Studies on Gastric Cancer Associated Molecular Markers
|School||Nanjing Medical University|
|Course||Pathology and Pathophysiology|
|Keywords||Gastric cancer CDH1 Resequencing miRNA Microsatellite BAT25|
ObjectiveThe aims of this study were to study associated molecular markers of gastric cancer by CDH1 gene resequencing,miRNAs expression and MSI analysis.1.Resequencing anlysis of CDH1 geneDNAs were extracted from patients’ tissues,corresonding normal mucosa,and peripheral blood leucocytes of healthy donors.PCR was peoformed with 27 resequencing primer pairs of CDH1 gene synthesized by Applied Biosystems,respectively.The PCR products were detected by agarose electrophoresis.The purified PCR products were subjected to sequencing PCR reactions,in which purify of the products was made with a NaAC/EDTA method.After denaturated with Hi-Di formamide,the PCR products were sequenced via capillary electrophoresis on an ABI 3100-Avant Genetic Analyzer.The data of sequence were blasted with CDH1 gene reference sequence with SeqScape v 2.1.1 software（Applied Biosystems）to look for variant sites.We investigated 11 gene variant sites in CDH1 gene from patients with sporadic gastric cancer.Among these variants,intronl-49G＞T is a novel mutation site.Statistical analysis showed that there was a statistically significant correlation between intronl-1030 site A,-284 site A allele and tumor differentiation.We also found 8 novel germline mutations in 3 samples with familial gastric cancer history.All these new mutation sites locate in the intron adjacent to coding regions.2.Expression of miR-21,miR-145 and miR-155Total RNA was extracted from paraffin-embedded gastric cancer tissues and corresponding normal mucosa.Reverse transcription reactions were done with special looped primer from Applied Biosystems.The expression of miR-21、miR-145 and miR-155 were then detected by real time PCR with a reference gene U6.We calculatedΔCt values of each sample and analized on 2-ΔCtby t-test statistical method.The results showed that the expression of miR-21 and miR-155 in gastric cancer tissues was higher significantly than that in adjacent normal tissues,respectively.The expression of miR-145 in most of cancer tissues was lower,but not statistically,than its expression in adjacent normal tissues.In addition,we found that the expression of miR-21 and miR-145 were higher than the expression of miR-155 in cancer tissues and normal tissues.The expression of miR-155 in gastric tissues is relatively low.3.Study on microsatellite instabilityTotal RNA was extracted from frozen gastric cancer tissues and the corresponding normal mucosa.Multiple fluorescent PCR reactions were done with 5 primer pairs including D2S123、D17S250、D5S346、Bat-25 and Bat-26 in one reaction tube.The PCR products were denatured and detected via capillary electrophoresis on an ABI 3100-Avant Genetic Analyzer.MSI was blasted and judged using GeneMapper 3.0 software.The rate of MSI in tested sporadic gastric cancer was 35.1%which was similar to previous report.Among these 5 loci,BAT25 was most sensitive （85.0%）,next was BAT26（45.0%）,and D17S250 was most unsensitive （5.0%）.MSI gastric cancer and MSS gastric cancer showed a different distribution in the different ages of population.Patients with MSI gastric cancer were significant higher in the group over 65 years as compared with those less 65 years.MSI gastric cancer was correlated significantly with lower hMSH2 expression.No significant difference between MSI gastric cancer and MSS gastric cancer was found to relate to sex, differetation,classification,lymphoid node metastasis,clinical staging, TNM staging,and Hp infection. 4.Investigation of BAT25 sequence in healthy populationWe extracted 431 DNAs from healthy people and detected BAT25 polymorphism by direct sequencing technique.We found that BAT-25 of Chinese people was quasi-mononucleotide in the size of the poly（T）tracts. We suggested that when BAT25 was used as a microsatellite analysis site, tumor samples should be done simultaneously with paired normal samples for the sake of correct microsatellite instability diagnoses.ConclusionResequencing whole gene technique could screen more mutations in cancer correlated gene.In patients with sporadic gastric cancer and people with familiar gastric cancer history,we detected 19 allele variations of CDH1 gene contained 9 novel mutations in all.-1030A allele and -284A allele were correlated to low differentiation of sporadic gastric cancer. CDH1 gene mutation rate in the subjects with familiar gastric cancer were higher than in the patients with sporadic cancer.More variations in CDH1 are still needed to be studied.Our study showed that the up-regulation of miR-21 and miR-155 were associated with the gastic carcinogenesis,whereas the down-regulation of miR-145 may partly play some roles.Formalin-fixed paraffin-embedded specimens could act as satisfactory resources for miRNA study.Multiple fluorescent PCR with 5 MS loci method was useful in detecting MSI in gastric cancer.Low expression of hMSH2 was correlated with MSI in gastric cancer.BAT25 was the most sensitive locus in MSI analysis in our study,and we confirmed that Bat25 of Chinese people was quasimonomorphic.