Research On Detection Methods Of Biomarkers Of Gastric Cancer Based On Nanoparticles

Gastric cancer incidence and mortality rank first among all kinds of tumors in China. Most patients were diagnosed too late for curative treatment. Early gastric cancer, whereby disease is limited to mucosa and submucosa, confers a survival rate of greater than 95% in 5 years in many centres. So, it is necessary to develop a minimal invasive method to identify gastric cancer at an early stage. Cancer cells shed circulating, tumor specific DNA (cirDNA) into various body fluids. These molecules of DNA harbor genetic alterations that can be harnessed for diagnostic purposes. Circulating nucleic acids are an important emerging biomarker in cancer diagnostics as well as a non-invasive diagnostic solution for a wide range of clinical disorders. Methylation and microRNA (miRNA) are hot spots in epigenetics. On the other hand, genetic single nucleotide polymorphisms (SNPs) are the most frequent forms of sequence variations in the human genome. Both genetic and epigenetic events influence gene expression with dramatic effects on proliferation, survival and invasion. Tumor susceptibility is determined by individual genetic characteristics, and early detection is the key to improve the efficacy and increase the survival rate of gastric cancer patients. Therefore, further study on gastric cancer etiology and pathogenesis to identify associated molecular markers and screen high-risk groups to take effective prevention measures has great significance. With the rapid advances in nanotechnology, magnetic nanoparticles (MNPs) have been widely applied in the detection of various biological signals because that they can be easily collected under the external magnetic field, furthermore, they have the characteristics of high surface area. Combination detection of E-cadherin cirDNA methylation, miR-21 relative expression and SNPs associated with gastric cancer was performed using MNPs, highlighting the molecular alterations that occur in gastric carcinogenesis could provide new clinical tools for early diagnosis of gastric cancer. Finally, we explored a SNP genotyping method based on Pd nanoparticles. The main research contents are as follows:1. Study on promoter methylation status of cirDNA E-cadherin gene in gastric cancerMNPs can improve efficiency of DNA extraction due to their easy separation property. MNPs have the incomparable advantages over the traditional DNA extraction method. There are a small amount of circulating DNA (cirDNA) in the plasma. Extraction productivity directly influenced detection results. We selected suitable MNPs to extract cirDNA in the plasma of 32 gastric cancer patients and 10 healthy controls. Maldi-TOF was employed to investigate the promoter methylation of E-cadherin gene. Gastric cancer plasma yielded higher assay sensitivity for detection of methylation of E-cadherin gene than healthy control plasma. There was a proportion of hypermethylation in low/low-moderate differentiated patients than in moderate differentiated ones. Ulcer type patients have a significantly higher frequency of hypermethylation than non-ulcer type patients (P<0.05). Promoter methylation of E-cadherin gene in plasma cirDNA may play a vital role in the pathogenesis of gastric cancer, which was clinically significant for being an adjunct early diagnostic tool of gastric cancer.2. Detection of relative expression level of miR-21 in gastric cancer based on MNPs and chemiluminescenceRecently, miRNAs, a class of small, regulatory, non-coding RNA molecules, display aberrant expression patterns and functional abnormalities in human diseases including cancers. Circulating miRNAs have close correlation with disease prognosis, indicating that the cell-free miRNAs serve as informative biomarkers for diverse gastric pathological status. miR-21 may be important in the initiation and progression of gastric cancers as an oncogenic miRNA (oncomiR). Practical implication of miRNAs as biomarkers for diagnosis of human gastric cancer was discussed. We established gastric cancer related miR-21 detection method based on MNPs and chemiluminescence. The proposed method was applied to detect relative expression level of miR-21 in gastric cancer patients and control serum samples (normalized relative to miR-16) and evaluate their diagnostic values. miR-21 was significantly overexpressed in human gastric cancer bloods. miR-21 had great diagnostic power for gastric cancer against healthy group and showed potentials as biomarkers in clinical application.3. Study on the association between SNPs and gastric cancer susceptibility based on MNPsIn this study, by using a high-throughput SNP genotyping method based on MNPs and dual-color fluorescence hybridization, COX-2 gene promoter region-765G>C polymorphism and BCL-2 gene-938C>A polymorphism of 118 gastric cancer cases and 120 normal controls samples in northern Jiangsu were genotyped. The results showed that:For COX-2-765G>C, there are 59.3% wildtype,33.1% heterozygous type and 7.6% mutant type in gastric cancer patients; 72.5% wild type,23.3% heterozygous type and 4.2% mutant type in controls, respectively. For BCL-2-938C>A, there are 51.7% wild type,44.1% heterozygous type and 4.2%mutant type in gastric cancer patients; 38.3% wild type,51.7% heterozygous type and 10% mutant type in controls, respectively. COX-2-765G>C and BCL-2-938>A polymorphisms have an effect on the risk of developing gastric cancer in northern Jiangsu population. Carriers with COX-2 -765GC and-765CC genotypes had a significantly higher risk of gastric cancer than that with COX-2 -765GG genotype in gastric cancer group (OR=1.808,95%CI:1.050-3.113, P<0.05). Namely individuals carrying the -765GC or-765CC genotype can significantly increase the gastric cancer risk and the-765C allele is associated with increased susceptibility to gastric cancer in northern Jiangsu of China. The individual risk of gastric cancer carrying BCL-2-938CC genotype was 3 times that carrying AA genotype (OR CC vs. AA= 3.185,95%CI:0.103-0.955, P< 0.05). In addition, as C allele OR value rising significantly, the BCL-2 -938C>A locus have allele "dosage-effect" relationship with genetic susceptibility of gastric cancer. In-depth study showed that:There is an obvious multiplicative interaction effect between COX-2 -765CC/GC type and BCL-2-938CC genotype, respectively, in the risk of gastric cancer. When compared with COX-2-765GG and BCL-2-938AA genotype, carriers with COX-2-765CC/GC and BCL-2-938CC genotypes had a significantly increased risk for developing gastric cancer (OR=10,95%CI:1.280-78.117, i><0.05; OR=4.667,95% CI:1.137-19.154, P<0.05). The results revealed a statistically significant difference indicating that COX-2 gene and BCL-2 gene have obvious synergies in gastric cancer incidence.4. Detection of SNP based on both "Pd label Ag stain" and sandwich hybridization systemWithin the last decade, precious metal nanomaterials represented by Au have been broadly used as label and become a hot field in biological analysis due to their good physical chemistry properties such as optical and catalysis. Palladium nanoparticles (Pd NPs), as one of the important precious metals, are of excellent catalytic property. However, there have been hardly any reports on using Pd NPs as the labels to detect DNA to this day. There are many advantages of Pd NPs. It is cheaper than Au and more stable than Fluorescent molecular marker. It can catalyze Co or Ni to create magnetic signal, which can amplify signal and improve the detection sensitivity. In this study, we explored a DNA detection method based on "Pd label Ag stain" and sandwich hybridization system. We firstly prepared MUA@Pd NPs in-situ and conjugated them with DNA, and then genotyped COX-2 promoter -765G>C SNP by menas of sandwich hybridization system. The Pd-labeled signal was amplified by silver staining. The results were verified by DNA direct sequencing.