Expressions And Function Of SUSD2 In Hepatocellular Carcinoma

IntroductionHepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. It is the third leading cause of cancer death in worldwide. However, HCC patients present with advanced disease stage, even with radical resection, most of patients develop metastasis and recurrence within 5 years of surgery. In general, HCC is associated with genome instability and mutation. Accumulation of disorder on gene expression and protein modulation promotes occurrence and development of HCC. Recently, sorafenib, the mixed kinase inhibitor, has been approved for treatment in HCC. Although clinical research on HCC has taken a great progress, the pathogenesis of HCC remain poor. However, due to lack of early diagnosis methods and the result of surgical resection is not satisfactory, emphasizing the need for understanding the mechanism on pathogenesis and new biomakers of HCC patients.Recently, the human SUSD2 has been shown is located on chromosome 22 and encodes an 822-amino acid type I membrane protein containing somatomedin B, AMOP, von Willebrand factor type D, and Sushidomains, which are frequently found in molecules playing important roles in cell-cell and cell-matrix adhesion. Currently, SUSD2 has been reported to be dysregulated in non-small cell lung cancer,breast cancer and colon cancer,accumulating evidences suggested that reduced expression of SUSD2 plays a key role in tumorigenesis,however, little is known about the effect of SUSD2 expression on HCC patients and prognosis.In this study, we examined the expression pattern of SUSD2 in HCC patients, and the correlation between its expression levels with clinicopathological variables of HCC. Furthermore, the biological function was studied by up-regulated and down-regulated the expression of SUSD2 in HCC cell lines.Methods1、Two HCC related tissue specimens of microarray data GSE17856, GSE45144 were analyzed by the bioinformatics software Qlucore Omics Explorer (QOE). Co-differential ly expressed genes of the two microarray were got by crossing two sets of data. The gene function and pathway analysis of co-differentially expressed genes were analyzed by using bioinformatics software DAVID. A new HCC related gene was determined by literature from co-differentially expressed genes.2、8 pairs of fresh HCC tissues and adjacent nonmalignant liver tissue were collected from patients to investigate the expression pattern of SUSD2 by using Western blotting and qRT-PCR.3、Compared the expression of SUSD2 in HCC tissues and normal liver tissues by immunohistochemistry using tissue microarray. Examined the subcellular localization of the SUSD2 protein. ROC analysis was applied to determine cut-off scores for tumor "high expression". Discovered the relationship between SUSD2 and clinicopathological parameters of HCC patients.4、To investigate the function of SUSD2 in the tumorigenesis of HCC, we changed the expression level of SUSD2 in HepG2 cells and SMMC7721 cells, we up-regulated SUSD2 in HepG2 cells by the transient transfection of pcDNA3.1-SUSD2 and down-regulated expression of SUSD2 in SMMC7721 cells by the transient transfection of psimHl-SUSD2. Western blotting tested SUSD2 protein level of the transient transfection HCC cell lines.5、To explore whether SUSD2 affects the growth of HCC cells, we conducted cell proliferation by using CCK-8 assays.6、To examine whether SUSD2 expression influences the cell apoptosis of HCC cells, we conducted cell apoptosis using Annexin V-FITC/PI double staining by flow cytometry.7^ We conducted wound-healing assay and transwell assays to examine whether SUSD2 expression influence the ability of HCC cells on cell migration and invasion.Results1, Bioinformatics analysis of HCC related genes:There were 383 co-differentially expressed genes in GSE17856 and GSE45144 screened by QOE, of which 171 genes were upregulated,182 genes were down regulated. We used DAVID to find the functional annotation and pathway of the differentially expressed genes and found that these genes were mainly associated with the cell adhesion, cell adhesion, oxidation reduction, p53 signaling pathways and Jak-STAT pathways. SLJSD2,a new HCC related gene, was determined by literature from co-differentially expressed genes.2, Western blot showed that 7/8 HCCs displayed reduced level of SUSD2 compared with the adjacent normal liver tissues. Similar results for mRNA expression were observed using qRT-PCR. The results showed that in 7of the 8 sample pairs, mRNA fold changes were less than 1 between HCC and adjacent normalliver tissue which indicated the SUSD2 mRNA expression was downregulated in HCC tissues compared to the adjacent normal liver tissues(P＜ 0.05).3、We further examined the expression and subcellular localization of the SUSD2 protein by IHC in a TMA, which including 180 cases of HCC and 16 cases of normal liver tissue. SUSD2 is primarily expressed in the cytoplasm within tumor cells. According to ROC analysis, expression percentage for SUSD2 above the critical value 52.5% was defined as positivity. The positive expression of SUSD2 was detected in 16/16 (100%) of non-cancerous adjacent tissues. However, the high expression of SUSD2 was only detected in 37.8% (68 of 180) HCC cases and the remaining 62.2% (112 of 180) were scored as having no or low SUSD2 expression (P= 0.000). The results demonstrated that low expression of SUSD2 was positively correlated with tumoradvanced clinical stage (%2= 30.244, P< 0.05), histological grade (X2= 5.198, P< 0.05),pT status(X2= 33.175, P< 0.05), pN status (X2= 4.785, P< 0.05)and,pM status (x2= 4.620, P< 0.05). However, there was no statistically significant correlations between SUSD2 expression and other clinicopathologic features, such as patient gender (X2= 1.093, P> 0.05), age at diagnosis (X2= 0.31,P> 0.05).4、Western blotting revealed that pcDNA3.1-SUSD2 transduction up-regulated SUSD2 in HepG2 cells at the protein level and psi-mHl-SUSD2 transduction down-regulated SUSD2 in SMMC-7721 cells at the protein level.5、CCK-8 assays showed that up-regulation of SUSD2 significantly inhibited the HepG2/SUSD2 proliferation (P< 0.05). On the contrary with the up-regulation results, down-regulation of SUSD2 significantly increased the growth of SMMC7721/KD cells in comparison with control cells(P< 0.05).6、The results of Annexin V-FITC/PI double staining by flow cytometry indicated that HCC cells with SUSD2 overexpression promoted cell apoptosis(.P< 0.05). Furthermore, we carried out a similar experiment and found that SUSD2 knock down significantly reduced cell apoptosis compared with cells treated with scramble shRNA (P< 0.05).7% Wound-healing assay showed that SUSD2 knock down significantly increase cellular migration of SMMC7721 cells (P< 0.05). And SUSD2 up-regulation significantly decreased cellular migration of HepG2 cells. Next, we carried out an experiment to compare the ability of SUSD2 on invasion. SUSD2 up-regulation significantly decreased invasion of HepG2 cells (P< 0.05). SUSD2 down-regulation significantly increased invasion of SMMC7721 cells (P< 0.05).ConclusionWe found the new HCC related gene SUSD2 by using bioinformatics tools for analyzing microarray array data. In the present study, we reported for the first time the SUSD2 expression levels of both the mRNA and protein were markedly reduced in the majority of HCC tissues, when compared with their paired adjacent normal liver tissues. The expression of SUSD2 was positively correlated with HCC patients’ clinicopathologic features. To investigate the possibility that SUSD2 could suppress the tumorigenesis of HCC, we altered the expression of SUSD2 in HCC cell lines by up-regulation and down-regulation. As expected, up-regulation of SUSD2 in HepG2 cells significantly inhibited the cell proliferation, migration, invasion and promoted cell apoptosis compared with those of the cells transfected with empty vectors. Consistently, down-regulation of SUSD2 in SMMC7721 cells enhanced the cell proliferation, migration, invasion and decreased cell apoptosis compared with those of the cells transfected with empty vectors. The major finding from this study provide evidence that SUSD2 may play as a tumor suppressor in HCC and a potentially effective biomaker. Our research provides information for further research on the molecular mechanisms of SUSD2 in tumorigenesis in HCC.