Genetic Instability Of PINX1, Caspase-8 Gene And Their Relationship With Clinical Pathological Behaviors Of Gastric Carcinoma In Chinese

BackgroundThe malignant tumors have threatened human health and lives severely.One of the most important reasons is that the microinvasion or micrometastasis exists at the time of surgery and can not be controlled.Therefore it is very important to clarify the mechanism of invasion and metastasis,and quite significant to establish some definite molecular marker and target for the therapy.It has been investigated that the expression of anti-oncoprotein becomes low with the occurrence and metastasis of tumor,then the growth and metastasis of tumor cell are out of control.The main reason that the expression of anti-oncoprotein reduces is anti-oncogenes inactivity.To investigate the mechanism of genetic inactivity,scientists did lots of research on many anti-oncogenes such as P53,P16,et al,and found that the genetic instability,the representative of microsatellite instability and loss of heterozygosity,might be one important factor that leads to genetic mutation,the anti-oncogenes function maladjustment, and the tumor occurrence.Microsatellite instability(MSI)and loss of heterozygosity(LOH),the alteration in length and strength of short tandem repeat sequences are important molecular characteristics of many human tumors.Many researches indicated that MSI or LOH of anti-oncogenes plays an important role in occurrence of sporadic tumor.MSI was first found in tumors of the hereditary non-polyposis colon carcinoma(HNPCC)syndrome,and then it is found in many other kinds of tumors,such as colon cancer,gastric cancer, carcinoma of endometrium,breast cancer,prostatic cancer and pancreatic cancer,et al. Many studies suggested that the occurrence of MSI increase the frequence of mutation,and moreover,it lead to mutation of genes which has great relationship with tumor which may be an important mechanism of tumor occurrence.According to Knudson's two-hit hypothesis of TSG(tumor suppressor gene)inactivation,the common chromosomal region of LOH is a potential site harbouring TSG,thus LOH is regarded as a valuable molecular genetic marker to find TSG.PINX1 which locus in 8p23,compared of 7 exons,has two transforms incoding 174 aa and 328 aa,is a telomerase inhibitor,is also a new tumor suppressor based on recent researches.A new series of research about the Pinx1 have been studied,especially the correlation between Pinx1 expression and telomerase activity in tumorgenesis and development,and found that it has lower expression in tumor tissue than in normal tissue. Emi.M reported that there was a high frequence of LOH in colorectal carcinomal and hepatocellular carcinomas on 8p.But there was little research about genetic instability in gastric carcinoma,nor in Chinese people.Several lines of evidence indicate that deregulation of apoptosis is involved in the mechanisms of cancer development,a family of cysteine proteases that cleave the substrates at aspartate residues,known as caspases,plays a central role in the activation and propagation of death signaling.Caspase-8 activation plays a central role in the initiation phase of apoptosis.Domestic literature mainly limited in mechanism of signal transduction and their application of related medicine.Caspase-8,locus 2q33-34,which is a region of gene deletion easily in neuroblastoma,indicating that it is a candidate tumor suppressor.In human cancer tissues,somatic mutations of caspase—8 gene have been reported in colon cancers,suggesting the possibility that caspase-8 might be mutated widely in other human cancers.Another research suggests that caspase-8 gene mutation may affect the pathogenesis of gastric carcinoma,especially at the late stage of gastric carcinogenesis. These data indicate that caspase-8 mutation may contribute to the pathogenesis of both colorectal and gastric cancers.But genetic instability of caspase-8 in gastric carcinoma has not been reported.ObjectiveTo investigate MSI and LOH of locus D8S277 of PINX1 gene,D2S116 of caspase-8 gene,their effect on the expression of PINX1 and caspase-8,which would reveal the function mechanism of anti-oncogene and provide experimental basis for metastasis mechanism of cancer.Methods(1)Phenol-chloroform Method for extracting DNA from paraffin-embedded gastric carcinoma tissues.(2)Polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)and displaying band by ordinary silver stain to analyze the genetic instability of the locuses.(3)Expression of protein was detected by Envision immunohistochemistry.(4)Leica-Qwin computer imaging techniques was used to analyze Expression of protein.(5)SPSS statistic software analyzed the relationship among experimental results.ResultsExpression of PINX1 protein and genetic instability of PINX1 gene in Chinese people with gastric carcinoma①The frequency of LOH was significantly higher in the cases with lymph node metastasis than in those without metastasis(18.6%vs 0.00%,P＜0.01),and was significantly higher in the cases at TNM stageⅢ+Ⅳthan in those at stageⅠ+Ⅱ(21.4% vs 2.9%,P＜0.01).②The frequency of MSI was significantly lower in the cases with lymph node metastasis than in those without metastasis(10.00%vs 30.00%, P＜0.05Fisher's exact test);and was significantly higher in the cases well-differentiated than those moderately differentiated(60.00%vs 9.80%,P＜0.05),and those poorly differentiated (60.00%vs 17.41%,P＜0.05);and was significantly lower in the cases infiltrating out of serous membrane than those infiltrating mucosa and muscular layer(4.55%vs 50.00%, P＜0.01),and than those infiltrating serous membrane(4.55%vs 35.71%,P＜0.01).③The positive rate of PINX1 protein was significantly higher in the cases without lymph node metastasis than in those with lymph node metastasis(80.00%vs 50.00%,P＜0.01);and was significantly higher in the cases at TNM stageⅠ+Ⅱthan those at stageⅢ+Ⅳ;and was significantly lower in the cases between 40-60 years old than those above 60 years old (43.75%vs 65.52%,P＜0.05).The frequency of LOH was significantly lower in the cases with positive expression of PINX1 protein than those negative(1.92%vs 31.58%,P＜0.01).Genetic instability of caspase-8 gene in Chinese with gastric carcinomaNone of 90 gastric carcinomas showed LOH or MSI at locus D2S116.Conclusions①The genetic instability of PINX1 gene may paly a major role in tumor development and MSI and LOH regulate the development of gastric carcinoma through different pathways.LOH palys a major role in negative expression of PINX1,so can be regarded as a sign of gastric carcinoma development and deterioration.MSI may affect the prognosis and tumor turnover.The influence of MSI and LOH of locus D8S277 to PINX1 express indicate that PINX1 is not only a tumor suppressor,but also a tumor metastasis suppressor.②There is rare loss of hererozygosity or microsatellite instability in locus D2S116 of caspase-8 gene in gastric carcinoma.The genetic instability of caspase-8 gene may not be the major factor that affects the prognosis of gastric carcinoma.Among different malignant tumors,the mechanism of how the genetic instability of caspase-8 suppresses the carcinoma also could be distinct.