The Study Of Microsatellite Alteration Of RASSF1A Gene, Mismatch Repair Enzyme And Development HPV16 In Cervical Pathological Changes

Objective:Recent cloned Ras association domain family 1A gene has been considered as a new candidated antioncogen,which is located on human chromosome 3p21.3. RASSF1A is found to be in activated in several major human cancers,which implicates its significance in carcinogenesis. Microsatelliter alterations is the main mechanism resulting in activation,which takes part in the signal trancduction of cell apoptosis,stabalization of microtubular and proceeding of mitosis,as well as inhibits the growth of cells and induces cell apoptosis as down regulatory factor of Ras and then promote the occurrence and development of tumor.MS(Imicrosatellite instability,MSI)plays an important role in the tumorigenesis and development of colorectal carcinoma. MSI means repeated sequence lose or gain in DNA genome,which is also called copy error.Up to now,there are 9 mismatch repair genes.Among them hMLH1 and hMSH2 are the most important mismatch repair genes,their principal role is keeping the integrity and stability of genetic substance and true of duplication.If these genes were defective which result in MSI and probably led to the instability of genome and tumorigenesis of tumor.High risk human papillomavirus especially HPV16 have direct bearing on the occurrence of cervical carcinomas.Investigating the presence of MSI in chronic cervicitis,CINI, CINII～CINIII,well～moderately differentiated squamous carcinoma of the cervix,poorly-differentiated squamous carcinoma of the cervix with methods of molecular biology,on the basis of this,studying the low expression of hMLH1 (human mutl homologue 1)and hMSH2 (human muts homologue 2)protein and HPV16 infection in order to investigate the relationship of MSI,MMR(hMLH1 and hMSH2) and the infection of HPV16 in cervical carcinoma.Methods: 1 Material: 119 biopsy under colposcope or surgically resected cervical pathological changes cases.There are 16 cases were chronic cervicitis ,20 cases were CINI, 31 cases were CINII~CINIII ( include noninvasive cervical carcinomas) and 52 cases were carcinoma of cervix uteri.Histological type: All were squamous carcinoma;Grading: 36 cases were well-differentiated and moderately-differentiated squamous carcinoma of the cervix,16 cases were poorly-differentiated squamous carcinoma of the cervix;Stage of disease was determined according to FIGO criteria in 2000: 37 cases were in stage I~II,15 cases were in stage III ~IV. And adjacent normal cervical tissue (If selects material time the suspicious cancer takes the specimen edge which the surgery excises)were chosen as comparision.All cases were flesh and biopsy or surgically resected,keeping in liquid nitrogen or 4% paraformaldehyde.All cases were not treated by any medical therapy and proved by pathology.2 The detection of MSI:To select 3 MSI loci: D3S2382E,RH91127 and SHGC-56838 ,detecting the presence of MSI in cervical pathological changes cases with polymerase chain reaction-single-strand conformation polymorphism(PCR-SSCP) comparing with adjacent normal tissue. The DNA of cervical cancer was extracted according to the procedure of extracting box. Then DNA and homologous primer wereused to amplify in PCR reaction system at different conditions. The amplified production was carried through electrophoresis at native polyacrylamide gel electrophoresis, silver staining and the outcome was analyzed. Determination of result: MSI was characterized by shift and/or gain of electrophoretic bands.Evaluation was performed by comparison with the own normal tissue, tissues were scored as exhibiting MSI (+)if there was a moving or change of quantity or density (the cases of MSI+ were carried out again).3 To investigate the protein of hMLH1 and hMSH2 in cervical pathological changes cases with immuno histochemical method and study the relationship between hMLH1,hMSH2 and the cervical pathological changes level and MSI of RASSF1A gene in molecular.4 HPV16 infection of their cervical tissues were detected.Polymerase chain reaction (PCR) technique was used to detect the infection state of HPV16. Using PCR method, Determination of result: Evaluation was performed by comparison with the HPV16(+).5 Statistical analysis: To detect the MSI, the negative expression of hMLH1,hMSH2 protein and the infectionstate of HPV16 in the cervical pathological changes. Data analysis was carried out by using SPSS14.0 stastistical software package, and performed using Pearson's Chi-square test and Fisher's Exact test.p < 0.05 was considered statistically significant.In the consistency of MSI,the low expression of hMLH1,hMSH2 protein and the infection state of HPV16,statistical analysis was performed by spearman rankcorrelation checking.Results: 1 The presence of MSI in cervical pathological changes cases and adjacent normal tissues: In adjacent normal tissues,no MSI was found.But in 119 cervical pathological changes cases (16 chronic cervicitis cases,20 CINI cases,31 CINII~CINIII cases , 36 well-differentiated squamous carcinoma and moderately-differentiated squamous carcinoma of the cervix cases , 16 poorly -differentiated squamous carcinoma of the cervix cases,including 37 stageI ~stageII cases,15 stage III~stage IV cases ),the expression of MSI are follow: D3S2832E : 0,0.0%,22.6%,13.9%,43.8%,including stageI ~stageII13.5%(5/37),stage III~stage IV 46.7%;RH91127: 0,0.0%,19.4%,11.1%,43.8%,including stageI ~stageII 13.5%,stage III~stage IV 40.0%;SHGC-56838 : 0,0.0%,19.4%,11.1%,43.8%,including stageI ~stageII 10.8%,stage III~stage IV 46.7%.We checked microsatellite alteration at D3S2832E,RH91127 and SHGC-56838 using SPSS14.0 Chi-square (χ2-test) and Fisher exact probabities,Comparing chronic cervicitis cases with CIN,CIN with cervical carcinoma ,the difference of each group had no statistically significant(P > 0.05). Comparing cervical carcinoma with chronic cervicitis,CINII~CINIII with CINI ,well-differentiated and moderately-differentiated squamous carcinoma of the cervix cases with poorly-differentiated squamous carcinoma of the cervix cases,stageI ~stageII with stage III~stage IV,the difference of each group had marked significance(P<0.05).2 The low expression rate of hMLH1 and hMSH2 in adjacent normal tissues and cervical pathological changes cases.In 119 cervical pathological changes cases(16 chronic cervicitis cases,20 CINI cases,31 CINII~CINIII cases,36 well-differentiated squamous carcinoma and moderately -differentiated squamous carcinoma of the cervix cases ,16 poorly-differentiated squamous carcinoma of the cervix cases,including 37 stageI ~stageII cases,15 stage III~stage IV cases ), the rates of low expression hMLH1were: 18.8%,20.0%, 48.4%,44.4%,75%,including stageI ~stageII 43.2%,stage III~stage IV 80.0%,and the rates of hMSH2 were 12.5%,10.0%,41.9%,27.8%,62.5%, including stageI ~stageII 24.3%,stage III~stage IV73.3%. Comparing cervical carcinoma with chronic cervicitis, CINII~CINIII with CINI,well-differentiated and moderately -differentiated squamous carcinoma of the cervix cases with poorly -differentiated squamous carcinoma of the cervix cases,stageI ~stageII with stage III~stage IV,the difference of each group had marked significance(P<0.05).3 The positive rate of HPV16 in patients with cervical cancer was significantly increased than that in patients with CIN or cervisitis tissues,and the difference of each group no significance(P>0.05).But between CINII~CINIII with CINI the difference of this group had marked significance(P<0.05).4 Relationship of MSI ,MMR(hMLH1 and hMSH2) and HPV16HPV16 DNA was detected in 16 of 1 (6.3%) cases of cervicitis, the rates of MSI and MMR were 0 and 31.5% . In CIN HPV16 DNA was detected in 22 of 51 (43.1%), the rates of MSI and MMR were 37.3% and 66.7%. In cervical cancer HPV16 DNA was detected in 37 of 52 (71.2%), the rates of MSI and MMR were 65.4% and 92.3%. The rates of the three groups were all higher in cervical cancer than in CIN and cervicitis. Spearman rank correlation checking indicated the consistency is satisfactorily between MSI ,HPV16 DNA and the negative expression of MMR ,the result is r=1,P=0.002,it is well correlation.Conclusion: 1. MSI probably is the paths in the tumorigenesis of genesis of cervical carcinoma.That means there would be a type cervical carcinoma by the path of MSI. 2. In cervical carcinoma, 3 microsatellite loci (D3S2832E, RH91127 and SHGC-56838 ) were sensitive in the investigation of MSI. 3. In tumorigenesis and development of MSI cervical carcinoma ,there were probably low expression of hMLH1 and hMSH2 in the meantime.Suspecting the low expression of HMLH1 and hMSH2 were important mechanism in MSI cervical carcinoma,but the exact mechanism of cervical carcinoma pathway needed to be studied.4. The positive rate of HPV16 in patients with cervical cancer was significantly increased than that in patients with CIN or normal cervical tissues. 5. In MSI ,HPV16 DNA and the negative expression of MMR ,it is well correlation. The rates of the three groups were all higher in cervical cancerthan in CIN and cervicitis...