Elucidating the Molecular Basis of Thyroid Tumorigenesis

Abstract

Elucidating the Molecular Basis of Thyroid Tumorigenesis Base Excision Repair (BER) pathway is an important pathway involved in DNA repair mechanisms. PARP1, APEX1, OGG1 and XRCC1 are important genes of BER pathway. Polymorphism in these genes have been frequently reported in different cancers including thyroid cancer. The present study was carried out to evaluate the association of these genes with thyroid cancer at DNA, RNA and protein level. Tissues samples and blood samples were collected from different hospitals of Pakistan. To amplify specific regions of DNA to study polymorphisms, polymerase chain reaction using Allele Specific Amplification Refractory Mutation System (ARMS-PCR) was used followed by sequence analysis. To study the expression of these BER pathways genes at mRNA level, Real Time PCR was performed. Expression was also checked at protein level using Immunohistochemistry in cancer patients. In the last phase of this study, in vitro analysis of XRCC1 was carried out using shRNA to prepare knockdown model of XRCC1 gene. ARMS-PCR followed by sequencing was performed for polymorphism analysis of genes PARP1, APEX1, OGG1 and XRCC1 in 456 thyroid cancer patients and 400 healthy controls. Three polymorphisms of each gene were analyzed for their possible association with thyroid cancer. In gene PARP1, Homozygous mutant genotype (CC) of first SNP (Val762Ala, rs1136414) showed significant association with thyroid cancer, it increased the risk of thyroid cancer 1.30 folds (OR=1.30; 95% CI=0.99- 1.71; p=0.05). In second SNP (Ala284Ala, rs1805414), heterozygous (CT) showed an association with thyroid cancer and it significantly decreased the risk of thyroid cancer (OR=0.80; 95% CI=0.65-1.00; p=0.05). In third SNP (Asp81Asp, rs1805404) of PARP1, homozygous mutant genotype (TT) showed an association with thyroid cancer and it significantly decreased the risk of thyroid cancer (OR=0.63; 95% CI=0.40-1.00; p=0.05). In rs1130409 of APEX1 gene polymorphism, heterozygous and homozygous mutant were significantly associated with thyroid cancer (OR=2.44; 95% CI=1.84-3.23; p=0.0001 and OR=0.41; 95% CI=0.31-0.54; p=0.0001, respectively). In polymorphism rs1803118 of APEX1, heterozygous (CT) showed a highly significant association with thyroid cancer and it significantly decreased the risk of thyroid cancer (OR=0.32; 95% CI=0.24-0.43; p=0.0001). In OGG1, xii heterozygous (CG) genotype of rs1052133 showed highly significant association with thyroid cancer with significant decreased risk of thyroid cancer and played a protective role (OR=0.40; 95% CI=0.30-0.54; p=0.). In rs159153 polymorphism of OGG1, homozygous mutant genotype (TT) showed highly significance association with thyroid cancer and it significantly decreased the risk of thyroid cancer (OR=2.21; 95% CI=1.68-2.92; p=0.0001). In the case of XRCC1 polymorphism analysis, homozygous mutant genotype (AA) showed highly significant association with thyroid cancer and significantly decreased the risk of thyroid cancer (OR=0.17; 95% CI= 0.10-0.31; p=0.0001). In rs25487 polymorphism of gene XRCC1, heterozygous (GA) and homozygous mutant genotypes (AA) showed highly significant association with thyroid cancer. Heterozygous mutant significantly decreased the risk of thyroid cancer while homozygous mutant (AA) of the same SNP significantly increased the risk of thyroid cancer almost equal to 5 folds ((OR=0.24; 95% CI= 0.17-0.34; p=0.0001 and OR=4.80; 2.54-9.07; p=0.0001 respectively). In rs1799782 of XRCC1, homozygous mutant showed an association with thyroid cancer and decreased the risk of thyroid cancer (OR=0.55; 95% CI= 0.38-0.81; p=0.002). For expression analysis at mRNA level, 96 histopathological confirmed thyroid cancer tissue samples along with 96 adjacent non-cancerous tissues as controls were used in this study. Mean expression of PARP1 transcript in control tissues was higher as compared to tumor tissues and the difference was statistically highly significant (p=0.008). In case of APEX1, mean expression of APEX1 transcript in controls tissues was downregulated compared to tumor tissues, but the difference was statistically non-significant (p=0.51). In expression of OGG1, expression of OGG1 transcripts in controls tissues was higher compared to tumor tissues and this difference was statistically highly significant (p=0.02). In XRCC1 mean expression of XRCC1 transcript in controls tissues was higher compared to tumor tissues, but the difference was statistically non-significant (p=0.33). Immunohistochemical analysis of PARP1, APEX1, OGG1 and XRCC1 showed relative immunoreactive intensity in case of PARP1 gene 50% tumor tissues showed weak immunoreactive intensity, 37% showed moderate and 12% tumor tissues showed strong immunoreactive intensity. In case of APEX1 gene, 62.5% tumor tissues showed weak immunoreactive intensity, 25% moderate and 12.5% tumor tissues showed strong immunoreactive intensity. In case of of OGG1 gene, it was found that 75% tumor tissues showed weak immunoreactive xiii intensity, 12.5% tumor tissues showed moderate and 12.5% tumor tissues showed strong immunoreactive intensity. In case of XRCC1 gene, 50% tumor tissues showed weak immunoreactive intensity, 25% moderate and 2% tumor tissues showed strong immunoreactive intensity. In third part of research, in vitro analysis of XRCC1 gene was carried out in thyroid cancer cell line using short hairpin (shRNA). For this purpose, shRNA was designed and constructed and knockdown model of XRCC1 gene was prepared. Knockdown was confirmed using real time PCR. Knockdown cells showed significantly decreased expression of XRCC1 gene. MTT cell toxicity and metabolic activity assay showed significant reduced proliferation process in thyroid cancer cell line. Flow cytometric cell viability assay showed reduction in number of living cells in which shRNA for XRCC1 was used. In vitro results showed that XRCC1 may be used as target for anticancer strategies and diagnosis in future.