| PurposeHepatocellular carcinoma(HCC),the most common primary(90%)malignancy of the liver,being ranked the fifth most common tumor,currently responsible for a third of cancer-related deaths on a global scale.Moreover,the cancer statistic of 2015 conducted in China shows that Hepatocellular carcinoma has been the third most common cancer and the second leading cause of cancer-related death.Hence,liver cancer has become one of the seriousest health hazards to Chinese people.The molecular mechanisms of hepatocarcinogenesis is very complex comprising of multiple genetic and epigenetic alterations,chromosomal aberrations,gene mutations and altered molecular pathways,and the molecular mechanisms of hepatocarcinogenesis have not yet been clarified clearly.To confront this dire situation,exploration of effective genes that closely associated with HCC and researching the function of these genes in HCC is crucial,and it would help us to identify novel molecular markers for early diagnosis as well as new therapeutic targets to improve the outcome of patients with HCC.SMOC2(Secreted modular calcium binding proteins 2)is a recently identified matricellular protein that belongs to the SPARC protein family.Numerous studies have revealed that SMOC2 plays a important role in maintaining tissue microenvironmental stability and regulating a variety of cellular functions,such as cell-cycle regulation,cell proliferation.However,the molecular function of SMOC2 in cancer is poorly explored.Several recent microarray studies reported SMOC2 is significantly downregulated in various tumors,including ovarian cancer,pancreatic cancer,and so on.Such fndings suggest that SMOC2 may have an anti-oncogenes role in the development and progression of cancers.Even though the function of SMOC2 has been investigated in several types of cancer,the detailed functional role of SMOC2 in human HCC has not been reported.In the present study,we investigated the expression and prognostic value of SMOC2 in primary HCC.Additionally,we investigated the relationship between SMOC2 and HCC Clinicopathological parameters.We further assess various biologica aspects of SMOC2 in hepatocarcinogenesis using HCC cell lines.The purpose of this study was to investigate the clinical significance and biological function of SMOC2 in human hepatocellular carcinoma.MethodsExperimental methods: Real-time quantitative polymerase chain reaction(RT-PCR)was conducted to detect SMOC2 transcriptional level in HCC tissues samples.Western blotting was used to evaluate the level of SMOC2 expression in HCC cell lines and primary HCC clinical specimens,and its correlation with HCC tumor characteristics,clinical parameters and prognosis of patients was discussed through immunohistochemical analyses.A battery of methods(cell proliferation and colony formation assays,cell migration and invasion assays,cell cycle and apoptosis assays,Tumorigenicity assays)were employed to assess various biologica aspects of SMOC2 in Bel-7402 and HepG2 cells which stable overexpression of SMOC2 using a lentiviral vector.Statistical methods: MannWhitney U-test was used to evaluate differences in SMOC2 mRNA transcriptional level or protein expression levels between HCC tumors and their paired adjacent non-tumor tissue samples.Correlations between SMOC2 expression level and the clinical characteristics of patients were analyzed using the Pearson’s chi-square test(χ2).Survival curves for OS and DFS were calculated using the Kaplan-Meier method and their differences evaluated by the log-rank test.A Cox proportional hazards regression model was used in univariate and multivariate analyses to explore the effect of SMOC2 expression and clinicopathological variables of patients on OS and DFS.The factors included in multivariate analysis were the one which considered statistically significant in univariate analysis.A two-tailed unpaired Student t-test was used to assess differences in cell proliferation rates,colony formation,cell migration and invasion,cell cycle distribution and apoptotic frequency between LV-SMOC2 and LV-NC HCC cells.Results1.SMOC2 mRNA transcription level(n=40,P < 0.0001)and protein expression level(n=40,P =0.0101)were significantly downregulated in human HCC tissues compared to the matched normal tissues.Meanwhile,SMOC2 protein expression was lower in five HCC cell lines tested(Bel-7402,HepG2,QGY-7701,SK-Hep1,SMMC-7721)compared to the normal hepatic cell line L02.2.SMOC2 positive staining was predominantly located in the cytoplasm.Chi-square analyses suggested that SMOC2 expression was significantly associated with tumor size(χ2=9.156,P=0.002),tumor number(χ~2=4.896,P=0.027)and tumor-nodes-metastasis(TNM)stage(χ~2=16.356,P < 0.001).Moreover,Low SMOC2 expression was also observed significantly more frequently in cases with distant metastases(χ~2=6.141,P=0.013).Kaplan–Meier survival curves revealed that high SMOC2 expression was associated with improved overall survival(OS)and disease-free survival(DFS)in HCC patients.Multivariate analysis indicated that SMOC2 expression was an independent prognostic marker for the OS and DFS of patients.3.Subgroup analysis showed that the high and low SMOC2 groups had significantly different outcomes in the patient subgroup with serum AFP≤25 ng/m L or>400ng/m L(P<0.05)and subgroup with a tumor size<5cm or ≥5cm(P<0.05).Similar results were obtained for the TNM stage I-II subgroup(P<0.05).4.Functional analyses(cell proliferation and colony formation assays,cell migration and invasion assays,cell cycle and apoptosis assays)demonstrated stable overexpression of SMOC2 using a lentiviral vector significantly inhibited cell proliferation,colony formation,migration and invasion,and induced G0/G1 phase arrest in HCC cells in vitro.5.Experiments with a mouse model showed that SMOC2 overexpression in the HCC cells significantly delayed tumor growth compared with the negative control cells.Accordingly,the means and standard deviations of tumor volume and tumor weight in the SMOC2 overexpressed group at the end of observation were significantly smaller than that of the control group(P<0.05).ConclusionSMOC2 might play an anti-oncogenic role in the development of HCC and could be an effective prognostic indicator and a novel therapeutic target for HCC.Moreover,it could provide evidence for tertiary prevention. |
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