CXCL6 Is Upregulated In Hepatocellular Carcinoma And Promotes Proliferation And Invasion Of Liver Cancer Cells

Backgrounds:Hepatocellular carcinoma after resection in the typical recurrence have two representative forms:First, the primary tumor metastasis(Generally occurs in primary tumor resection or liver transplantation after 2 years,as the early relapse);The second, recurrence(After 2 years of treatment of the primary tumor, as the long-term recurrence). Currently the diagnosis of primary hepatocellular carcinoma recurrence and metastasis in clinical imaging method is mainly for the application and combination of serological detection method, including periodic review of B-ultrasound, and periodic review patients with positive preoperative alpha-fetoprotein (AFP) serum AFP. The postoperative examination showed that the recurrence rate of AFP was significantly higher than that of patients who had no significant decrease in preoperative AFP levels,persistent positive alpha-fetoprotein in hepatocellular carcinoma suggested residual stoma activity. Serum AFP alone for early diagnosis of recurrent liver cancer is lower than B-ultrasound, B-ultrasound diagnosis of early and lower than CT. The combination of the three organic use will help early diagnosis. In addition to conventional liver cancer after resection, including chest X-ray film and the whole body bone imaging ECT. There is no clear evidence that positron emission tomography (PET-CT) for early diagnosis of liver cancer recurrence and metastasis. The detection of HCC metastasis and recurrence of the method is clearly difficult to meet the current ideal of early diagnosis and treatment of clinical needs. Studies have shown that the induction of a tumor-promoting signal for sustained release of the surrounding microenvironment of the liver with impaired hepatic fibrosis and cirrhosis is associated with long-term recurrence; Different molecular subgroups of the HCC microenvironment have been identified as being associated with poor prognosis in patients with HCC. In early tumors(<2 cm of the tumor, no vascular invasion or extrahepatic spread), the gene expression profile encoded by non-neoplastic tissue adjacent to hepatocellular carcinoma is predictive of clinical outcome, which may be more convincing than the genetic description of tumor cells themselves. Therefore, it is important to find new treatment targets for prevention of HCC proliferation and invasion.Inflammation as a major feature of cancer in recent years, has been recognized by everyone. In the past studies, it was found that inflammation is closely related to the development and progression of tumors, and has a profound impact on tumor growth, invasion and metastasis. Chemokines and their receptors are an important component of inflammatory microenvironment.A large number of studies have confirmed the existing literature, and chemokine receptor plays a very important role in promoting tumorigenesis, development and metastasis. Chemotaxis-induced chemotaxis of cancer cells by tumor-derived chemokines is an important step in tumor metastasis. The role of chemokines in the promotion of liver cancer metastasis has also been more and more attention.Chemotactic cytokines (chemokines) were reported to play important roles in promoting metastasis of HCC. Chemotaxis of cancer cells leading by tumor-derived chemokines is an important step during tumor metastasis. In other word, chemokines and their receptors play crucial role in tumor invasion and metastasis. CXC chemokines are divided into pro-angiogenic(ELR+) and anti-angiogenic(ELR-) according to whether with a Glu-Leu-Arg (ELR) N-terminal motif[5]. The granulocyte chemotactic protein 2 (GCP-2) which also known as CXCL6 belongs to ELR+CXC chemokines. Similar to other CXC ELR+chemokines, CXCL6 was demonstrated to facilitate tumor growth in animal experiment. Besides, CXCL6 has been illustrated as important mediators of progression in several solid tumors[6]. CXCL6 was found up-regulated in solid tumors, including small cell lung cancer, melanoma and colorectal cancer. Among common HCC cell lines, MHCC97H or HCCLM3 cells characterized with high metastatic capacity and HepG2 or SMMC7721 cells with low metastatic capacity[7]. Levels of CXCL6 in MHCC97H or HCCLM3 were higher than those from HepG2 or SMMC7721 cells. This result suggests that CXCL6 may facilitate HCC cell invasion[8]. However, further studies about the role of CXCL6 on the invasion and growth of HCC cells remain insufficient. Moreover, the significance of CXCL6 in predicting prognosis of HCC patients remains unclear.Objectives:The aim of this study was to further explore and validate the effect of CXCL6 on proliferation and invasion of hepatocellular carcinoma cells in HCC. To analyze the relationship between the expression of CXCL6 in clinical specimens and prognosis of tumor size and vascular invasion, overall survival rate (OS) and relapse-free survival rate (RFS). To study the difference of expression of CXCL6 in MHCC97L, MHCC97H, HCCLM3, HepG2 and SMMC 7721 cell lines. And in vitro cell proliferation assay was performed by targeting MHCC97H and HCCLM3 cell lines that silenced CXCL6.Then we study the SMMC-7721 which was transfected with lenti-CXCL6 vector have higher invasion and migration abilities than control or not. The MMP9 was knockdown using SiRNA manner in HCC cells transfected with lenti-vector or lenti-CXCL6,and we observe the change of HCC proliferation and invasion ability. It is expected to provide predictors and therapeutic targets for the early diagnosis and treatment of HCC recurrence and metastasis.Materials and methods:1.We collected 196 liver cancer samples from 2005 to 2011 and followed up to December 2013. The expression of CXCL6 on tissue microarray was detected by immunohistochemistry, including 212 HCC samples. Complete follow-up data included patient age, sex, tumor size, degree of cirrhosis, local invasion, TNM stage, grade, tumor microsatellite, vascular invasion, and thrombosis. We used SPSS 16.0 software to analyze the correlation between CXCL6 expression and clinicopathological features in patients with hepatocellular carcinoma. In addition, the association between CXCL6 and overall survival and recurrence-free survival in patients with hepatocellular carcinoma was analyzed by the product limit method (Kaplan-Meier method) using SPSS software.2.The expression levels of CXCL6 in HCC and adjacent normal liver tissues were compared by real-time quantitative polymerase chain reaction (PCR) and Western blot.3.SiRNA was transfected into MHCC97H and HCCLM3 cells to evaluate the effect of CXCL6 by Lipofectamine2000 reagent. The knockout effect of siRNA in hepatocarcinoma cells was detected by qPCR and immunoblotting. We also designed an in vitro invasion assay to test the invasion of MHCC97H and HCCLM3 cells after silencing CXCL6.4.In vitro cell proliferation assay was performed using siRNA targeting MHCC97H and HCCLM3 cell lines that silenced CXCL6.5.The expression of CXCL6 in HCC cells was further investigated by lentivirus transfection..6.After the MMP9 was knockdown using SiRNA manner in HCC cells transfected with lenti-vector or lenti-CXCL6, we study the pro-invasion effect of CXCL6 in SMMC-7721 cell.Results:1. By analyzing 178 available paired tissues, we found that CXCL6 was elevated in 71.91% (128/178) of HCC patients, but was down-regulated in 14.04%(25/178) of HCC patients.2. By statistical analysis, we found that the expression of CXCL6 was closely related with tumor size and vascular invasion which are important clinicopathological parameters of HCC progression.3. Our results showed that the CXCL6 mRNA and protein expression level in HCC tissues were significantly higher than that in CNL tissues. Our results illustrated that CXCL6 is up-regulated in HCC tissues compared with CNL tissues.4. We detected the CXCL6 protein expression in eight HCC cell lines and non-HCC cell line (Lo2, immortal liver cell) and found CXCL6 was highly expressed in MHCC97L, MHCC97H and HCCLM3 cells, but down-regulated in HepG2 and SMMC7721 cells. Moreover, the mRNA expression level of CXCL6 in HCC cell lines was generally consistent with protein expression status.5. Through in vitro invasion assays, we concluded that silencing of CXCL6 significantly down-regulated the invasion capacities of MHCC97H and HCCLM3 cells6. We carried out in vitro cell proliferation assay in MHCC97H and HCCLM3 cells which were transfected with siRNA targeted CXCL6. The CCK-8 assay showed that silencing of CXCL6 significantly inhibited HCC cell proliferation in vitro.7. The results showed that the SMMC-7721 which was transfected with Ienti-CXCL6 vector had higher invasion and migration abilities than control.8. The results showed that the pro-invasion effect of CXCL6 in SMMC-7721 cell was inhibited by knockdown of MMP9.Conclusions:1. CXCL6 is up-regulated in HCC tissues compared with CNL tissues, CXCL6 have higher metastatic capacity In hepatocellular carcinoma cells with high metastatic potential; The expression of CXCL6 was positively correlated with the malignant potential of HCC; CXCL6 may play role in facilitating motility of HCC cells.2. Overexpression of CXCL6 promotes HCC cell migration and invasion.3. CXCL6 promotes HCC cell invasion via MMP9 pathway.4. CXCL6 may act as a potential independent prognostic factor for HCC and a new target for preventing HCC progression.