Expression Of ADAMTS5 In Hepatocellular Carcinoma And Its Function

Background:Hepatocellular carcinoma(HCC)is the most common histological subtype in primary liver cancers, with high incidence and mortality rate. As it is prone to metastasis and recurrence,the prognosis of HCC patients remains poor. Therefore, a more profound knowledge of the potentional molecular mechanisms regulated by ADAMTSs would contribute a better understanding of the real impact of these metalloproteases in HCC, which may provide novel strategies in diagnosis and treatment in HCC.A Disintegrin-like and Metalloproteinase with Thrombospondin-1 Motifs 5(ADAMTS5), together with Matrix Metalloproteinases( MMPs) and A Disintegrin and Metalloproteinases( ADAMs), belong to Zn2+-dependent metalloprotease family. Aberrant expression of ADAMTS5 plays an important role in carcinogenesis and tumor progression, and the structure of ADAMTS5 protein shows that it could function as an anti-angiogenic protein independent of its proteoglycanase activity. Therefore, more mechanistic and functional studies are necessary to fully understand how ADAMTS5 can influence carcinogenesis and tumor progression in HCC.rs2830585 is a nonsynonymous single-nucleotide polymorphism(ns SNP), mapping to The Thrombospondin-1 Motif repeat1(TSR1) domain of ADAMTS5. By application of SNPs 3D software, it shows effect of rs2830585 on ADAMTS5’s function by changing amino acid sequence of TSR1 domain, which is crucial for anti-angiogenic activity of ADAMTS5. Therefore, it is of great significance to study the impacts of ADAMTS5 rs2830585 polymorphism on the risk of hepatocellular carcinoma, which is characterized as primary malignant tumor with abundant angiogenesis.Objective1. To study the expression of ADAMTS5 gene product and its relationship with prognosis in patients with HCC.2. Clarify molecular mechanisms regulated by ADAMTSs in carcinogenesis and tumor progression of HCC.3. Confirm the relationship between ADAMTS5 rs2830585 single nucleotide polymorphism and genetic susceptibility to HCC.Methods:Part I:1. Using tissue microarray and immunohistochemistry to analyze expression of ADAMTS5, CD34 and Vascular Endothelial Growth Factor(VEGF) in HCC.2. Comparing ADAMTS5 expression between HCC and distant paracarcinomatous tissues by Mc Nemar’s test.3. Evaluating the association of ADAMTS5 expression with clinicopathological characteristics by chi-square test or Fisher’s exact probability test.4. Assessing association of ADAMTS5, VEGF, Microvessel Density(MVD), and tumor size by Spearman’s rank correlation coefficient.5. Assessing the association of ADAMTS5 expression with overall survival(OS) of patients by survival analysis.6. Assessing expression of ADAMTS5 protein in normal liver cell line and different HCC cell lines by Western Blot.Part II:1. Varifying effect of ADAMTS5 expression in HCC on VEGF secretion, with Enzyme-linked Immunosorbent Assay(ELISA) to generations of stable ADAMTS5- expressed HCC cell lines.2. Detect effect of HCC cell culture-conditioned medium on Human Umbilical Vein Endothelial Cell(HUVEC) migration and Matrigel tube formation, by HUVEC migration assay and Matrigel tube formation assay.3. Varify effects of ADAMTS5 on HCC tumor growth and angiogenesis in vivo by xenograft assay.Part III:1. Clinical data of 220 HCC paients and 220 healthy patients without HCC were collected and survival data of HCC paients were obtained by follow- up.2. With informed consent, the peripheral blood DNA of patients were extracted, and then used to test genotype rs2830585 polymorphism.3. SPSS software version 19.0. was performed to analyse the relationship between ADAMTS5 rs2830585 single nucleotide polymorphism and genetic susceptibility to HCC.Results:Part I:1. The expression of ADAMTS5 was significantly lower in HCC tissues versus paracarcinomatous tissues(Mc Nemar’s test, p<0.05).2. ADAMTS5 positive expression was found to be significantly associated with tumor stage(p<0.01).3. ADAMTS5 positive expression was found to be related with decreased MVD(r=-0.530, p<0.001), VEGF( r=-0.640, p<0.001) and tumor size(r=-0.361, p<0.05) than negative expression patients.4. HCC Patients with positive ADAMTS5 expression had remarkably better OS compared to those with negative ADAMTS5 expression(29.56 versus 12.96, p<0.001). ADAMTS5 expression was an independent prognostic factor for OS of HCC patients(p<0.05).5. The level of ADAMTS5 expression was higher in normal liver L02 cells than in HCC cell lines.Part II:1. VEGF expression level in tumor cell culture-conditioned medium derived from ADAMTS5-expressed HCC cell lines was significantly lower compared with the control group(p<0.001).2. The capillary tube formation capacity and migration capacity of HUVECs were significantly suppressed in the ADAMTS5 group compared to the control group(p<0.01). However, the reduced capacity of HUVECs were largely restored by recombinantion of VEGF(p<0.01).3. Tumor xenografts generated from HCC cells with high ADAMTS5 expression grew more slowly than those generated from control cells(p < 0.01). Immunohistochemistry analysis showed that high expression of ADAMTS5 in xenografts inhibited angiogenesis and VEGF expression.Part III:1. Logistic regression analyses indicated that, in comparison with the G allele of rs2830585, the A allele was associated with a significantly decreased risk of HCC(adjusted OR, 0.348; 95%CI 0.236-0.512; P <0.001). Individuals with the rs2830585 GA genotype had a lower risk for developing HCC compared with individuals having the rs2830585 GG genotype(adjusted OR, 0.339; 95%CI, 0.044-11.535; P<0.001).2. Stratified analysis showed no statistically significant interaction between rs2830585 genotypes and stratification factors.3. Correlation analysis showed no statistically significant interaction between rs2830585 genotypes and Clinicopathological Characteristic of HCC Patients.Conclusion:1. ADAMTS5 was downregulated in HCC,and the high expression of ADAMTS5 was correlated with good prognosis of HCC.2. ADAMTS5 suppressed tumor growth and angiogenesis by downregulating the secretion of VEGF.3. ADAMTS5 rs2830585 polymorphism was significantly associated with HCC susceptibility in Chinese Han population, and the ADAMTS5 rs2830585 A allele was significantly associated with decreased risk of HCC.