile, the incremental expression of Ang II and AT1R in HCC tissues suggested that Ang II and AT1R were important in tumor angiogenesis and metastasis [38]. However, the increased expression of AT2R in HCC tissues was not as obvious as that of AT1R, considering that the increased AT2R expression was related to high expression of Ang II. Furthermore, GRK2 expression in HCC tissues was clearly decreased compared with that in tumor-adjacent and normal liver tissues, which indicated that GRK2 had negative effects on the proliferation, migration and invasion of HCC cells.

In conclusion, TNF-α and Ang II are important bioactive factors that promote the proliferation, migration and invasion of HCC cells, primarily by suppressing GRK2 expression. Additionally, the differential regulation of relevant receptors TNFR1, AT1R and AT2R can influence the formation and progression of HCC. Therefore, the present study provides novel ideas for the prevention and treatment of HCC through studying the interaction and probable mechanism of different bioactive factors associated with HCC.