Effects Of Osteopontin On Hepatocellular Carcinoma Invasion, Metastasis And Experimental Studies On Antisense Therapy Targeting Osteopontin

Hepatocellular carcinoma (HCC) is the sixth prevalent malignant tumor, which is ranked the third of tumor-related death in the world and is the second in China. HCC recurrence and metastasis lead to the failure of treatment. Surgery remains the best treatment for HCC. However, the rate of HCC recurrence and metastasis is 61．5% after radical resection and is 43．5% even for small HCC. Multi-genes and multi-factors are involved in the processes of HCC recurrence and metastasis. Elucidating molecular mechanisms of HCC recurrence/metastasis and searching for effective target therapies are the promising pathway to improve the survival. OPN is the secreted glycoprotein promoting cell chemotaxis, adhesion and migration. Previous studies indicate OPN expression is associated with HCC progression. However, the mechanisms of OPN promoting HCC recurrence/metastasis and which factors increase HCC OPN expression are poorly understood.Part one: Osteopontin expression in hepatocellular carcinoma (HCC)cell lines with different metastatic potential and HCC samplesTo study the relationships between OPN expression and HCC cell lines with different metastatic potential and HCC recurrence/metastasis after surgery. Immunocytochemistry, RT-PCR, Western Blot and ELISA were applied to detect OPN expression of HCC cell lines SMMC-7721, MHCC97-L and HCCLM6 with different metastastic potential. Immunohistochemistry of OPN was performed in the tissue arrays containing 200 HCC samples. We found that OPN expression was leveled with increasing metastatic potential of cell lines. OPN expression of samples was not associated with age, sex, serum AFP level, tumor size, cell differentiation, tumor capsule, lymphatic node metastasis while it was significantly associated with HCC TNM classification, portal vein tumor thrombi and HCC recurrence/metastasis after surgery. OPN expression detected in 66% HCC samples of patients with recurrence/metastasis after surgery was significantly higher than 37% samples of patients without recurrence /metastasis after surgery(P<0．05) . OPN expression is the indicator of HCC recurrence/metastasis after surgery.Part two: Mechanisms of osteopontin promoting hepatocellular carcinomainvasion and metastasisTo study the mechanisms of OPN promoting HCC malignant phenotypes. HCC cell line SMMC-7721 cells with low invasion and no metastatic potential were transfected by pcDNA 3．1(-)/OPN plasmid while cells transfected with mock plasmid were controlled group. OPN expression was testified by RT-PCR, Western Blot and ELISA. Functional assays in vitro were performed to observe changes of malignant phenotypes after transfection. SMMC-7721 cells stably expressing high level of OPN were established by plasmid transfection and G418 screening while cells transfected with mock plasmid were used as control. Differences of metastasis-related genes expression between two groups were compared by gene chips. Real-time PCR and ELISA testified the different expression of MMP-2 and uPA in the gene chips. OPN expression of SMMC-7721 cells was elevated after transient transfection. Functional assays in vitro indicated that SMMC-7721 cells after transfection showed higher adhesive, migrant and invasive capabilities(cell adhesion rate: 75．33%±10．59% vs 57．34%±2．52%; cells of outer surface in migrant assay: 14．33±2．51 vs 6．34±1．53; cells in invasive assay: 8．23±1．53 vs 4．12±1．29) while the ability of cell proliferation was similar. 88 metastasis-related genes were up-regulated in SMMC-7721 cells with high OPN expression while 6 genes were down-regulated. Real-time PCR and ELISA found the level of MMP-2 and uPA expression were higher than the controlled group while MMP-9 level did not increase. OPN might enhance the expression levels of MMP-2, uPA and other genes to promote malignant phenotypes of SMMC-7721 cells.Part three: Mechanisms of osteopontin high expression in hepatocellular carcinoma cell line HCCLM6 with high metastatic potential.To study the relative mechanisms of OPN high expression in HCC cell line HCCLM6 with high metastatic potential. Firstly, Genomic DNA was extracted and OPN promoter amplified by PCR was applied as probe. Using Protein/DNA array, transcription factor activity profiles of binding with OPN promoter in cell lines SMMC-7721 and HCCLM6 of different OPN expression, different metastatic potential were examined. 23 different transcription factors activities were associated with cell line OPN expression. Transcription factor c-myb activity was confirmed by electrophoretic mobility shift assays (EMSA) in the two cell lines. Real-time PCR detected c-myb mRNA expression in cell lines with different OPN expression levels. Small interference RNA (siRNA) inhibited c-myb expression. We found that c-myb siRNA reduced expression level of c-myb and OPN in HCCLM6 cells and also inhibited HCCLM6 cells invasive ability in vitro. Secondly, using informatics, we found that microRNA(miRAN)-96 maybe participate regulating OPN translation processes. Real-time PCR examined the transcriptional level of miRNA-96 in cell lines. OPN expression was determined after increasing or decreasing miRNA-16 level by miRNA-96 mimics and miRNA-96 inhibitor in HCCLM6 cells. We found that miRNA-16 level was different in cell lines with different metastatic potential. OPN expression was associated with miRNA-96 level in HCCLM6 cells. Decreasing miRNA-96 level significantly suppressed OPN expression and inhibited the invasive ability of HCCLM6 cells in vitro.Part four: Antisene oligonucleotides targeting osteopontin suppress hepatocellular carcinoma invasion and metastasisTo evaluate the effects of 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides (ASOs) targeting OPN on HCC invasion and metastasis and study the underlying mechanisms. ASOs were delivered to HCC cell line HCCLM6 with high metastatic potential in the form of complexes with Lipofectamine. HCCLM6 cells were treated with OPN ASOs while base-randomized oligonucleotides (ASOs-C) and Lipofectamine alone were applied as controls. OPN expression was detected by RT-PCR, Western blot and ELISA. MTT, Flow cytometer, trans-well migrant/invasive assays were performed after HCCLM6 cells were treated with ASOs. MMP-2 and uPA level were measured by ELISA. 36 nude mice with orthotropic implantation of HCCLM6 cells were randomly equally assigned to three groups as the following: OPN ASOs treatment group, ASOs-C controlled group and injection of physiological saline(NS) group. Drug administration was started at the second day after tumor implantation. 50mg/kg OPN ASOs, ASOs-C or 0．2ml NS was injected through intraperitoneum(i.p.) once every three days for 12 times. After six weeks, 6 mice randomly selected from each experimental group were sacrificed to observe the growth and metastasis of HCCLM6．OPN expression was assessed by immunohistochemistry. Pulmonary metastases were detected by H.E. stains. Blood samples were collected for routine blood analysis and detecting AST, ALT level in serum. Another 6 mice in each group were maintained to evaluate life span. We found that in vitro, expression of OPN in HCCLM6 cells was significantly suppressed by OPN ASOs while ASOs-C and Lipofectamine did not affect(P<0．05) . HCCLM6 cells treated by 400 nM OPN ASO for 24h showed in significantly decreased migrant and invasive abilities. The same concentration of OPN ASOs also significantly reduced the levels of MMP2 and uPA expression. OPN ASOs did not affect HCCLM6 cells growth or promote cell apoptosis. The incidence of lung metastasis of HCCLM6 treated by OPN ASO in nude mice was significantly inhibited (2/6) compared with the groups treated by ASOs-C(6/6) and NS(6/6) . The tumor weight and the survival of nude mice with HCCLM6 were similar among three groups. ASOs had no apparent hepatic and blood toxicities. OPN ASOs may serve as a novel therapeutic strategy for inhibiting HCC invasion and metastasis.Conclusions1．OPN expression is associated with the invasive and metastatic abilities of HCC cell lines. OPN expression is associated with HCC TNM classification, portal vein tumor thrombi, HCC recurrence/metastasis after surgery.2．OPN promotes malignant phenotypes of SMMC-7721 cells with low invasive capability. Increasing OPN expression results in changes of many tumor metastasis-related genes. OPN may increase the expression level of MMP-2, uPA and other genes to enhance the malignant phenotypes of HCC.3．Transcription factor c-myb is correlated with OPN expression in HCCLM6 cells with high metastatic potential. MiRNA-96 level is also associated with OPN expression. Increasing c-myb or miRNA-96 level promote the expression of OPN, underlying new targets of regulating OPN expression.4．Targeting OPN antisense oligonucleotides significantly inhibit the invasion and metastasis of HCCLM6 with high metastatic potential, implicating a novel drug for anti-HCC recurrence/metastasis. The potential application of this work1．OPN is the new therapeutic target of anti-recurrence/metastasis for HCC.2．c-myb or miRNA-96 maybe the new targets of regulating OPN expression.The novelty of this work1．Demonstrate that OPN increases MMP-2, uPA and other genes expression to promote HCC malignant phenotypes.2．Explore the mechanisms of OPN high expression and find that c-myb and miRNA-96 promote OPN expression in HCC.3．Find that targeting OPN antisense oligonucleotides inhibit HCC invasion and metastasis.