IL-17 predicts the effect of TACE combined with apatinib in hepatocellular carcinoma

2.1 Ethics statement

The present study was performed with the approval of the Ethics Committee of Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science (approval number 2018-056). All aspects of the study complied with the Declaration of Helsinki. In addition, all participants signed the informed consent.

2.2 Patients and tissues

All HCC samples were collected from 175 patients who had undergone curative resections from primary HCC. There were 97 patients had undergone TACE 1–2 months after resection (TACE group), whereas 78 patients taken an additional apatinib (TACE + Apatinib), and their clinical data are shown in Table 1.

Inclusion criteria: (1) No chemotherapy contraindications, mainly, there are no obvious obstacles in organ function; (2) BCLC liver cancer stage A, B or C stage; (3) liver function Child grade A or B; KPS score > 60 points; (4) the surgeon could not accept or reject the surgical resection, and did not receive other treatment before surgery.

Exclusion criteria: (1) Have chemotherapy contraindications; (2) severe blood coagulation; disabled person; (3) heart, lung and other important organ dysfunction; (4) stop taking medicine; more than 1 month; (5) there is a hepatic artery-portal vein or hepatic vein or portal vein trunk or left and right main tumor thrombus; (6) massive ascites or refractory peritoneal effusion.

Human HCC cell lines SK-Hep1 (HTB-52, Manassas, USA) was cultured in DMEM medium (D6046, Sigma Aldrich, USA) which was added into 10% fetal bovine serum (10099-141, Gibco, USA) at 37°C and 5% CO₂. Si-control and Si-IL-17 were synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China), and were directly transferred into cells by Lipofectaminetrademark 2000 transfection reagent (11668019, Invitrogen, USA).

2.4 Western blot

Cell lysates were separated by SDS-page and then transferred to PVDF membrane. Primary antibody was selected as follows: anti-IL-17 (ab79056, abcam, UK), or anti-E-cadherin (ab1416, abcam, UK), or anti-vimentin (ab92547, abcam, UK) or anti-GAPDH (ab9484, 1 : 3000, abcam, UK). And second antibody was selected as follows: goat anti-rabbit (ab150077, 1 : 1000). Primary antibody was incubated overnight at 4°C and second antibody was incubated for 1 hour at room temperature.

2.5 Transwell assay

Transwell experiment was used to determine migration and invasion ability of cells. Matrigel (BD354248, Becton Dickinson, USA) is the material we use for the Transwell assay, and all operations refer to the instructions. After membrane was dried, it was stained with crystal violet for 20 minutes, and were counted under a microscope.

2.6 Animal experiments

5×10⁶/0.2 ml logarithmic phase of human SK-Hep1 cells were taken. 1-week-old nude mice (male/female = 1 : 1, quality certificate: NO.0003286) that were adaptive for feeding (room temperature of 20–24°C, half day and night, air humidity of 60%) were selected. Middle axillary lateral skin of nude mouse was selected as cell inoculation site. Si-control SK-Hep1 (5 mice), Si-IL-17 (5 mice). Mice were sacrificed and tumor tissue were taken out after 14 days after cell inoculation.

2.7 Immunochemistry

Levels of IL-17, E-cadherin, Vimentin and CD34 protein in tissue were measured as described previously by immunochemistry [21]. And the primary antibody was described as western blot. CD34 (ab81289, abcam, UK). Finally, 5 fields per slice were photographed and scored: IL-17 and vimentin located at cytoplasm, E-cadherin at cell membrane; color score: no color was negative (0), faint yellow or yellow was weak positive (1), yellow was positive (2), brown-yellow or tan was strongly positive (3). Two pathology deputy chief physicians blinded to diagnosis all color scoring in stained tissue. At the same time, count the proportion of positive cells in each field of view. Immunohistochemical score of protein = Color score×Positive cell ratio. In this study, Immunohistochemical score of protein > 1.5 was considered to be high expression.

2.8 Statistical analysis

Data were analyzed by SPSS 20.0 and presented as mean±SD. Student’s t test, χ² test and Fisher’s exact test were used to compare the differences between groups. Pearson method was used to analyze the correlation between two indicators. Cox regression model was used to test univariate and multivariate analysis for survival times of HCC patients. Survival curves of HCC patients was drawn by Kaplan-Meier method. Log-rank test was used to compare differences of survival curves. P < 0.05 indicated significant difference.

3.1 IL-17 was related with the migration and invasion of HCC cell in vitro

We established IL-17 knockdown SK-Hep1 cell line by transfecting Si-IL-17, and investigated the effect of IL-17 expression level on migration and invasion of SK-Hep1 cells in vitro by transwell assay. As showed in Fig. 1A, knockdown of IL-17 significantly increased E-cadherin protein expression and decrease Vimentin protein expression, which were two proteins closely related to the epithelial-mesenchymal transition (EMT) ability of the cancer cells. Moreover, the results of transwell assay showed that down-regulation of IL-17 expression not only reduced the migration ability of SK-Hep1 cells (Fig. 1B), but also reduced its invasive ability (Fig. 1C). These data suggested that IL-17 promoted the SK-Hep1 cell migration and invasion.

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Fig.1

Down-regulation of IL-17 expression inhibited the migration and invasion of SK-Hep1 cell in vitro. A, Down-regulation of IL-17 expression leads to up-regulation of E-cadherin protein and down-regulation of Vimentin protein expression in SK-Hep1 cells; B-C, IL-17 was down regulated in SK-Hep1 cells and promoted cell migration (B) and invasion (C). Each experiment was repeated three times independently. Bar = 50 μm.

Nude mice are the most commonly used animal models for studying the biological functions of tumor cells. We established an animal model of HCC xenografts in nude mice by subcutaneous injection of SK-Hep1 cells with Si-control or Si-IL-17. After 2 weeks, the mice were sacrificed and tumor tissues were obtained. Immunohistochemistry was used to detect the expression of IL-17 and CD34 protein in tumor tissues. As showed in Fig. 2A, the expression of IL-17 and CD34 protein in Si-IL-17 group were significantly lower than that in Si-control group. Calculating the number of MVD in tumor tissue according to the expression of CD34, and we found that the MVD of tumor tissue in Si-IL-17 group were significantly lower than that in Si-control group (Fig. 2B). In addition, we also obtained mouse peripheral blood through the orbital venous plexus and separated the plasma before the mice were sacrificed. We measured the content of VEGF in serum and found that the level of serum VEGF in Si-IL-17 group was significantly lower than that in Si-control group (Fig. 2C). It indicated that IL-17 was associated with angiogenesis in HCC tumor tissue.

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Fig.2

Inhibition of IL-17 expression can reduce tumor microvessel density in nude mice. A) Immunohistochemistry was used to detect the expression of IL-17 and CD34 protein in tumor tissues of nude mice injected with SK-Hep1 cells; B) Microvessel density (B) or plasma VEGF level (C) of tumor tissue in nude mice inoculated with SK-Hep1 Si-IL-17 or control cells via subcutaneous injection. At least 5 nude mice per group. Bar = 100 μm.

Immunohistochemistry was used to measure IL-17, E-cadherin, Vimentin and CD34 protein expression in 175 cases HCC tissue (Fig. 3A), and the Pearson method was used to analyze the correlation between IL-17 and E-cadherin, Vimentin or CD34 protein expression. And we found that IL-17 protein expression was negative correlation with E-cadherin protein expression (Fig. 3B), was positive correlation with Vimentin protein (Fig. 3C), and was positive correlation with MVD of HCC tumor tissues (Fig. 3D) which were calculated according to the expression of CD34. These results indicated that IL-17 protein expression was related the invasion and angiogenesis in HCC tumor tissue.

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Fig.3

IL-17 protein expression was related to E-cadherin, Vimentin and CD34 protein in tumor tissues with HCC. A) Immunohistochemical detection of IL-17, E-cadherin, Vimentin and CD34 protein expression in tumor tissue with HCC. Case 1 with high expression of IL-17, Vimentin and CD34 protein, and low E-cadherin expression. Case 2 with low expression of IL-17, Vimentin and CD34 protein, and high E-cadherin expression; B-D) The expression of IL-17 protein in tumor tissues with HCC was positively or negatively correlated with the expression of E-cadherin (B), Vimentin (C) and CD34 protein (D). Bar = 100 μm.

Adjuvant TACE is one of the most commonly used methods for the treatment of HCC, and apatinib is a common drug for postoperative patients with HCC. However, the effect of TACE combined with apatinib in the treatment of patients after HCC surgery is inconsistent. As showed in Fig. 4, we found that although TACE combined with apatinib treatment did not significantly improve overall survival within 5 years of all HCC patients and patients with high IL-17 expression, it could significantly improve overall survival within 5 years of patients with low IL-17 expression.

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Fig.4

Prognostic significance of postoperative adjuvant TACE combined with apatinib treatment according to IL-17 expression. A-C) Kaplan-Meier was used to analyle the prognostic significance of TACE combined with apatinib treatment in all HCC patients (A), in low IL-17 expression HCC patients (B) or in high IL-17 expression HCC patients (C). MS = Median survival (month).

In addition, the factors influencing the survival times of HCC patients with different IL-17 expression were analyzed by COX regression model. The results showed that liver cirrhosis, tumor differentiation, BCLC stage, cancer cell metastasis and apatinib therapy were significantly associated with overall survival within 5 years of HCC patients with low IL-17 expression who accepted adjuvant TACE (Table 2), and GGT, liver cirrhosis, tumor differentiation, BCLC stage and cancer cell metastasis were significantly associated with overall survival within 5 years of HCC patients with high IL-17 expression who accepted adjuvant TACE (Table 3).