Relative Study On Microsatellite Instability Of FHIT And Expression Of Mismatch Repair Gene In CIN And Cervical Cancer

Objective: In the world, cervical cancer is one of the most common causes of death in female malignant tumor. The infection of high risk Human papilloma virus (HPV) is one of the main nosogeneses. However, the infection of HPV can't result in cervical cancer alone, genetic change played a role in the carcinogenesis of cervical cancer. Its carcinogenesis is the result of multiple factors and steps. For the past few years, some research discovered that genetic mutation of DNA repair gene resulted in functional depression and deprivation of DNA mismatch repair system, which caused instability of genetic material, its main appearance is microsatellite instability (MSI) that led to carcinogenesis. The carcinogenesis and development involve in activation or inactivation of many genes. Except for the infection of HPV, genetic alteration contributed to the carcinogenesis of cervix, too. Up to now, little research has been paid to the collaborating study about hMLH1, hMSH2, MSI and occurrence of cervical carcinoma. The aim of my study is to investigate the MSI, negative expression of mismatch repair gene of hMLH1, hMSH2 in the cervical intraepithelial neoplasia and cervical cancer, through which to discover the important role in the cervical cancer. The study will provide new thinking of malignant transformation from CIN to cervical cancer, provide new direction of carcinogenesis and development of cervical cancer and provide theoretic basis for further exploring mechanism of cervix molecular tumorigenesis and for early diagnosis, right therapy and prognostic judgement of cervical carcinoma.Method:1 Specimens collection: 42samples CINâ… , 24samples CINâ…¡~CINâ…¢and 56 samples squamous carcinoma of the cervix were collected in the out-patient clinic and hospital room of the Affiliated Second Hospital of Hebei medical university from June, 2006 to June, 2007. A part of pathological tissue of cervix was obtained from patients of operation or biopsy and the same time a part of normal tissue of cervix of the same patient was obtained, which was used in own control. To draw venous blood 3ml in order to make own control, if the homologous normal tissue can not be obtained.We separated the pathological cervix into two parts. One part and the homologous normal tissue were thrown into liquid nitrogen and the other part was thrown into 4% paraformaldehyde. The entire specimen was confirmed by pathology. All the specimens were not received radiotherapy and chemotherapy.2 The detection of MSI: we analyzed MSI in 42 samples CINâ… , 24samples CINâ…¡~CINâ…¢and 56 samples squamous carcinoma of the cervix using the polymerase chain reaction for amplification with 2 polymorphic microsatellite repeats on chromosome 3p14.2. The DNA of cervical cancer was extracted according to the procedure of extracting box. Then DNA and homologous primer were used to amplify in PCR reaction system at different conditions. The amplified production was carried through electrophoresis at native polyacrylamide gel electrophoresis, ethidium bromide staining and the outcome was analyzed. Determination of result: Evaluation was performed by comparison with the own normal tissue, tissues were scored as exhibiting MSI (+) if there were increase, impairment or moving (the cases of MSI (+) were carried out again).3 The negative expression of hMLH1, hMSH2 protein in the different cervical tissue by immunohistology.4 Statistical method: We use SPSS13.0 to carry out statistic analysis and the condition of MSI in different pathological tissues, different locus, clinical stage and pathological grade of cervical cancer, the negative expression of hMLH1, hMSH2 protein in the CINâ… , CINâ…¡~CINâ…¢, squamous carcinoma of the cervix using Chi-Square test or Fisher's exact propability. The correlation of MSI and the negative expression of hMLH1, hMSH2 protein using spearman rank correlation. There have statistic discrepancy if P>0.05.Results:1 The condition of MSI1.1 Condition of microsatellite instability in the three types of pathological tissue of cervix In 122 different cervical tissue cases, the total frequency of MSI was 9.02% (11/122), the total frequency of MSI in 42 CINâ… cases was 0.00% (0/42), the total frequency of MSI in CINâ…¡~â…¢cases was 4.17%(1/24), the total frequency of MSI in cervical cancer cases was 17.86%(10/56). There statistic discrepancy was found among the three groups (P<0.05). There no statistic discrepancy was found between the group of CINâ… and the group of CINâ…¡~â…¢(P>0.05). Between the two groups and the group of cervical cancer, there had statistic discrepancy (P<0.05).1.2 Difference of MSI frequency between two loci of microsatelliteIn 122 different cervical tissue cases, the rate of MSI (+) on the two loci (D3S1300 and D3S1234) was 4.00% (5/122), 4.76% (6/122), respectively. In the group of CINâ…¡~â…¢, the only one case display MSI (+) on the loci of D3S1300. In the group of cervical cancer, there ten cases display MSI (+) , in which four cases on the locus of D3S1300, the others on the loci of D3S1234. There had no statistic discrepancy between the two loci (P>0.05). In the group of cervical cancer,the rate of MSI (+) on the locus of D3S1300 and D3S1234 were 7.14%(4/56) , 10.71% (6/56), respectively. There no statistic discrepancy was found between the two loci (P>0.05).1.3 Relationship between microsatellite instability and clinical stage of cervical carcinomaThere were two cases display MSI (+) in the stage I (2/22, 9.09%), there were four cases display MSI (+) in the stageâ…¡(4/28, 14.28%), there were four cases display MSI (+) in the stageâ…¢~â…£(4/6, 66.67%). The frequency of MSI showed distinguished statistic difference in different clinical stages (P<0.05). There no statistic discrepancy was found between the stageâ… and stageâ…¡(P>0.05). There had distinguished statistic discrepancy between the stageâ…¢~â…£and stageâ… ~â…¡(P<0.05).1.4 Relationship between microsatellite instability and histology differentiated types of cervical carcinomaThere were four cases display MSI (+) in 22 cases of well-differentiated squamous cell carcinoma (4/22, 18.18%), two cases display MSI (+) in 16 cases of moderately differentiated squamous cell carcinoma (2/16, 12.50%), there were four cases display MSI (+) in 18 cases of poorly differentiated squamous cell carcinoma (4/18, 22.22%). The frequency of MSI showed no statistic difference in the histology differentiated types of cervical carcinoma (P>0.05).2 The negative expression of mismatch repair gene of hMLH1, hMSH2 protein in different cervical tissue2.1 The negative expression of hMLH1 protein in different cervical tissueThe result of immunohistochemistry dyeing indicated that the negative expression of hMLH1 protein gradually augmented with the headway of the cervical lesion, the rates of negative expression of group of CINâ… , CINâ…¡~â…¢, cervical cancer were 14.3%(6/42), 25%(6/24), 42.9% (24/56), respectively. Chi- Square test indicated that the three groups had significant statistic difference(P<0.05);There had no statistic difference between the group of CINâ… and the group of CINâ…¡~â…¢(P>0.05); There had significant statistic difference between the groups of CINâ… , CINâ…¡~â…¢and the group of cervical cancer (P<0.05).2.2 The negative expression of hMSH2 protein in different cervical tissueThe result of immunohistochemistry dyeing indicated that the negative expression of hMSH2 protein gradually augmented with the headway of the cervical lesion, the rates of group of CINâ… , CINâ…¡~â…¢, cervical cancer were 11.9% (5/42), 16.7% (4/24), 28.6% (16/56), respectively. Chi-Square test indicated that the three groups had no statistic significance (P>0.05).3 The correlation of MSI and the negative expression of hMLH1, hMSH2 protein in the cervical cancer3.1 The correlation of MSI and the negative expression of hMLH1 protein in the cervical cancerNine of ten cases of MSI (+) fellowing negative expression of hMLH1. Spearman rank correlation indicated that MSI and the negative expression of hMLH1 had the positive correlation (P<0.05).3.2 The correlation of MSI and the negative expression of hMSH2 protein in the cervical cancerFour of ten cases of MSI (+) fellowing negative expression of hMSH2. Spearman rank correlation indicated that MSI and the negative expression of hMSH2 had no correlation (P>0.05).Conclusions:1 The rate of MSI(+) gradually elevated with the headway of the cervical lesion, and the rate of MSI(+) in the cervical cancer is more than in the CIN, which indicated the MSI of FHIT is the event of advanced stage in the CIN. The detection of MSI of FHIT can be regarded as an important index of reference and the detection of MSI of FHIT is helpful to the early diagnosis and screening of cervical cancer. It is significant to the prognosis of cervical cancer.2 With the advance of clinical stage of cervical carcinoma, the rate of MSI (+) became higher and higher. This indicated that MSI played a role in the development of cervical cancer and MSI is the event of nonage to the squamous cell cancer of cervix in the course of malignant advancement.3 There no relation between MSI and histology differentiated types of cervical carcinoma. This indicated MSI is the event of nonage to the squamous cell cancer of cervix.4 The negative expression of hMLH1 protein gradually augmented with the headway of the cervical lesion. There had positive correlation between MSI and the negative expression of hMLH1 protein, which be regarded as basic reason of MSI.5 The negative expression of hMSH2 gradually augmented with the headway of the cervical lesion. There had no correlation between MSI and the negative expression of hMSH2 protein and the negative expression of hMSH2 be regarded is independent with MSI, but it is still regarded as the risk factor of early cervical cancer.