摘要 :
Objectives:
My main objective in this study as a clinical master degree student was to reconstruct and expressed the wild and mutated-type (E545A) of p110α plasmids.
Methodology:
The wild-type of p110α plasmid was...
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Objectives:
My main objective in this study as a clinical master degree student was to reconstruct and expressed the wild and mutated-type (E545A) of p110α plasmids.
Methodology:
The wild-type of p110α plasmid was constructed by cloning the p110α gene (PIK3CA) from the normal cell genomic DNA by PCR.The clone p110α genes and the pcDNA3.1 plasmid were digested using the restriction enzymes Asc-1 and Sma-1.The digested fragments of p110α genes and pcDNA3.1 plasmid were then ligated using T4 DNA ligase.The recombinant DNA was then introduced into Escherichia Coli and finally transfected into HEK293A cells.
The mutated-type (E545A) of p110α gene was induced using the QuickChange Lighting Site-Directed mutagenesis kit.
Results:
We successfully reconstruct and expressed the wild-type of p110α plasmid.The correct nucleotides sequence of the PIK3CA gene was confirmed by genesequencing.Overexpression of the p110α proteins in HEK293A were confirmed by western blot.
The E545A mutation was successfully induced and confirmed by genesequencing.Downstream upregulation of the PI3K pathway in transfected HEK293 Aharbouring mutated p110α gene was confirmed by western blot showing overexpression of the AKT/PKB level.
Discussion and Conclusion:
The PIK3CA gene is mutated on average in 15% of human cancers with mutations clustering predominantly within three hotspots: E542K,E545Kand H1047R.
The E545A mutation although not considered as a hotspot mutation have been reported to contribute up to 88% of the total of PIK3CA mutation identified in ovarian cancers and associated with various other types of cancers.
We found in our department that 33%(36 cases out of 110) of patients with Gastrointestinal Stromal Tumour (GIST) also harbours this mutation.We strongly believed that tumour cells harbouring both KIT and E545A mutations are resistant to the receptor tyrosine kinase inhibitor-Imatinib.The reconstructed plasmids will be used in studying Imatinib resistance in GIST.
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摘要 :
Background：
Gastric Cancer is one of the most common malignant tumor and second leading cause ofdeath worldwide.Gastric cancer in general has seen steady decline over the decade.Thisdecline is observed in all other types of ...
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Background：
Gastric Cancer is one of the most common malignant tumor and second leading cause ofdeath worldwide.Gastric cancer in general has seen steady decline over the decade.Thisdecline is observed in all other types of Gastric Cancers except，cancers in Gastric Cardia.Incidence of Gastric Cardia Adenocarcinoma (GCA) is found to be in increasing trend.Occurrence of GCA varies among different ethnic groups and geographical regions.
Micro RNAs (miRNAs) are non-coding RNAs with the function of post translationalregulation of gene expressions.miRNAs play important roles in the regulation of allbiological processes at the cellular and molecular level.Any alteration causing abnormality inmiRNAs may lead to the disturbances in gene expression which in turn may result inoncogenesis.Single Nucleotide Polymorphisms (SNPs) are the most common form of geneticvariation.SNPs in human population are supposed to be responsible factors for the individualvariation in disease susceptibility as well as physical appearance.
Aim of the study：
To find out whether hsa-miR-34b/c rs4938723 T＞C，hsa-miR-423 rs6505162 C＞A，pre-miR-125a rs 12975333 G＞T and pri-miR-124-1 rs531564 C＞G polymorphisms are associated withthe risk of GCA.
Material and method330 cases and 608 controls were selected according to the inclusion criteria of the study fromthe two affiliated hospitals of Jiangsu University，Zhenjiang，Jiangsu，P.R.China.Informedand signed consents were obtained from all the subjects.2ml Sample of whole blood wascollected from all subjects and buffy coat was extracted from it which was stored in ultra lowtemperature refrigerator.Genomic DNA was extracted from the stored buffy coat usingQIAamp DNA Blood Mini Kit (Qiagen，Berlin，Germany).Extracted DNA were amplifiedby Polymerase Chain Reaction (PCR) and Ligation Detection Reaction (LDR) was used todetermine the genotypes of hsa-miR-34b/c rs4938723 T＞C，pri-miR-124-1 rs531564 C＞G,pre-miR-125a rs12975333 G＞T and hsa-miR-423 rs6505162 C＞A.
Result：
Analysis of Demographic Characteristics of patients using chi square test and student t-testshowed that rate of tobacco use was significantly high in cases than in controls(p=0.006).There were no significant differences in the rest of the demographic characteristicsbetween cases and control subjects.The primary information for selected fourpolymorphisms were similar to Chinese population in SNP database except for pre-miR-125ars12975333 G＞T which was not available.
Comparative analysis for pre-miR-125a rs12975333 G＞T polymorphism was not possible asall genotypes were CC homozygote in all subjects.In the stratified analysis of hsa-miR-423rs6505162 C＞A polymorphism and risk of GCA，when CC homozygote genotype was takenas a reference in stratification analysis of has-miR-423 rs6505162 C＞A，male patientsshowed significantly decreased risk of GCA in CA heterozygote genotype (p =0.030) and inCA+AA variants (p=0.042) whereas female patients showed significantly increased risk ofGCA in AA genotype (p=0.037) and in AA vs.CA+CC variants (p=0.040).For rest of thecharacteristic findings were not significant.
Logistic analysis of hsa-miR-34b/c rs4938723 T＞C，pri-miR-124-1 rs531564 C＞G，and hsa-miR-423 rs6505162 C＞A polymorphisms and risk of GCA along with stratified analysis ofhsa-miR-34b/c rs4938723 T＞C and pri-miR-124-1 rs531564 C＞G polymorphisms and risk ofGCA by all demographic characteristic did not revealed any significant findings.
Conclusion：
SNPs in CA and CA+AA variants of has-miR-423 rs6505162 C＞A polymorphisms areassociated with decreased risk of GCA in male patients whereas AA and CA+CC variants areassociated with significantly increased risk in female patients.However，similar and largescale studies are warranted to validate the findings of this study.
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