Expression Of HMSH2,hMLH1 And P53 In Sporadic Colorectal Carcinoma

Objective To detect the expression of mutated mismatch repair gene(hMSH2 and hMLH1) and p53 gene, then observed the relationship betweenhMSH2, hMLHl gene mutation and mutant p53 gene. To investigate the roleof mutated mismatch repair gene hMSH2 and hMLHl in the carcinogenesisand development of sporadic colorectal carcinoma.Methods Tumor DNA and DNA from corresponding normal neoplastic tissue were extracted using hydroxybenzene phenol-chlorofrom method with protein K digestion. We analysed the normal and tumor genomic DNA for hMSH2 mutation in exon 5, 6 and 12, hMLHl mutation in exon 12, 14 and 16, p53 mutation in exon 5 6,7 and 8 using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and EB staining. PCR was performed using 5.0μ1 of genomic DNA, 10×buffer 5.0μl, 25mmol/L Mgcl₂ 4.0μl, 1.0μl of each primer, dNTPs 1.0μl, Taq DNA polymerase 2.0 μl and double distilled water 31.0μl, the total volume was 50.μl. The product of PCR which showed abnormal strand was sequenced directly.Results1. The mutation rate of mismatch repair gene was 15. 55%(7/45), and the mutation rate of hMSH2 and hMLHl gene in SCC was 2. 22% (1/45), 13. 33%(6/45) respectively.2. The mutation of hMSH2 and (or) hMLH1 expression was observed in5 of 13 right-sided SCC, and 3 of 32 left-sided SCC. Loss of hMSH2 and (or) hMLHl expression was more frequently associated with right-sided location (p<0. 05). The mutation rate of hMSH2 and (or) hMLHl in male and female was 14.28% (4/28) and 17.65% (3/17) respectively, and there is no significantly difference between them (/?> 0. 05). The mutated hMSH2 and (or) hMLHl expression was noted in 2 of 15 well-differentiated cases, and in 3 of 21 moderately-differentiated cases, and in 2 of 9 poorly-differentiated cases (p>0. 05) . In addition, the mutated hMSH2 and (or) hMLHl expression was observed in 4 of 24 Dukes' A or B cases and 3 of 21 Dukes' C or D cases.3. The rate of mutated of p53 gene in sporadic colorectal carcinoma was 48.89% (22/45) ,which is significantly higher than normal mucosa. The p53 gene mutation in sporadic colorectal carcinoma is not related to tumor differentiation and clinical stage.4. The frequency of mutated MMR (hMSH2 and (or) hMLHl) in mutated p53 patients 27. 27% was significantly higher than in un-mutated p53 patients 4.35% (p<0.05) .Conclusion There is a subset of SCC displaying MMR defective. Furthermore, there is no difference between the mutation of MMR gene and clinical grade and stages. Mutated hMLHl expression is significantly higher than hMSH2, suggesting that hMLHl gene plays more important role in SCC than hMSH2 does. In addition, loss of hMSH2 and (or) hMLHl expression is more frequently associated with right-sided location. p53 gene has a high mutation rate in sporadic colorectal carcinoma, and there is a closely relationship between mutation of MMR gene and mutation of p53 gene, so MMR mutation may be prompter for p53 gene mutation.