| Hepatocelluar carcinoma (HCC) is one of the most common cancers all over the world. Liver cancer in men is the fifth most frequently diagnosed cancer worldwide but the second most frequent cause of cancer death. An estimated748,300new liver cancer cases and695,900cancer deaths occurred worldwide. The overall survival of patients with HCC remains unsatisfactory because of a high incidence of recurrence and metastasis. Thus, it’s very important to study the mechanisms of invasion and metastasis of HCC.Previous studies showed that SAPK interacting protein1(SIN1) is essential for early embryonic development and is the key regulator of Akt which play an important role in various pathological conditions such as cancer. SIN1was identified as a key TORC2component and regulator of the Akt pathway that positively controls Akt-Ser473phosphorylation and activation. However, the biologic function and clinical significance of SIN1in HCC remains unknown. Therefore, we carried out the current study to explore the potential role of SIN1in HCC by determining its expression in human HCC tissues as well as cell lines. Furthermore, the biological function and the underlying molecular mechanisms of SIN1in HCC invasion and metastasis were also investigated. Our result was shown below:1. SIN1expression was significantly elevated in human HCC tissues and HCC cell lines. The expression of SIN1mRNA was highly elevated in HCC tissues compared with ANLTs and the medium fold was2.2(range:1.0-20.1). Increased SIN1mRNA was also evidenced in thrombus than that in ANLTs, with the medium fold of4.02(range:2.9-5.1). Moreover, the results of semi-quantitative RT-PCR, showing the significantly elevated SIN1mRNA in HCC tissues than ANLTs, also confirmed these results. Consistent with mRNA expression, the western blot results showed that the expression of SIN1protein in HCC tissues was also significantly higher than that in the corresponding ANLTs (0.80±0.11vs0.22±0.07; P<0.01). The SIN1protein levels in ANLTs were higher than those in normal liver tissues, however the difference was not statistically significant (P>0.05). We compared SIN1expression in three special types of HCCs:solitary large hepatocellular carcinoma (SLHCC), small hepatocellular carcinoma (SHCC), and nodular hepatocellular carcinoma (NHCC). The results showd that the SIN1mRNA level in SLHCC was lower than those in NHCC. Both of qRT-PCR and Western blot showed the significantly lower levels of SIN1in SLHCC than those in NHCC, but no significant differences between levels of SIN1in SLHCC and SHCC were found (P>0.05). Furthermore, we examined the SIN1expression in six liver cell lines, including a normal liver cell line (LO2cells) and five HCC cell lines with different metastastic potential (HepG2, MHCC97-L, MHCC97-H, SMMC-7721and HCCLM3). Either real-time qRT-PCR or Western blot showed that the SIN1expression levels were significantly higher in HCC cell lines than those in the normal liver cells (LO2). We also compared the expression of SIN1in4HCC cell lines (HepG2, MHCC97-L, MHCC97-H, and HCCLM3) and found that HCCLM3and MHCC97-H with strongest metastatic abilities had the highest mRNA and protein expression levels of SIN1, followed by MHCC97-L, and then HepG2cells.2. Correlations of SIN1expression with clinicopathologic characteristics and prognosis of HCC:Immunohistochemistry showed that SIN1expression was significantly higher in HCC than in ANLTs. We also found that SIN1expression negatively correlates with capsular formation (P=0.037) and positively correlates with vascular invasion (P=0.023),tumor numbers (P=0.012), and TNM stage of HCC (P=0.020). According to the immunohistochemistry results, all HCC patients were divided into two groups:high expression group in which SIN1expression scored as2+or3+(n=39); low expression group in which SIN1expression scored as1+or negative (n=21).High expression group had the lower overall survival rates and disease-free survival rates than low expression group (P=0.012and P=0.001, respectively). In addition, results from the multivariate cox regression analysis indicate that high SIN1expression (relative risk,1.373; P=0.046) together with vascular invasion (relative risk,2.536; P=0.038), tumor numbers (relative risk,1.687; P=0.048), and TNM stage (relative risk,2.236; P=0.037) were independent prognostic factors for overall survival of HCC patients.3. SIN1promotes invasion and metastasis of HCC cells in vitro:We employed the wound healing and Transwell assays to analyze the function of SIN1in HCC cell invasion and migration. The wound healing assay showed that HCCLM3SIN1-RNAi cells closed much slower than that of HCCLM3NC cells (45%versus95%, P<0.01). Meanwhile, the closure of MHCC97-HSIN1-RNAi was also significantly slower than that of MHCC97-HNC (49%versus97%, P<0.01),suggesting a role for SIN1in the regulation of HCC cell migration.To further confirm the results, we performed a transwell assay and the results showed that the numbers of HCCLM3SIN-RNAi cells which passed through the matrigel was significantly less than that of HCCLM3NC cells (56±13versus161±18, P <0.01). The results obtained from MHCC97-HSIN1-RNAi cells and MHCC97-HNC cells were consistent with the results obtained from HCCLM3cells (41±11versus97±17,P<0.01). 4. SIN1promotes invasion and metastasis of HCC cells in vivo:The HCC metastatic mouse model was constructed by using HCCLM3NC and HCCLM3SIN1-RNAi cells. The results showed that the knockdown of SIN1in HCCLM3cells significantly reduce the distant metastasis in vivo model. The numbers of intrahepatic metastatic nodules were counted and analysed with Student’s t-test. The results suggest that SIN1plays an important role in invasion and metastasis of HCC.5. SIN1facilitates Epithelial-Mesenchymal Transition in HCC:SIN1is identified as a key regulator in the Akt pathway by controlling Akt-Ser473phosphorylation and activation, which is very important for Epithelial-Mesenchymal Transition (EMT). Since AKT activation up-regulates snail expression and induces EMT, we hyopothesize that SIN1facilitates EMT in HCC by regulating Akt-Ser473phosphorylation and activation. To test this hypothesis, we investigated the expression level of Akt and p-Akt in HCCLM3SIN1-RNAi, MHCCLM3SIN1-RNAi, HCCLM3NC and MHCC97-HNC cells and the results showed that the depletion of SIN1inhibited Akt phosphorylation and activation in HCC. The expression of Snail, Vimentin, MMP9and N-cadherin was significantly lower in HCCLM3SIN1-RNAi cells than in HCCLM3NC cells. In contrast, the expression of E-cadherin was much higher in HCCLM3SIN1-RNAi cells than in HCCLM3NC cells. Similar to HCCLM3cells, down-regulation of SIN1in MHCC97-H cells reduced Snail, Vimentin, MMP9and N-cadherin expression but increased E-cadherin expression. To confirm these results, immunohistochemistry was used to investigate the expression of SIN1, Snail, Vimentin, N-cadherin, MMP9, and E-cadherin in the same HCC tissue. The results showed that Snail, Vimentin, N-cadherin, MMP9had positive correlation with SIN1, however, E-cadherin had negative correlation with SIN1.In conclusion, our study has shown for the first time that SIN1is overexpressed in HCC and its overexpression is significantly correlates with a poor prognosis of HCC. SIN1expression negatively correlates with capsular formation and positively correlates with vascular invasion, tumor numbers, capsular formation, and TNM stage of HCC. Furthermore, we have demonstrated that SIN1plays an important role in HCC invasion and metastasis by facilitating EMT. |
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