To investigate the relationship between HCC and the signaling pathway molecules of TLR2, a total of 20 paired fresh liver carcinoma tissues and pericarcinoma tissues were collected from patients undergoing curative resection without consideration of recurrence and/or metastasis at the Mengchao Hepatobiliary Hospital of Fujian Medical University (Fujian, China) between June 2010 and July 2016. Clinical and pathological diagnosis of HCC met the criteria of The American Association for the Study of Liver Diseases (https://www.aasld.org). Tumor stage was determined according to Barcelona Clinic Liver Cancer (BCLC) staging classification (12). Primary HCC and matched peritumoral tissues were defined by pathological sampling specification; all tumor tissues excised by hepatectomy were trimmed by an experienced pathologist to remove as much normal liver tissue as possible. Generally the pericarcinoma tissues were located 2 cm beyond the cancer tissues. The final obtained tumor tissues had at least 80% HCC cells as confirmed by H&E staining. Subsequently, the specimens were dissected and then frozen in liquid nitrogen for DNA extraction and subsequent experiments. Serum from patients with HCC was additionally preserved prior to undergoing curative resection. Additionally, the serum of normal healthy individuals was obtained from donors. The serum samples were centrifuged for 5 min at 300 × g at 4°C and were frozen at −80°C. The study was approved by the Ethics Committee of the Mengchao Hepatobiliary Hospital of Fujian Medical University (study license no. 2016-007-01). Informed consent was obtained from patients and health recruits.

The human hepatocellular carcinoma cell line, B76/Huh7, was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The B76/Huh7 cells were cultured in DMEM medium (Hyclone; Thermo, Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Gibco; Thermo, Fisher Scientific, Inc.) in a humidified incubator with 5% CO₂ at 37°C.

To determine the association between HCC and TLR2 as well as TLR2 downstream factors, the HCC tumor and peritumoral tissues were dissociated and 2×10⁶ cells were analyzed by FACS Verse (FCM; BD Biosciences, San Jose, CA, USA). FlowJo 7.6.1(BD Biosciences, San Jose, CA, USA) was used for flow cytometric analysis (Engine 2.79000OS version: Windows 7 Java Version: 14.1-b02). Suspensions of 2×10⁶ B76/Huh7 cells cultured in DMEM, in serum from patients with HCC, in serum from healthy individuals or in 10% FBS were also used to assess TLR2 protein expression by FCM. The cells were collected and washed twice with PBS, stained with 5 µl anti-human TLR2-phycoerythrin (PE) antibody (Product #12-9922-41; 1:1,000; eBioscience™; Thermo Fisher Scientific, Inc.) at 4°C for 30 min away from light. After washing twice with PBS, the cells were collected by low speed centrifugation (1,000 × g; 5 min) at 4°C, suspended in 500 µl PBS and analyzed by FCM. As a negative control an immunoglobulin G2a-PE antibody was used.

The B76/Huh7 cells were seeded at 2×10⁵ cells/well into 6-well dishes and cultured overnight until they reached 70–80% confluence. The cells were treated with 1 µg/µl Pam3CSK4 (Invitrogen; Thermo Fisher Scientific, Inc.) for 72 h to stimulate the cells. Total cellular protein was extracted for western blotting 24 h after stimulation. Another set of B76/Huh7 cells were transfected with pcDH-TLR2-GFP-PMV which was designed and constructed by United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province. To create this plasmid, human TLR2 cDNA was PCR amplified and ligated into a Xho1 and BamH digested pcDH-CMV-EF-PURD-GFP vector, and insertion was confirmed by a sequencing company. Additionally, another set of cells was transfected with a GFP-expressing pcDNA-MyD88 plasmid which was provided by another laboratory. The transfected B76/Huh7 cells were seeded at 2×10⁵ cells/well into 6-well dishes and cultured overnight until they reached 70–80% confluence. Lipofectamine™ 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection of the B76/Huh7 cells according to the manufacturer's protocol. A plasmid DNA (pcDH-TLR2-GFP-PMV, pcDNA-MyD88-GFP or scrambled plasmid) was added to reach a total DNA of 2 µg per transfection. The transfected cells were split equally into two 6-well dishes, and cells were incubated for 48 h. In the second 6-well dishes, 24 h after transfection, the cells were treated with Pam3CSK4 for 5 h in 3 wells each. Green fluorescence was emitted after successful transfection. Images were observed and captured using a fluorescence microscope (Axiocam 506; Carl Zeiss) 48 h after transfection. Images of the cells were composed from white brand images and green fluorescence images.

The B76/Huh7 cells were seeded at 2×10⁵ cells/well into 6-well dishes and cultured overnight until they reached 70–80% confluence. Three groups: TLR2-siRNA-transfected groups; scramble control group; and the blank group were treated in parallel. The sequences for the siRNAs are shown in Table I. Transfections were also performed with Lipofectamine™ 3000 reagent. Transfected cells were incubated for 24 h and harvested for reverse transcription-quantitative (RT-q)PCR. In regards to the other cells, 48 h after transfection, these cells were collected for western blotting.

Total RNA was extracted from fresh liver HCC tissues and cell lines using Trizol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, 1 µg of total RNA was reverse-transcribed using GoScript™ Reverse Transcription mix, Oligo(dT) (Promega Corporation, Madison, WI, USA). For qPCR analysis, single-stranded cDNA was synthesized using oligo(dT) primer in a 20 µl reaction mixture. The primer sequences used are presented in Table II. The PCR thermocycling conditions were: 95°C for 2 min; and 40 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. 18S rRNA was amplified as a control to normalize expression levels. The level of each mRNA was expressed as a ratio relative to the housekeeper gene using the 2^−ΔΔCq method (13). Experiments were repeated at least three times for each sample to ensure the reproducibility of the results.

Cells were harvested and lysed in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) for 20 min. The lysate was centrifugated at 17,000 × g at 4°C for 30 min and protein concentration was determined using a bicinchoninic acid assay. Cell lysates were resolved using a 12% gel using SDS-PAGE, and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% non-fat milk and then blots were probed with antibodies against TLR2 (cat. no. 12276; dilution 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), MyD88 (cat. no. 50010; dilution 1:1,000; Cell Signaling Technology), NF-κB (cat. no. 8242; dilution 1:1,000; Cell Signaling Technology) or GAPDH (cat. no. sc-47724; dilution 1:8,000; Santa Cruz Biotechnology, Inc.) overnight at 4°C. GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as a loading control with the same operation. After washing the membrane three times with PBST for 10 min each wash, the membrane was incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (cat. no. sc-2357; 1:5,000; Santa Cruz Biotechnology, Inc. or cat. no. sc-516102; dilution 1:5,000; Santa Cruz Biotechnology, Inc.). After washing three times with PBST for 10 min each wash, the protein bands were visualized using enhanced chemiluminescence. Densitometry analysis (Bio-Rad Laboratories) was performed using GAPDH as a control.

Proliferation of B76/Huh7 cells was measured using Cell Counting Kit-8 (CCK-8) assay (Phygene, Shanghai, China) at 0, 24, 48 and 72 h following treatment with 5 µg/ml TLR2 agonist Pam3CSK4. Cells were cultured in 96-well cell plates (1.5×10⁵/well; 6 wells per condition) overnight, after which the cells were transfected or treated as described above. After transfection, a sample of the transfected cells was collected as the 0 h sample, while the other cells were further cultured for 24, 48 or 72 h. At the end of each treatment period, CCK-8 reagent was added to the culture medium at a concentration of 5 mg/ml and incubated for 4 h at 37°C. The supernatant was then removed, and cells were mixed with 100 µl/well CCK-8 solution. The absorption was measured using a microplate reader (Spectra Max M5; Molecular Devices, LLC, Sunnyvale, CA, USA) at 450 nm. The supernatant was collected, and ELISA kits were used to measure IL-6, IL-8, IFN-β, TNF-α production (R&D Systems China Co., Ltd., Shanghai, China) 24, 48 and 72 h after transfection.

A total of 48 h after transfection, the cells were trypsinized, collected, washed twice with cold PBS and 1×10⁶ cells were resuspended in 100 µl of Annexin V binding buffer. A total of 5 µl APC Annexin V and 5 µl 7-AAD (BD Biosciences) was added into the cell suspension. The cells were gently vortexed and incubated for 15 min at room temperature in the dark. A total of 400 µl binding buffer was added to each tube and analyzed by flow cytometry.

Statistical analysis was performed using GraphPad 6.0 (GraphPad Software Inc, La Jolla, CA, USA). Data from at least three experiments are expressed as mean value ± standard deviation and were interpreted using one-way ANOVA followed by Bonferroni or Dunnett test where relevant. All comparisons were determined using a repeated measures one-way ANOVA between the experimental group and the control group. P<0.05 was considered to indicate a statistically significant difference.

To examine the role of TLR2 in HCC progression, semi-quantitative RT-PCR and FCM were used. The results showed that TLR2 expression in the peritumoral tissues was significantly higher compared with that noted in the tumor tissues (Fig. 1A and B). The protein expression level of TLR2 was determined using western blotting, and similar to the PCR results, protein expression of TLR2 was significantly higher in the peritumoral tissue compared with that noted in the tumor tissue (Fig. 1C). Our complementary study of 141 samples showed the expression levels of TLR2 in the tissues of liver and no differences were found for different stage and prognosis (Table SI).

To distinguish whether the serum of patients with HCC was able to interfere with the TLR2 signaling pathway in HCC cells through the activation of Pam3CSK4, the TLR2 expression in B76/Huh7 cells was detected by FCM after 48 h of culturing with the serum of HCC patients (Ca), with the serum of normal healthy individuals (Normal) or FBS. The results showed that the expression of TLR2 decreased in the cells treated with serum from HCC patients when compared with cells treated with the serum from healthy individuals (Fig. 2A). Transcription of various inflammatory cytokines such as IL-8 and TNF-α was altered, as determined by qPCR. Compared with the cells treated with FBS, expression of inflammatory cytokines was decreased in cells treated with serum from patients with HCC (Ca) and in cells treated with serum from healthy individuals (Normal). Inflammatory cytokine levels of TLR2-mediated activation of cells treated with the serum of HCC patients and serum from healthy individuals were lower than in the cells treated with FBS especially following the activation of Pam3CSK4 (PSK), confirming efficient inverse feedback (Fig. 2B). This suggests that some component of the serum from patients with HCC interfered with the TLR2 signaling pathway through Pam3CSK4 activation in liver cells and reduced the TLR2-mediated immune inflammation. Yet, this substance remains unknown and further experiments are warranted.

To confirm the findings with TLR2 in vitro, a TLR2 agonist, Pam3CSK4, was used to stimulate B76/Huh7 cells. Following treatment with the agonist, the cells were assayed by qPCR, western blotting and FCM (Fig. S1). The protein expression levels of signaling pathway proteins, including MyD88, IRAK1, TRAF6 and NF-κB were significantly increased 48 h after stimulation with Pam3CSK4 (Fig. S2A). The relative protein levels of the signaling molecules were also increased (Fig. S2B).

To detect the efficiency of TLR2-overexpression plasmid and whether the pathway was activated by TLR2 agonist, B76/Huh7 cells were divided into six groups: TLR2-overexpression plasmid group; scramble group and the blank group with or without stimulation with Pam3CSK4. The results demonstrated that the expression of molecules in the TLR2 signaling pathway, such as TLR2 and MyD88 in the TLR2-overexpression groups were significantly higher than in the scramble groups compared with the control groups, particularly when stimulated with Pam3CSK4, both at the protein level (Fig. 3A) and gene level 48 h after transfection (Fig. 3B). Images of B76/Huh7 cells transfected with the plasmids were obtained using a fluorescence microscope at the same time (Fig. S3).

To detect the efficiency of MyD88-overexpression plasmid, B76/Huh7 cells were also divided into six groups; MyD88-overexpression plasmid group; scramble group; and the blank group with or without Pam3CSK4 stimulation. In comparison with the scramble groups and the control groups, MyD88-overexpression plasmid group also demonstrated higher expression of TLR2 and MyD88 both at the protein level (Fig. 4A) and gene level (Fig. 4B) 48 h after transfection, particularly when stimulated with Pam3CSK4. There was no consistent corresponding alterations in the expression of NF-κB following transfection with either of the two plasmids (Figs. 3 and 4). Images of transfected cells with the plasmids in B76/Huh7 cells were obtained using a fluorescence microscope (Fig. S4).

The functional experiments showed that overexpression of TLR2 and MyD88 significantly inhibited the proliferation of B76/Huh7 cells (Fig. 6A and B) and significantly increased apoptosis of B76/Huh7 cells (Fig. 7A and B).

To detect the efficiency of TLR2-siRNA or MyD88-siRNA, cells were divided into five groups per a target RNA: siRNA1 group, siRNA2 group, siRNA3 group, scramble group and the blank group. The results of the qPCR and western blotting demonstrated that the expression of protein and messenger RNA (mRNA) of TLR2 or MyD88 in the siRNA groups was significantly decreased compared with the blank groups (P<0.05). It was observed that all six different siRNAs significantly reduced expression of TLR2 and MyD88 at the protein (Fig. 5A) and gene expression level (Fig. 5B). The results also showed that the expression of NF-κB did not significantly differ at the gene and protein level. The siRNAs with the largest decrease in the expression levels for both TLR2 and MyD88 were used for the subsequent experiments. Knockdown of TLR2 or MyD88 significantly increased proliferation (Fig. 6C and D) and decreased apoptosis significantly (Fig. 7A and B) in the B76/Huh7 cells.

To investigate the role of cytokines through the activation of the TLR2 signaling pathway, IL-6, IL-8, TNF-α and IFN-β concentrations were assayed in the supernatant of cells using ELISA, 24, 48 and 72 h after transfection of the TLR2-overexpression-plasmid or MyD88-overexpression-plasmid in B76/Huh7 cell line. The results showed that IL-6 and TNF-α expression was decreased and IL-8 and IFN-β secretion was increased slowly between 24 and 48 h in the TLR2-overexpressing cells and following stimulation with Pam3CSK4 in the supernatants of the B76/Huh7 cells. IL-6 and TNF-α secretion was decreased in the MyD88-overexpressing cells and following stimulation with Pam3CSK4 between the 24 and 48 h timepoints in the supernatants of B76/Huh7 cells. IL-6 and TNF-α secretion was increased in the MyD88-overexpressing cells and Pam3CSK4 stimulated cells between 48 and 72 h. But IL-8 secretion was increased slowly between 24 and 72 h in the supernatants of the B76/Huh7 cells and IFN-β was decreased slightly in quantitative terms between 24 h and 72 h in the supernatants of B76/Huh7 cells (Fig. 8). Next it was determined whether the function of the TLR2 pathway was associated with IL-6, IL-8, TNF-α, and IFN-β in B76/Huh7 cells lines. The results showed that the proinflammatory cytokines, such as IL-6 and TNF-α were decreased and anti-inflammatory cytokines, including IL-8 and IFN-β increased when the cells were transfected with the TLR2 plasmid in B76/Huh7 cells. IL-6 and TNF-α secretion was increased between 48 and 72 h suggesting that additional MyD88 pathways were also stimulated in B76/Huh7 cells.