The Functional Role Of Metastasis-Favoring Protein GOLPH2 In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the commonest and worst-prognostic carcinoma in the world. It takes the third place of cancer-related death globally and the second in China (more than half of the HCC cases are in China). Though some of the patients have long term survival through early diagnosis and comprehensive therapy, recurrence and metastasis of tumor has been the biggest barrier for further improvement of treatment. The total 5-year survival was still no more than 5% and the 5-year recurrent rate was as high as 60% after curative resection. However, prediction and intervention of metastatic recurrence are still not yet available at this moment. Therefore it is urgently needed to investgate the molecular basis underlying HCC metastasis.Only in this way, better prognosis and longer survival might be further achieved.In our previous studies, by comparing the gene expression profiles between 40 HCCs with and without intrahepatic metastasesusing cDNA micorarray with 9180 genes, we found that the gene expression profiling of primary HCCs was very similar to that of their corresponding metastases, however, a 153 genes'signature was found significantly different between HCCs with and without metastasis(p<0.001). We proposed for the first time a new hypothesis that genes favoring HCC metastasis progression-were likely initiated from the primary tumors (Ye QH, et al. Nat Med 2003), which established a possible basis for early prediction, prevention and treatment of HCC metastasis.Recently, the laser capture microdissection (LCM), gene expression array and SNP array were introduced to explore the genomic aberration of paired HCCs with extrahepatic metastasis as well as HCCs with and without metastasis on mRNA and DNA levels. The new hypothesis that genes favoring HCC metastasis are initiated in primary tumors got further validated and some important candidate genes associated with HCC extrahepatic metastasis were disclosed. Among of them, type II Golgimembrane protein (GOLPH2) was found to be a leading gene between HCCs with lung metastasis and those without metastasis.It has been reported that GOLPH2 was elevated the HCC patients and was proposed as a serum marker for HCC. But its role in HCC carcinogenesis, invasion and metastasis progression is unclear. In this study, we will validatethe expression levels of GOLPH2 in different liver and HCC tissues useing RT-PCR, immunohistochemistry (IHC) and tissue microarray techonology and analyze its relationship with clinicopathological features and prognosis of HCC patients. Furthermore, we will probe into the biological behaviour changes of MHCC97H cells growth and invasiveness and the possible molecular mechanisms after knocking down its high endogenous GOLM1 level using lentiviral-mediated RNA interference.MethodsThe expressions of gene GOLM1 in 137 cases of different groups (incoluding 20 liver tissues of HCC with extrahepatic metastasis,41 liver tissues of HCC with intrahepatic metastasis,29 liver tissues of HCC without metastasis,36paratumor tissues of HCC,11nomal liver tissues) was verified by the means of RT-PCR. Meanwhile, we applied the method of immunohistochemistry to detect the expression of GOLPH2 protein in another independent tissue microarray (including:102 liver tissues of patients with hepatocellular carcinoma; 17 normal liver tissues), and analyze the correlation between the expression of GOLPH2 protein and clinicopathological features, patients tumor-free survival rate(TTR) and overall survival rate(OS).In the other hand,we used lentivirus-mediated RNAi to inhibit the expression of GOLM1 in order to obtain a stable MHCC97H cell line with low expression of GOLM1. RT-PCR and western-blot were introduced to validate interference effect. Furthermore, we observed changing of biological behavior in stable cell lines with RNA interference.ResultsThe results of RT-PCR showed that the expression of GOLM-1 in HCC tissues was up-regulated compared with adjacent tissues and normal liver tissue at mRNA levels (P <0.0001). Meanwhile, the expression of GOLM1 in patients with metastasis was significantly higher than the group without metastasis (P<0.05). Immunohistochemistry result showed that GOLPH2 mainly express in tumor tissues, while lowly or no express in paratumor and normal liver tissue. The time to recurrence rate(TTR) and overall survival rate(OS) of Patients with low GOLPH2 expression was significantly higher than patients with high expression (TTR:P=0.019;OS:P=0.007). Multivariate analysis indicates that GOLPH2 is a decision independent prognostic factors of hepatocellular carcinoma (P= 0.023) which indicated that it can be used to predict recurrence and overall survival of HCC patients in the future. Five stably transfected cells (MHCC97H-Lenti.GOLMli-1,MHCC97H,Lenti.GOLMli-3,MHCC97H-Lenti.GOLMli-2, MHCC97H-Lenti.GOLMli-4,MHCC97H-Lenti.GOLMli-5M) were obtained by lentivirus-mediated system. Real-time PCR assays showed that Lenti.GOLMli-1 (G1), Lenti.GOLMli-2(G2),Lenti.GOLMli-3 (G3), Lenti.GOLMli-4(G4), Lenti.GOLMli-5M (SCR) could suppress the Golml expression by 81.05%,36.22%,84.61%,9.25%,10.75%, respectively. The results of western blot further confirm this result. capacity of proliferation and colony formation were significantly suppressed in the stable infect MHCC97H cells(G1 and G3).ConclusionsGOLPH2 expression levels do relate to the hepatocellular carcinoma and its metastatic potential and prognosis; inhibition of GOLPH2 expression can significantly reduce the the capacity of HCC's cell proliferation and invasion, which suggests that GOLPH2 may play an important role in the occurrence and development of liver cancer and will be expected to become a new therapeutic targets and prognostic predictors.The potential application of this workGOLPH2 may be a new target of HCC and its metastatic potential therapy and a new prognostic predictors.The novelty of this workIt was the first time that GOLPH2 expression do relate to the hepatocellular carcinoma and its metastatic potential and prognosis; and inhibition of its expression can significantly reduce the malignant phenotype of hepatoma cells.