Study On Microsatellite Instability (MSI) And Expression Of HMLH1 In Gastric Carcinoma

Objective: Gastric carcinoma(GC) is the most commonmalignant tumor in digestive tract, and its death rate is at thefirst place in all kinds of cancer. Recently, people haveinvestigated gastric carcinoma by many ways, but there is noany index that can indicate its true pathogenesis. It is commonbelieved that the development of GC is multipath. Research hadrevealed that the absence or reduce of mismatch repair enzyme(MMR) had strong relationship with tumorigenesis. MMR isimportant enzyme in DNA repair system. Its function was tocorrect misincorporated nucleotides during DNA synthesis.There were nine kinds of mismatch repair enzyme in MMRkindred. Among them, human mut-l homologue1(hMLH1)protein and human mut-s homologue 2(hMSH2) protein , whoseexpression were always absent or reduced in many tumors ,arethe most important enzyme.Mcrosatellite(MS) is simple repetitive DNA sequencescomposed of 2 to 6 nucleotide repeat units, prone to occurreplication error of DNA polymerase. When the expression ofmismatch repair enzyme was low or diminished, Microsatelliteinstability(MSI) occured because the replication error could notbe repaired. MSI means the increase or decrease of simplenucleotides repeat units because of replication error. MSI couldincrease DNA genome instability and random mutationfrequency, giving rise to the changes of a series of tumor-relatedgenes and subsequently leading to tumor occurrence.This study used Western blotting and polymerase chainaction(PCR) methods to investigate the expression of hMLH1protein and MSI frequency in human gastric carcinoma andnormal mucosa. And to elucidate the relationship between themand occurrence of gastric carcinoma. Up to now, little researchhas been paid to the collaborating study about hMLH1,MSI andoccurrence of gastric carcinoma. So this study provide theoreticbasis for further exploring mechanism of gastric moleculartumorigenesis and for early diagnosis, right therapy andprognostic judgement of gastric carcinoma.Methods: 43 surgically resected gastric carcinoma andnomal gastric mucosa from carcinoma >10cm (next callednomal mucosa) cases from January, 2003 to March, 2005 werecollected from department of Gastrointestinal Surgery in HeBeimedical university.There was 33 males, 10 females. Among 43patients, there was 20 with high and middle differentiation glandcancer, 20 with low differentiation cancer, 3 with signet ring cell.Among them , cancers located in cardia, body and antrum were8,12 and 23,respectively. Stage Ⅰ-Ⅱand Stage Ⅲ-Ⅳwere18 and 25 cases.No patient underwent any anti-tumor treatmentbefore operation.Total protein and genomic DNA was extracted from gastriccarcinoma and nomal mucosa.The expression of hMLH1 proteinwas studied by Western blotting. Five microsatelliteloci(BAT-25, BAT-26, D2S123, D5S346, D17S250) wereanalyzed by polymerase chain action(PCR) methods.The Statistical analysis of expression of hMLH1 proteinusing two sample Student's-t test, significance was determinedif P<0.05. The difference of protein expression level betweengastric cancer and nomal mucosa, the difference of proteinexpression level, status of MSI in clinicopathologic featureswere evaluated by chi-squareχ~2 or Fisher's exact probabilitytest or Person related χ~2 test. P<0.05 was consideredstatistically significant. Pearson related coefficient C showsintimate degree.Result:1 The expression of hMLH1 in gastric carcinoma and adjacentnormal mucosaβactin protein: The molecular weight ofβactin protein is42kD. After SDS-PAGE electrophoresis and Westernhybridization, 21kD brown bands were found. βactin proteinwas detected when hMLH1 protein was explored and as thestandard for quantitative analysis of hMLH1 protein expression.The molecular weight of hMLH1 protein is 85kD. AfterSDS-PAGE electrophoresis and Western hybridization, 85kDbrown bands were found. The bands were analyzed by gelimage analyzing system Lab Work 4.5 software. The proteinexpression was quantitative data (named IOD), and the relativeIOD was used for the comparation of protein expression. Thelow expression rate of hMLH1 protein was 44.18%(19/43) ingastric carcinoma, significantly differed from adjacent normalmucosa(0/43)(P<0.05). The low expression rate of hMLH1protein in gastric antrum was 65.21% (15/23),significantlyhigher than that in gastric Cardia (2/8) and body(2/12)(P=0.003). While, the low expression rate of hMLH1 protein inwell differentiated adenocarcinoma was 65.00% (13/20) ,significantly different from that in poorly differentiatedadenocarcinoma(5/20,25.00%) and signet-ring cellcarcinoma(1/3) (P<0.05). expression rate of hMLH1 protein hadstrong ralationship with tumor location and histologydifferentiated types(C was 0.804 and 0.707).No significantdiscrepancy was found in tumor size, with or not with serousmembrane invasion, with or not with lymph node metastasis andTNM stage considering the low expression rate of hMLH1protein.2 MSI status of gastric carcinomaIn 43 gastric carcinoma cases, the total frenquency of MSIwas 48.84%(21/43). The MSI frenquency in 5 loci (BAT-25,BAT-26, D2S123, D5S346,D17S250) were 20.93%(9/43),32.56%(14/43),30.23%(13/43),20.93%(9/43) 20.93%(9/43),respectively. And no significant discrepancy was found betweenthe 5 loci. The cancers were classified in MSI-High(MSI-H,≥2loci), MSI-Low(MSI-L,only 1 loci) and microsatellitestable(MSS) by standard. And the number of MSI-H, MSI-L andMSS were 14,7 and 22.3 The relationship between the expression of hMLH1 proteinand MSIThe low expression rate of hMLH1 protein was92.85%(13/14) in MSI-H, In MSI-L, the low expression rate ofhMLH1 protein was 71.42%(5/7), significantly differed fromMSS4.54%(1/22) (P<0.05). The expression of hMLH1 proteinhad strong ralationship with MSI(C was 0.969).4 The correlation between MSI and the clinicpathologicalcharacterThe MSI frenquency in gastric antrum (15/23,65.22%)wassignificantly higher than that in gastric Cardia (3/8) andbody(3/12) (P=0.021). While, the MSI frenquency in welldifferentiated adenocarcinoma(14/20,70.00%) was significantlydifferent from that in poorly differentiatedadenocarcinoma(6/20)and signet-ring cell carcinoma(1/3) (P<0.05). At the same time,in stageⅠ-Ⅱof gastric carcinoma the MSI frenquency was66.67% (12/18), significantly higher than that in stage Ⅲ-Ⅳ(9/25,36.00%) (P<0.05). MSI had strong ralationship withtumor location, histology differentiated types and TNM stage(Person related coeffcient C was 0.63,0.715 and 0.515).Nosignificant discrepancy was found in tumor size, with or notwith serous membrane invasion and with or not with lymphnode metastasis considering the MSI frenquency.Conclusion:1 There was probably the MSI and MSS pathway in...