Clinical Significance And Preliminary Analysis Of Biological Functions Of THBS2 In Hepatocellular Carcinoma

Primary liver cancer(PLC,referred to as liver cancer)is one of the most prevalent malignant tumors worldwide.Its incidence ranks sixth among all cancer types,and its mortality rate ranks fourth among cancer-related deaths.Ninety per-cent of PLC cases have the form of hepatocellular carcinoma(HCC).Globally,50%of liver cancer cases and related deaths occur in China.Currently,radical resection of the tumor is the most frequently used form of treatment,but high recurrence rates seriously impact patient prognosis.Clinically,patients with sim-ilar clinical and pathological features of PLC experience varying disease-free sur-vival(DFS)time following radical resection,suggesting that genetic abnormali-ties may represent a contributing factor to disease prognosis.Protein functionality can be affected by altered genetic profiles.In this study,we selected 9 paired spec-imens of HCC from patients who underwent radical resection and displayed dis-parate postoperative DFS.Moreover,440 high-throughput protein chips were screened to identify relevant differentially expressed proteins,including THBS2.Subsequently,a meta-analysis of THBS2 expression in malignant tumors was per-formed,and its prognostic potential was assessed.Finally,the expression of THBS2 was correlated with clinical characteristics and prognosis of HCC patients,and the effects of THBS2 on HCC cell proliferation,migration,and invasion were assessed in vitro.Part ?:Identification of THBS2 as a potential marker of recurrence and metastasis of liver cancer by high-throughput protein chips screeningObjective:To identify biomarkers that affect the recurrence and metastasis in HCC patients after radical resection.Methods:Eighteen HCC patients who underwent radical hepatectomy were divided into two groups,9 patients each.Group A included subjects with DFS longer than 3 years and group B included subjects with DFS shorter than 1.5 years.The clinical and pathological features of the two groups were comparable.Fol?lowing the extraction of proteins from the surgically removed tumor-tissue,the expression of 440 cancer-associated proteins was examined using protein chips(RayBiotech,GSH-CAA-440).The data were analyzed and explored by means of proteomics.Results:The median DFS in group A,58.0 months,was significantly longer than in group B,15.5 months(P<0.001).According to the volcano plot,31 of the 440 analyzed proteins were differentially expressed,with 25 of them highly ex-pressed in group B(the recurrence group).Five most upregulated proteins were,in the order of expression,MIP-1b,TnMP-2,MIP-1a,THBS2,and AFP.Heat map analysis showed that the expression pattern of THBS2 was very similar to that of AFP.GO function and KEGG enrichment analysis indicated that the main regu-latory proteins of tumor metastasis-associated pathways included,among others,MIP-1a,MIP-1b,THBS1,THBS2,VCAM-1,and ICAM-2.Analysis of protein interactions demonstrated that MIP-1a,MIP-1b,THBS1,and THBS2 were all lo-cated in the critical nodes of the protein interaction network.The average scan signal of the THBS2 in group B was 32969.11,and 5875.96 in group A.The 5.5 higher expression of THBS2 in group B was statistically significant(t=2.9,P=0.019).The expression of THBS1 protein was 1.5-fold higher in group B than in group A(t=2.434,P=0.027).Conclusion:Proteomics analysis identified THBS2 as a potential biomarker of HCC recurrence and metastasis after radical resection of the tumor.Part ?:Meta-analysis of THBS2 expressions in malignant tumors and its prognostic value.Objective:It is not enough to judge whether THBS2 can be used as an ef-fective prognostic marker based on the results of a single study.Thus,a meta-analysis of THBS2 expression in malignant tumors was performed,and its prog-nostic potential was assessed.Methods:A meta-analysis of published diagnostic data was accomplished by searching Pub-Med,the Cochrane system review database,and EM-BASE platforms.Statal 5.0 software to evaluate the results.Hazard ratios(HRs)and 95%confidence intervals(Cls)were used to determine the correlation between THBS2 expression and patient prognosis.Heterogeneity was assessed using Q and I2 tests.Publication bias was assessed by funnel plot and the Egger linear regression method.Results:The meta-analysis of OS of 2664 patients with malignant tumors across the 13 studies showed that the risk of an adverse survival prognosis in pa-tients with elevated THBS2 expression were 1.49-fold higher than patients with low THBS2 expression(95%Cl:1.34-1.66,P<0.001).The four documents in-cluded 6 groups of data.The meta-analysis of disease-free survival(DFS)in 5,000 patients with malignant tumors suggested that the total combined HRs and 95%CIs were 1.22(1.11-1.35),(P<0.001).Increased THBS2 expression was signif-icantly associated with an adverse prognosis in cancer patients.Conclusion:High expression of THBS2 is significantly correlated with poor survival in patients with malignant tumors.Thus,THBS2 represents a predictor of OS and DFS in these patients.Part ?:Correlation between THBS2 expression and clinicopathologi-cal features and prognosis of HCC patientsObjective:By expanding sample size,this study aims to investigate the cor-relation between THBS2 expression and clinicopathological features and progno-sis in 129 HCC patients.Methods:THBS2 expression in cancerous and matched paracancerous tis-sues obtained from 20 HCC patients was assessed by ELISA.Moreover,the cor-relation between THBS2 expression and clinicopathological features and progno-sis was analyzed statistically in 129 HCC patients.Results:Expression of THBS2 in the cancerous tissues was significantly higher than that in the matched paracancerous tissues in 20 HCC patients(Z=-3.696,P<0.001).In the 129 HCC patients,THBS2 expression in the cancerous tissue was correlated with the number of tumors(P=0.018)and the BCLC stage(P=0.013).The expressions of THBS2 in the groups with recurrence and metastasis time?12m,>12m and ?24m,and>24m were statistically significant.The higher the THBS2 expression level,the shorter the recurrence and metastasis time(?2=11.758,P<0.05).Kaplan-Meier survival analysis showed that the me-dian DFS and OS of patients with high THBS2 expression were shorter than those with low THBS2(P=0.032,P=0.036).The patients with a maximum tumor diam-eter greater than 5 cm and high expression of THBS2 had an earlier recurrence,shorter time to metastasis after radical surgery,and worse prognosis(P=0.002).The results of life table and log-rank analyses showed that the 3-year,4-year and 5-year survival rates of the high THBS2 expression group were lower than those of the low expression group(P<0.05).The COX univariate analysis showed that the high expression of THBS2,number of tumors>1,tumor thrombus,high de-gree of pathological malignancy(low/medium-low differentiation),BCLC stage,and late TNM stage were the factors resulting in the poor prognosis of DFS and OS for HCC patients after surgery(P<0.05).The number of tumors was an in-dependent risk factor for DFS and OS in HCC patients(P<0.001).Conclusion:High THBS2 expression in HCC tissue indicates a higher risk of recurrence and metastasis,shorter median DFS and OS,and worse survival prognosis.Part ?:Effects of THBS2 overexpression on the proliferation,migra-tion,and invasion of liver cancer cells in vitroObjective:To investigate the effects of overexpression of the THBS2 gene on the biological behaviors of hepatoma cells HepG2 and SMMC-7721.Methods:THBS2 expressions in a normal human hepatocyte line LO2,and four human hepatoma cell lines,HepG2,SMMC-7721,MHCC-97H and Huh-7,were assessed by qRT-PCR and Western blot analysis.Additionally,HepG2 and SMMC-7721 cells were stably transfected with the constructed THBS2 overex-pressing recombinant lentiviral vector,and the effect of THBS2 overexpression on cell proliferation,migration,and invasion was determined by CCK-8 test,col-ony formation assay,scratch method,and Transwell assay.Results:The qRT-PCR and Western blot results showed that the expression of THBS2 mRNA and protein in HepG2,SMMC-7721,MHCC-97H and Huh-7 hepatoma cell lines was higher than that in LO2 normal liver cells(P<0.05).The CCK-8 test and colony formation assay showed that the overexpression of THBS2 in hepatoma cells significantly enhanced their proliferation(P<0.05);the scratch and Transwell assays showed that the overexpression of THBS2 in hepatoma cells significantly enhanced their migration and invasiveness(P<0.05).Conclusion:The THBS2 expression is high in human hepatoma cells.THBS2 overexpression significantly enhances the proliferation,migration,and invasion of HepG2,SMMC-7721 hepatoma cell lines,indicating that THBS2 is associated with the malignant biological behaviors of hepatoma cells.