CX3CR1 regulates hepatocellular carcinoma(HCC) metastasis via PI3K/AKT pathway

Background Fractalkine receptor CX3CR1 is differentially expressed in hepatocellular carcinoma and its function is unknown. As a special case of chemokine family, CX3CR1 only binds to its unique ligand CX3CL1, and CX3CL1 also only binds to its only receptor CX3CR1, the unique matching between ligands and receptors is not found in other chemokines, and this specicity is essential for diagnosis, prognosis and clinical target therapy. Methods CX3CR1 expression was analyzed by immunohistochemistry, migration and invasion abilities of SMMC-7721 cells were detected by wound-healing and transwell after overexpressing CX3CR1. In addition to overexpression experiments, interfering RNA siCX3CR1 was used for reverse validation in HepG2 cells. To further clarify the mechanism of CX3CR1 regulating HCC metastasis, the classical PI3K/AKT pathway were detected by Western blot. Results The CX3CR1 levels were positive correlated with pathological TNM stage, meanwhile a negative correlation between high expression of CX3CR1 and survival rate was found among patients at stageII-III. In our study, overexpression of CX3CR1 promoted migration and invasion ability of SMMC-7721 cells, whereas knockout of CX3CR1 inhibited these abilities of HepG2 cells. Mechanistically, CX3CR1 regulates the metastasis of HCC cells via PI3K/AKT pathway, and the PI3K inhibitor LY294002 could stop the promoting effect of CX3CR1 on SMMC-7721 cells. Conclusion CX3CR1 regulated HCC cell metastasis via PI3K/AKT signaling and this effect might be a new target for clinical diagnosis and treatment.


Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world [1]. Although there are many methods to treat hepatocellular carcinoma, such as surgery, radiotherapy and chemotherapy [2][3][4], but many patients have lost the opportunity of surgical treatment because the early symptoms of primary hepatocellular carcinoma are not obvious [5]. The use of radiotherapy and chemotherapy is also limited because of its toxic and side effects. Therefore, it is particularly important to explore the mechanism of the occurrence and development of hepatocellular carcinoma and nd new treatment methods.
Chemokines are 8-10KD proteins which can regulate leukocyte migration to infected sites by binding to their speci c chemokine-receptors [6]. Chemokine receptor 1 (CX 3 -C motif chemokine receptor 1, CX 3 CR1) is the only known member of CX 3 C family. It is mainly expressed on the surface of T cells, natural killer cells, monocytes and vascular endothelial cells [7][8][9][10]. More and more studies have shown that chemokines and their receptors play an important role in the growth, invasion and metastasis of tumors.
It has been con rmed that CX 3 CR1 is involved in regulating the occurrence and development of breast cancer [11], prostate cancer [12], pancreatic cancer [13], ovarian carcinoma [14] and other tumors [15], but there are few reports about its role and mechanism in hepatocellular carcinoma.
In this study, the effects of overexpression of CX 3 CR1 on the migration and invasion of human hepatocellular carcinoma cells were explored in two aspects: overexpress CX 3 CR1 in low-expression cell lines and inhibit CX 3 CR1 in high-expression cell lines. The molecular mechanism of CX 3 CR1 was preliminarily explored to provide a new basis for the treatment and prognosis of hepatocellular carcinoma.

Plasmid transfection
The SMMC-7721 cells in logarithmic growth phase were inoculated into 96-well plate, 24-well plate, 6-well plate and culture dish respectively. When the cell density reached 60%, the complete DMEM medium was replaced with medium without FBS nor penicillin-streptomycin. The mixture of 1μg/ml CX 3 CR1 overexpression plasmid, 1μl/ml Lipo2000 (Cat.No.11668019, Invitrogen, California, USA), 100ug/ml medium without FBS was incubated at room temperature for 30 minutes, then the mixture was added into the culture medium evenly. The medium without FBS was changed to complete medium after 4-6 hours. At the same time, the same amount of empty vector plasmid was added in the negative control group (NC group) to exclude the in uence of the plasmid itself on the experiment. The overexpression and empty plasmids were purchased from Genechem Co.,Ltd. (shanghai, China). 10nmol/ml LY294002 (Cat.No.HY-10108, MCE, China) was added in the SMMC-7721 cells after transfecting CX3CR1 over-expression plasmid.
Interfering RNA transfection HepG2 cells in logarithmic growth phase were inoculated into 96-well plate, 24-well plate, 6-well plate and culture dish respectively. When the cell density reached 50%, the mixture of 60μl/ml Buffer, 2.5μl/ml CX 3 CR1 interfering RNA and 6μl/ml transfection reagent was evenly divided into the medium. At the same time, the same amount of negative control interfering RNA was added to establish negative control group (NC group). The CX 3 CR1 and negative control interfering RNA were purchased from RIBOBIO Co.,Ltd. (Guangzhou, China).
Quantitative real-time polymerase chain reaction (qPCR) Quantitative real-time PCR was performed in a CFX-connect fast real-time PCR system (Bio-RAD, California, USA). The sample mixture was composed of 5μl SYBR (TaKaRa, Japan) 0.8μl primers 1μl cDNA and 3.2μl ddH 2 O. The following touch-down cycling conditions: 1) 95°C for 10min. 2) 95°C for 20s.

Wound-healing test
When the density of cells were reached to 80% in the 6-well plate after transfecting with plasmids or siRNA, the cells were scratched with a small pipette tip, and washed with PBS for 3 times, then replaced with the medium containing 2% serum. The cells were observed and photographed under the microscope at 0 hours, 24 hours and 48 hours. Then, the wound-healing width was recorded at the same observation point. The average wound-healing widthh of multiple observation points was calculated, and the scratch healing rate of each group was obtained.
Transwell 400μL culture medium with 2% serum containing 40000 SMMC-7721 cells or 60000 HepG2 cells was added into the upper chamber (matrix glue was used in invasion experiment, matrix glue and culture medium were 1:5 ratio, 50μL, Cat.No.356234, Corning, USA) and 700μL complete culture medium containing 10% serum was added into the lower chamber. Four parallel controls were set up in each group. After 24 hours, the cells on the lower side of chamber membrane were xed for 20 minutes with 500μL 4% polyformaldehyde. The cells were stained with 500μL crystal violet for 30 minutes. After washing and drying, the cells were counted under a microscope. At least 10 visual elds were observed in each chamber, and the average values were obtained after counting.
Statistical analysis SPSS 16.0 statistical software was used to analyze the experimental results. Each experiment was repeated three times. The differences between protein and mRNA expression were compared by t-test or Bonferroni-corrected t-test. The Log-rank(Mantel-Cox) test or Gehan-Breslow-Wilcoxon test was used to analyze difference between survival curves. The difference was statistically signi cant with *P < 0.05 **P<0.005 ***P<0.001.

CX 3 CR1 expression in HCC tissues is related to clinical stages and is a marker of poor prognosis
In order to clarify the relationship between CX 3 CR1 and the progression of HCC, we detected the expression level of CX 3 CR1 in 20 cases of hepatocellular carcinoma by immunohistochemistry between high-differentiation and low-differention groups, nding that the expression of CX 3 CR1 was uniform and higher in the low-differentiated group, but it was not uniform in the high-differentiated group, which had low-expression and high-expression regions. (Fig. 1a). Then we analyzed the mRNA expression data of CX 3 CR1 in 365 HCC tissues which were dowmloaded from The Human Protein Atlas. These results showed that the expression level of CX 3 CR1 was higher at stage than that at stage - (Fig. 1b). The correlation between CX 3 CR1 expression and prognosis of HCC patients at stage -was veri ed by Kaplan-Meier survival analysis, nding the patients with high expression of CX 3 CR1 had lower short-term survival rate (Gehan-Breslow-Wilcoxon test) in total-population (Fig. 1c) and male-popuation (Fig. 1d), the long-term survival rate (Log-rank(Mantel-Cox) test) was also lower in male-population but no difference in total-population. On the contrary, both short-term survival rate and long-term survival rate was no diffrence in female-population (Fig. 1e). In conclusion, there was a negative correlation between high expression of CX 3 CR 1 and survival rate was found among patients at stage -. CX 3 CR1 is expressed at low levels in SMMC-7721 cells and at high levels in HepG2 cells Western blot analyses were used to detect the mRNA expression and protein expression levels of CX 3 CR1 in different HCC cells compared with human normal hepatocytes LO2, nding that CX 3 CR1 expression levels were higher in HepG2 and lower in SMMC-7721 cells (Fig. 2a). Therefore, we choose HepG2 cell lines to verify interference experiments, and SMMC-7721 cells lines to verify overexpression experiments.
we downregulated CX 3 CR1 in HepG2 cells using siRNA and upregulated CX 3 CR1 in SMMC-7721 cells by transfecting CX 3 CR1-plasmid, the Q-PCR (Fig. 2b c and d) and western blot analyses showed these treatment were effective (Fig. 2e and f).

CX 3 CR1 regulates the migration and invasion ability of HCC cells
To explore the role of CX 3 CR1 in metastasis, Wound-healing test showed that the migration ability was promoted in SMMC-7721 cells after overexpressing CX 3 CR1 (Fig. 4a), and migraion ability of HepG2 was inhibited by knockout of CX 3 CR1 (Fig. 4b). Tanswell assays revealed that CX 3 CR1 overepression promoted the migraion and invasion of SMMC-7721 cells ( Fig. 3a and b), whereas knocked of CX 3 CR1 inhibited the migraion and invasion of HepG2 cells as compared with control cells (Fig. 3c and d).
Consistent with these results, the protein levels of matrix metalloproteinase [16][17] MMP-3 and MMP-9 were up-regulated in SMMC-7721 cells by overexpressing CX 3 CR1 (Fig. 4c), and the level of MMP-3 and MMP-9 were down-regulated in HepG2 by knocking out CX 3 CR1 (Fig. 4d). These ndings demonstrated that CX 3 CR1 regulated the migration and invasion of HCC cells. CX 3 CR1 regulates the migration and invasion of HCC cells via PI3K/AKT pathway PI3K/AKT pathway has been proved to play an important role in the process of chemokin-axis regulating tumor [18][19][20]. Western blot test was ued to verify whether PI3K/AKT signaling pathway was involved in regulating metastasis of hepatocellular carcinoma. The results revealed the phosphorylation of AKT(p-AKT) was up-regulated in SMMC-7721 cells by transfecting CX 3 CR1-plasmid compared with control group (Fig. 5a) , on the contrary, the phosphorylation-AKT level was down-regulated in the HepG2 cells after koncking out CX 3 CR1 (Fig. 5b). To investigate whether CX 3 CR1 was mediated through the PI3K/AKT pathway, we added the PI3K/AKT pathway inhibitor LY294002. Wound-healing test showed that LY294002 inhibitedd the migration ability of SMMC-7721 cells after ovexpressing CX 3 CR1 (Fig. 5c and d), and the migration and invasion-related protein MMP-9 also reduced by using LY294002 compared with over-expression CX 3 CR1 groups (Fig. 5e f and g), these results showed that CX 3 CR1 regulated the metastasis of HCC cells via PI3K/AKT pathway.

Discussion
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The death toll of HCC patients is as high as 745,000 [21]. The occurrence and development of HCC is a complex process involving multiple factors. Because of its initial symptoms, most patients have lost the best opportunity for surgical treatment [22]. With the continuous exploration and research, the chemokine axis is becoming increasingly important. More and more attention has been paid to the role of CXCR4 [23][24], CCL20 [25], CCL2-CCR2 [26], CCR4 [27] and other chemokine axes in the occurrence and development of hepatocellular carcinoma [28][29][30]. As a chemokine receptor, CX 3 CR1 has also been proved to be involved in regulating the biological effects of various tumors. In addition , tumor microenvironment, which consists of stromal cells, vascular endothelial cells, immune cells and extracellular matrix, is also an indispensable part of the process of tumorigenesis and development [31][32][33][34]. Chemokines and their receptors have been proved to play a very important role in tumor microenvironment. Studies have con rmed that chemokine axis CXCL9/10-CXCR3, CXCL12-CXCR4, CCL20-CCR6, CCL5-CCR5, CXCL5-CXCR2 and so on can regulate the functions of various immune cells in tumor microenvironment to further promote immune escape of tumor cells and angiogenesis of tumors to help metastasis and progress of tumors [35][36][37][38][39]. CX 3 CR1 has also been shown to play an important role in regulating tumor and tumor microenvironment [40]. Although there are many studies on CX 3 CR1 in tumors, the relationship between CX 3 CR1 and hepatocellular carcinoma is still unclear.
In this study, we found that CX 3 CR1 was differentially expressed in different stages of HCC and there was a signi cant negative correlation between higher-level CX 3 CR1 and prognosis in male-population. At the same time, CX 3 CR1 also showed differential expression in different hepatocellular carcinoma cell lines. In order to explore the direct relationship between CX 3 CR1 and biological characteristics of hepatocellular carcinoma cells, the CX 3 CR1 overexpression plasmid was transfected with SMMC-7721 cells, which expressed the CX 3 CR1 at low-level, on the contrary, the CX 3 CR1 interference RNA was transfected with HepG2 cells, which expressed the CX 3 CR1 at high-level. The migration and invasion ability of SMMC-7721 cells overexpressing CX 3 CR1 was signi cantly enhanced. At the same time, the levels of MMP-3 MMP-9 and p-AKT were signi cantly increased, and the migration and invasion ability of HepG2 cells inhibiting CX 3 CR1 was signi cantly weakened. At the same time, the levels of MMP-3 MMP-9 and p-AKT were signi cantly decreased. In conclusion, while overexpression of CX 3 CR1 in SMMC-7721 cells can promote its migration and invasion ability, interferencing with CX 3 CR1 can signi cantly inhibit the migration and invasion of HepG2 cells. Its mechanism may be related to activation of PI3K/AKT signaling pathways. Thus, CX 3 CR1 may become a potential new target for the treatment of hepatocellular carcinoma.

Conclusions
Our study reveal for the rst time the relationship between CX 3 CR1 and HCC metastasis. Here, we nd that CX 3 CR1 is signi cantly correlate with the survival of patients with hepatocellular carcinoma, and this phenomenon is more evident in male patients. CX 3 CR1 regulates migration and invasion ability of SMMC-7721 and HepG2 cells via PI3K/AKT pathway, and the PI3K inhibitor LY294002 reverse the promoting effect of CX 3 CR1 on SMMC-7721 cells. Thereby CX 3 CR1 regulates HCC cell metastasis via

Declarations
Ethics approval and consent to participate This study was approved by the Medical Research Ethics Committee of Chongqing Medical University, and informed consent was obtained from all patients.

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The trial was supported by the Natural Science Foundation of China (NSFC 81672103 to QS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.