| Gastric cancer is one of the most common cancers in the world, accounting for 8% of the total cancer cases and resulting in 10% of total mortality from cancer cases worldwide. Over 70% of new cases and deaths occur in developing countries, including Eastern Africa and China. Previous epidemiological investigations indicated that smoking, alcohol drinking, and Helicobacter pylori infection, may be the major risk factors for gastric cancer. However, only a small proportion of the people constantly exposed to these environmental factors eventually develop gastric cancer, indicating that host genetic factors may have critical functions in gastric carcmogenesis.A molecular epidemiological hospital based case-control study was thus conducted in this thesis study to investigate the independent roles of single nucleotide polymorphisms (SNPs) in base excision repair (BER) genes (APE1 and NEIL2), carcinogen metabolism gene (CYP2E1) and tumor suppressor pathway gene (MDM2) in causing gastric cancer. The aim was to evaluate the potential association between the single nucleotide polymorphisms (SNPs) in the mentioned genes and their susceptibility to gastric cancer in a Northern Jiangsu population. Also, the NEIL2 gene mRNA expression was associated with the studied NEIL2 SNP genotypes, to assess whether the NEIL2 genotypes have influence on the NEIL2 mRNA (hence protein) expression. Moreover, an SNP genotyping assay based on novel magnetic nanoparticles was used to genotype the NEIL2 SNP (rs804270) in order to assess the effectiveness of nanomaterials in designing new SNP detection assays.Five SNPs, namely; APE 1 (rs2275008), NEIL 2 (rs804270), MDM2 (rs2279744), and CYP 2E1 (rs2480256 and rs2031920) were genotyped using the single base extension (SBE) assay based on novel core-shell Fe3O4@SiO2@Au magnetic nanoparticles (MNPs) and TaqMan assays. SYBR Green Ⅰ(?) Quantitative Real-Time PCR (qPCR) was used to measure the NEIL2 mRNA in the gastric cancer cases, for its association with the studied NEIL2 genotypes. The TaqMan RT-PCR SNP genotyping assay was also used as confirmatory method.The X2 test was used to compare genotype frequency between patients and controls while unconditional logistic regression analysis was used to estimate odds ratios (OR) and 95% confidence intervals (CI) for genotype susceptibility to gastric cancer. The Student’s t-test was used to test the significance of the observed differences in the NEIL2 mRNA expression among the randomly selected NEIL2 genotypes. A total of 105 gastric cancer cases and 118 controls were involved in this thesis study.The Fe3O4@SiO2@Au nanoparticles were successfully fabricated and used to genotype the NEIL 2 (rs804270) SNP and results were confirmed by TaqMan RT-PCR SNP genotyping assays, meaning that the rapid development of biomedical nanotechnology works well in designing new SNP genotyping assays. Other SNPs; APE1 (rs2275008), MDM2 (rs2279744), and CYP 2E1 (rs2480256 and rs2031920) were successfully genotyped by TaqMan assays.Further, the odds of having gastric cancer for the AA genotype (mutant allele) in the APE1 (rs2275008) SNP was 5.49 times higher (95% CI=2.578-5069, p＜0.0001) as compared to the G/A (heterozygous) allele. The same was observed with the NEIL 2 SNP (rs804270) GG (mutant allele) OR 2.3 (95% CI=1.22-4.3, p=0.01), MDM2 SNP (rs2279744) GG (mutant allele) OR 14.7 (95% CI=5.6-8.15, p＜0.0001) and CYP 2E1 SNP (rs2031920) TT (mutant allele) OR 8.4 (95% CI=3.16-5.3, p＜0.0001). All these results were significant, except for the CYP 2E1 (rs2480256), meaning that subjects with mutant genotypes in the APE 1 (rs2275008), NEIL 2 (rs804270), MDM2 (rs2279744), and CYP 2E1 (rs2031920) SNPs were more likely to have gastric cancer as compared to subjects with heterozygous genotypes. Overall, genotype frequencies didn’t differ significantly between cases and controls in all five SNPs. The genotype frequencies in the cases and controls demonstrated Hardy-Weinberg proportions, meaning that the cases and controls truly represented a Chinese population. Moreover, there was differential expression of the NEIL2 mRNA among the studied genotypes, with low NEIL2 mRNA expression seen among the variant (GG) genotypes, meaning that the genotypes may also independently influence their respective gene mRNAexpression. In conclusion, findings from this thesis study suggest that the NEIL 2 (rs804270), APE 1 (rs2275008), MDM2 (rs2279744), and CYP 2E1 (rs2031920) mutant genotypes may play important roles in the development of gastric cancer among Chinese in the studied Northern Jiangsu population. Also, the Fe3O4@SiO2@Au nanoparticles were successfully fabricated and used to genotype the NEIL 2 (rs804270), confirming that the biomedical nanotechnology may work well in designing new SNP genotyping assays.Well designed large population-based studies will finally validate the findings in this thesis study and provide the rationale for using the identified gastric cancer susceptibility genotypes (alleles) in identifying at-risk subpopulations for primary prevention of gastric cancer. Knowledge of genotype and its susceptibility to gastric cancer should make it possible to develop individualized gastric cancer treatment (drug development) and prevention protocols that maximize the intended consequences, by specifically targeting individuals with variant alleles.The main innovations and breakthroughs in this thesis study are as follows:1. Promoter polymorphisms in some DNA repair, carcinogen metabolism and oncogenes genes have been reported to be associated with increased gastric cancer risk. However, prior to this thesis study, there had been no reports of studies that had examined associations between the NEIL2 (rs804270), APE1 (rs2275008), CYP2E1 (rs2031920) and MDM2 (rs2279744) promoter polymorphisms and gastric cancer risk. The association of the studied promoter polymorphisms with susceptibility to gastric cancer risk in this thesis study should be of value for future research in this field. The findings in this thesis study should therefore be validated in future population-based studies that include larger number of gastric cancer patients, with more detailed data on environmental exposure and clinicopathological characteristics.2. In this thesis study, differential expression of the NEIL2 mRNA was observed among the studied NEIL2 genotypes, with low mRNA expression seen in the variant (G/G) genotype, as compared to other genotypes (CC and C/G). This finding suggests that the genotypes may also independently influence their respective gene mRNA expression. This new finding also needs to be tested in future mechanistic studies.3. Also, the three-layered core-shell Fe3O4@SiO2@Au magnetic nanoparticles (MNPs) with low fluorescence background were successfully used to genotype the NEIL 2 (rs804270) SNP in this thesis study. The MNPs were fabricated and successfully used as DNA carriers in the SBE genotyping assay, after proper optimization. This was the first study to use this kind of MNPs to successfully genotype the SNPs, meaning that nanotechnology helps in designing new SNP genotyping assays. |
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