| Hepatocellular carcinoma (HCC) is one of the most common lethal malignancy.There are about500,000new patients were diagnozed with HCC each year. Its death rateis on the second place among malignancies in our country, more than half new livercancer patients worldwide occur in China each year. Today, although the tumor detectionrate has been significantly improved and clinical treatment strategies are increasinglysophisticated, the total time of HCC patients remains poor because of liver cancer is moreprone to recurrence and metastasis, so it is very important to identify novel indicators topredict and assess the liver cancer in the early phase.Chemokines are a hot topic in today’s medical research. CC chemokine receptor2(CCR2) is the specific receptor of monocyte chemoattractant protein-1(MCP-1/CCL2),meanwhile, it is also the receptors of CCL7, CCL8, CCL11, CCL12, CCL13. CCR2contains two isoforms, CCR2A and CCR2B, both of which were cut from the same gene,except for the difference of carboxy-terminal, and CCR2B is the major functional form.Newly researches indicate that, CCR2is abnoamally expressed in a variety of tumor cellssuch as prostate cancer, renal cell carcinoma, non-small cell lung cancer, myeloma cells, colorectal cancer, etc., moreover, it is associated with tumor invasion and metastasis.However, the relationship between CCR2and HCC remains to be clarified.ObjectiveFirst, investigate the expression of the CCR2in human HCC tissues and elucidate therelationship between CCR2and the clinical and pathological features of HCC. Second,understand the expression of the CCR2both in liver cancer cell lines with different invasiveabilities and normal liver cell line. Third, by using CCR2RNAi technique to disturb itsexpression liver cancer cell lines in vitro, and then elucidate the effects and possiblemechanisms on its invasiveness in different liver cancer cell lines.MethodsWe investigated the expression of the CCR2in HCC tissues and para-carcinomatissues in76cases and further analyze this problem by combining with theirclinicopathologic data. In vitro, we first detected the expression of CCR2protein inHepG2, SMMC-7721and MHCC97-H hematoma cell lines and HL-7702normal livercell line, then design and synthesis CCR2-specific small interfering RNA fragments(siRNA) and transfect MHCC97-H and HepG2hepatoma cell lines with specific siRNA,explore the change of CCR2protein and mRNA levels in the cells after transfection.Finally, by using Transwell chamber invasion experiment, we tested the invasivenesschanges of the MHCC-97H and HepG2hepatoma cell lines in vitro after CCR2-siRNAtransfection, and detect the changes of MMP-2protein expressions and its activity.ResultsImmunohistochemical staining results showed there were brown granular staining mainlyappeared in the cell membrane and scarcely in the cytoplasm. In HCC, CCR2staining showednegative expression in15(19.7%) patients, weakly positive in29cases (38.2%), moderatelypositive in12cases (15.8%) and strong expression in20patients (26.3%); while inpara-carcinoma tissue, only14(18.4%) cases showed weak positive, others were negative, ina word, the expression of CCR2in HCC tissues was significantly higher than that inpara-carcinoma tissues. Moreover, the expression of CCR2in HCC tissues was significantlyassociated with tumor diameter, portal vein thrombosis, metastasis, AJCC staging (χ2=12.41, P<0.001; χ2=7.476, P=0.006; χ2=7.227, P=0.007; χ2=20.711, P<0.001), and there wasno significant correlation with sex, age, tumor location, tumor volume, tumor differentiation,satellite lesions and AFP expression in HCC patients (χ2=0.215, P=0.643; χ2=0.038, P=0.845; χ2=0.974, P=0.323; χ2<0.01, P=1.000; χ2=3.665, P=0.056; χ2=2.951, P=0.086; χ2=0.026, P=0.873).Kaplan-Meier survival analysis demonstrated that the survival time of patients withhigh expression of CCR2was lower than that of patients with low expression of CCR2(χ2=27.133, P<0.01). Moreover, univariate Cox regression analysis indicated that highexpression of CCR2, metastasis, portal vein thrombosis, tumor size, satellite lesions,tumor number, AJCC TNM stage (P<0.01, P<0.01, P<0.01, P=0.004, P=0.023, P=0.028,P<0.01) were all risk factors for overall survival in HCC patients, while multivariate Coxregression analysis showed that highly expressed CCR2, tumor size, metastasis, portalvein thrombosis, tumor number (P=0.006, P=0.023, P <0.01, P=0.002, P=0.001)were independent risk factors for overall survival in patients.CCR2appeared to be highly expressed in HepG2, SMMC-7721and MHCC97-Hhepatoma cell lines, and the level of CCR2protein and mRNA expression wassignificantly reduced in the HepG2and MHCC97-H hematoma cell lines afterCCR2-siRNA transfection, moreover, the transwell chamber invasion assay showed thatthe number of cells that have penetrated the Transwell chamber membrane is significantlyreduced. Western-blot and ELLISA assay demonstrated that MMP-2expression and itsactivity was also significantly decreased in both the hepatoma cell lines afterCCR2-siRNA transfection.ConclusionCCR2expression is associated with the invasion and metastasis of HCC, and theinvasion of HepG2and MHCC97-H hepatoma cell lines can be effectively suppressed byCCR2-siRNA. Moreover, CCR2-siRNA transfection can also reduce the expression andactivity of MMP-2, and this process may be achieved through the regulation of matrixmetalloproteinases. |
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