Role Of CC Chemokine Receptor 1 (CCR1) In Hepatocellular Carcinoma Invasion And Metastasis

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide in terms of numbers of cases. HCC is one of most common malignancies in China, which accounts for over 40% of incidence of HCC in the world. HCC is also the second most common cause of death from cancer in China. Because of the difficulties in early diagnosis and the very poor prognosis, the relative 5-year survival of HCC is 5% to 6%. Although surgery achieves the best outcomes in well-selected candidates, the recurrence rate remains high after HCC resection. Even after small HCC resection, the 5-year recurrence rate after curative resection was as high as 60-70%. Recurrence and metastasis after resection remains an obstacle for more curative effect. For this reason, understanding the mechanisms that facilitate HCC cell invasion and metastasis, evaluation invasioness of HCC and searching for effective interventional target is of great importance. However, it remains elusive about the molecular biology underlying HCC invasion and metastasis.HCC cell invasion and metastasis are complicated processes involving several classes of proteins (such as celladhesion molecules, extracellular proteases, angiogenetic factors, cytokines, and growth factors). Among these factors, a family of chemoattractant cytokines called chemokines and their respective receptors have been suggested to be responsible for the metastatic potential of cancer cells. At first, chemokines were thought to play pivotal roles in inflammatory and immune responses by recruiting immune cells to specific sites through mediating the adhesion of cells to endothelial cells and initiation of trans-endothelial migration and tissue invasion, which seemed to be similar to the process of tumor cell invasion and metastasis. Recently, data have shown that many types of cancer cells can express specific chemokine receptors and respond to chemokine gradients. It has been reported that six human HCC cell lines constitutively and exclusively express CC chemokine receptor 1(CCR1), and it has been proposed that macrophage inflammatory protein (MIP)-1a (CCL3) utilized CCR1 to induce some uncertain signals in hepatoma cells. We have also observed previously that CCR1 is highly expressed in human HCC tumors compared with normal liver tissues and that the CCR1 expression level in human HCC is correlated with the level of HCC invasiveness. However, little is known about the detailed molecular mechanisms of CCR1-mediated HCC cell invasion.In the present study, the recently developed artificial microRNA (miRNA) technique was used to study the effect of CCR1 expression on HCC cell invasiveness and growth in vitro. Furthermore, the potential downstream molecular mechanisms of CCR1-mediated invasion were investigated. These results supported the view that CCR1 is crucial for HCC cell migration and invasion. Our results indicate that inhibition of CCR1 might be used therapeutically to suppress invasion and metastasis of HCC. Furthermore, the expression of CCR1 in hepatocellular carcinoma and its relationship with the prognosis of HCC after curative resection were analyzed by tissue microarray.Part I Introduction of exogenous mi RNA to silence CCR1 gene expression in HCC cells and cons truction of lentiviral vectorThe aim of this study is to silence CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) by introduction of exogenous miRNA and to construct a lentiviral vector of miRNA mediated RNA interference (RNAi) of CCR1 gene.Three double-stranded DNA vectors pcDNA6.2-GW/ EmGFP-miR (miRCCR1-85, miR-CCR1-206, and miR-CCR1-656) targeting the mRNA of human CCR1 and the mock vector were constructed then were transfected into human HCCLM3 cells with high metastatic potentials. Real-time PCR and Western blotting were used to quantify the mRNA and protein levels of CCR1, respectively. Three plasmids containing pre-miRNA sequences were constructed: miRCCR1-85, miR-CCR1-206, and miR-CCR1-656. Once recombinant plasmids were transfected into HCCLM3 cells, the expression of Emerald green fluorescent protein (EmGFP) could be detected directly by fluorescence microscopy. The resulting lentiviral vector containing CCR1 miRNA was named LV-miCCR1-656, and 293T cells were cotransfected. All virus stocks were produced by Lipofectamine2000-mediated transfection. The titer of virus was tested according to the expression level of GFP.A transfection efficiency of up to 70% was obtained in the current study. The results show that the HCCLM3 cell line strongly expressed high levels of CCR1. At 72 h after transfection, miRNA-mediated RNAi resulted in a significantly reduced level of CCR1 protein in miR-CCR1-206- and miR-CCR1-656-transfected cells. Compared with mock and miR-neg transfected HCCLM3 cells, the effect of RNAi on CCR1 was more marked in cells transfected with miR-CCR1-656 than in those transfected with miR-CCR1-206. Real-time quantitative PCR was also performed to evaluate the effect of miRNA-mediated RNAi on the mRNA level of CCR1. Forty-eight hours post-transfection, CCR1 mRNA levels in cells transfected with miR-CCR1-85, miR-CCR1-206 or miR-CCR1-656 were reduced, whereas CCR1 mRNA levels in miR-neg were unchanged. In agreement with the Western-blot results, the mRNA level in HCCLM3 cells transfected with miR-CCR1-656 was reduced by more than 80% compared with mock-transfected HCCLM3 cells. Therefore, miR-CCR1-656 has more efficient miRNA-mediated RNAi effects than miR-CCR1-85 and miR-CCR1-206, and achieved better suppression of CCR1 expression at both mRNA and protein levels. Lentivirus miRNA mediated RNAi vector of CCR1 LV-miCCR1-656 producing CCR1 miRNA was constructed successfully.Part II Downregulation of CCR1 in hibits human hepatocellularcarcinoma cell invasionWe have successfully silence CCR1 expression in the human HCC cell line HCCLM3 using artificial miRNA-mediated RNAi. To investigate the role of CCR1 in the invasiveness of hepatocellular carcinoma (HCC) cell line. The invasive potential of HCCLM3 cells was tested after CCR1 was knockdown. The effect of CCR1 downregulation on the proliferation of HCCLM3 cells was further analyzed. To determine if the reduced invasion ability of CCR1-silenced HCCLM3 cells was due to reduced MMP expression, gelatin zymography was used to detect MMP-2 and MMP-9 activity. Results show that transfection of HCCLM3 cells with miR-CCR1-85, miR-CCR1-206, and miR-CCR1-656 significantly reduced their ability to invade through the Matrigel coating. In particular, invasive activity was reduced by 80% in cells transfected with miRCCR1-656, which is correlated with the reduction in CCR1 expression. In HCCLM3 cells transfected with miR-CCR1-85, miR-CCR1-206, and miRCCR1-656, there was a small reduction in proliferation compared with that of mock- and miR-neg-transfected HCCLM3 cells. However, this difference was not significant, indicating that a relatively minor effect of downregulation of CCR1 expression on HCCLM3 cell growth. Transfection of HCCLM3 cells with miR-CCR1-85 or miR-CCR1-656 reduced MMP-2 activities but had little effect on MMP-9 activity. Furthermore, the MMP-2 activity of HCCLM3 cells transfected with miR-CCR1-656 was significantly lower than those of miR-CCR1-85- and miR-CCR1-206-transfected cells. These findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment.Part III The expression of CCR1 in hepatocellular carcinoma analyzed by tissue microarray and its relationship with prognosisPrevious studies have demonstrated that downregulation of CCR1 inhibits human hepatocellular carcinoma cell HCCLM3 invasion. The purpose of the present study was to investigate the expression of CCR1 in hepatocellular carcinoma tissue and its relationship with the prognosis of HCC after curative resection was analyzed by tissue microarray. 288 cases of HCC patients were enrolled with at least 5-year follow-up in this study. They all underwent initial surgical resection from January 1997 to December 2000 in Zhongshan Hospital, Liver Cancer Institute, Fudan University. The expression of CCR1 pattern in HCC tissue microarray was detected by immunohistochemistry analysis, and the relationship of CCR1 and postoperative survival time was also observed. The normal liver tissue microarray chip was set as control. Kaplan-Meier method, log-rank test and chi-square test were used to analyze the relationship of the angiogenesis agents with prognosis of HCC. Multivariate study with Cox's proportional hazard model was used to evaluate the prognosis-relative aspects.The expression of CCR1 was significantly higher in tumor tissues than those in non-tumor tissues. Meanwhile, the expression of CCR1 in the recurrence-group was significantly higher than those in the non-recurrence group. Compared with CCR1-negative group, the tumor-free survival rate was lower (P=0.0023) and the recurrent rate was higher (P=0.003) in CCR1-positive group. Simultaneously, the survival and tumor-free survival rates were significantly lower. Multivariate analysis showed that CCR1 was independent factors to the tumor-free survival rate and the survival rate after resection. The results of this study indicate that the expression of CCR1 was associated with tumor invasioness and high recurrent rate and mortality. CCR1 is a useful marker in the prediction of patient's outcome of postoperative and the patients with CCR1 positive were at higher risk of recurrence after resection and preventive treatment might improve the prognosis.Conclusions1. In the present study, we have shown for the first time that CCR1 gene expression knockdown using miRNA-mediated RNAi inhibited the invasiveness of human HCC cells. CCR1 might be a new therapeutic target of HCC.2. Our results showed that CCR1 has an important role in HCC cell invasion, which might be mediated by MMP-2.3. The expression of CCR1 was associated with high recurrent rate and mortality and CCR1 is a useful marker in the prediction of patient's outcome of postoperative...