| Background and Aims:Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the second most common cause of death from cancer in China. Metastasis is the major cause for dismal prognosis of HCC. For HCC cells, the ability to form metastasis phenotype is associated with the tumor microenvironment. Hypoxia is a feature of most solid tumors, and it is a negative prognostic factor owing to its contributions to epithelial-mesenchymal transition, vasculogenesis, invasiveness and metastasis. Chemokine are a superfamily of chemotactic cytokines of molecular weight8-14kDa. They bind to their receptors and regulate leukocyte trafficking to inflammation sites. Recently a growing body of evidence indicates that chemokines and their receptors play a key role in tumor growth, invasion, angiogenesis and metastasis. Although CCL2and CCR2have been reported to be associated with cancer metastasis, the role of other chemokines in cancer is poorly understood. We aimed to explore the status of CCL28in hypoxia-induced cell migration.Methods:The expression of HIF-la and CCL28were detected in50cases of hepatocellular carcinoma samples using real time PCR. Kaplan-Meier method, log-rank test and chi-square test were used to analyse the relationship of clinical characteristics with prognosis of HCC. In vitro, we used real time PCR, Western blot, and ELISAto detect the levels of HIF-1α and CCL28in HepG2and HCCLM3under hypoxia (1%O2); using siRNA and lipofectamine2000to seal up HIF-1α gene expression and detected the level of CCL28expression in HepG2/HCCLM3under hypoxia; using Trans-well and BD Matrigel to observe cellular mobility and invasion ability, using gelatin zymography to detect MMPs activity after CCL28siRNA transfection.Results:1) Real-time PCR showed that mRNA level of HIF-1α in hepatocellular carcinoma was0.0654±0.0988,29of50(58%) showed a high level of HIF-1αa; CCL28mRNA level in hepatocellular carcinoma was0.0254±0.0755, and23of50(46%) showed a high expression level. Spearman analysis indicated that CCL28expression strongly correlated with HIF-1α in hepatocellular carcinoma(r=0.595, p<0.001); high expression of HIF-1α and CCL28were both related with recurrence after surgery.2) In vitro, when cells HepG2and HCCLM3were subjected to hypoxia (1%O2), the mRNA levels of HIF-la and CCL28started to increase after6h hypoxia while the protein levels had a significant increase after12h hypoxia in a time-dependent manner; When HIF-1α siRNA was used to down-regulate HIF-1α expression, the protein level of CCL28under hypoxia was significant decreased (p<0.001);3) We used siRNA to inhibit CCL28expression in HCCLM3cells, and the mRNA and protein levels of CCL28were decreased. The number of invasion cells under hypoxia was significantly decreased as compared with control, and the activity of MMP-2was significantly decreased.Conclusions:1) Both of HIF-la and CCL28were highly expressed in hepatocellular carcinoma tissue, and their expression had a strong relationship. High level of HIF-la and CCL28were both related with recurrence after surgery.2) Hypoxia induced high expression of CCL28by up-regulated HIF-1α in a time-dependent manner.3) CCL28expression knockdown using siRNA inhibited the invasiveness of hypoxia-induced HCC cells invasion.4) CCL28played an important role in hypoxia-induced liver cancer cell invasion, which might be mediated by MMP-2. |
