The Expression Of OPN In HCC And Specific Glycosylation Aberrance Of OPN In HCC Tissues With Metastasis

Metastasis is the main reason of high mortality of hepatocellular carcinoma (HCC) patients, and unfortunately, metastasis is very common in the late stage of this disease. To investigate the molecular mechanism of HCC metastasis is the key to improve the therapeutic effect and prolong life span of patients. Osteopontin (OPN), a multifunctional calcium-binding protein with phosphorylated and glycosylated post-translational modifications, is widely involved in cell adhesion, cell migration, signaling transduction et al, together with its binding protein CD44. Recent publications showed that OPN play important roles not only in physiological processes such as bone formation and angiogenesis, but also in oncogenesis. The previous work of our institute discovered OPN gene was highly expressed in HCC tissue obtained from the petients with metastasis and expression level of OPN protein was related to cell invasion. While, the understanding of molecular mechanism underlying OPN-regulated HCC metastasis is still lacking.Nowadays, cumulative evidence show the aberrance of glycosylation level and glycan structure is closely related to cancer metastasis. OPN was a highly glycosylated protein, and its binding activity to receptor could be influenced when abnormally sialylated, which resulted in enhanced cell invasion. To investigate the HCC metastasis-related glycosylation change of OPN, this study used immunoprecipitation combined with lectin blot technology to comparatively analyze the glycosylation status of OPN from HCC tissues of the patients with and without metastasis. This study provided basic information for further research on molecular mechanism of OPN-regulated HCC metastasis. Part1Protein expression of OPN in HCC cell lines and tissue samplesReal-Time PCR, Westen Blot and Immunohistochemistry technologies were used to determine the mRNA and protein expression level of OPN in clinical tissue samples and several HCC cell lines with different metastatic potential (Hep3B, MHCC97L, MHCC97H, HCCLM3, HCCLM6). The results indicated the OPN expression was higher in HCC cell lines with high metastatic potential than that in HCC cell line with low metastatic potential (p<0.05), meanwhile OPN expression of HCC tissues from patients with metastasis also showed much higher than that in HCC tissues without metastasis (p<0.05). A conclusion can be drawn from the above results that the highly expression of OPN in HCC is related to HCC metastasis.Part2Specific glycosylation aberrance of OPN in clinical HCC tissues with metastasisOPN protein was enriched from HCC tissues with and without metastasis by immunoprecipitation, followed by specific glycan staining using lectin blot. The results showed that the lectin affinity of OPN to MAL, PHA-E, DSA and ConA was significantly decreased in HCC with metastasis than that in HCC without metastasis (p<0.05), which indicated that the inhibition of OPN glycosylation of a2,3-sialic acid, bisecting N-acetylglucosamine, multi-tennary structure and high-mannose type structure might be related to HCC metastasis.Conclusion1. Aberrantly high expression of OPN in HCC is related to its metastatic ability.2. The inhibition of OPN glycosylation of a2,3-sialic acid, bisecting N-acetylglucosamine, multi-tennary structure and high-mannose type structure may be related to HCC metastasis. Potential application value1. To indicate that OPN is a potential drug target for anti-HCC metastasis therapy.2. To reveal the HCC metastasis-related glycosylation change of OPN with the aim of providing basic information for further research on the molecular mechanism of OPN-regulated HCC metastasis.InnovationWe are the first to compare the glycosylation level and glycan structures of OPN between HCC tissues with and without metastasis.