The Expression Of Tumor Metastasis-Associated Genes S100A6, Rab5a In Cervical Cancer And Its Clinical Significance

Objective Diagnosis and treatment of lymph node metastasis of cervical cancer is key affecting factor of treatment. In order to study the mechanism of lymph node metastasis, we selected tumor metastasis-related genes of S100A6, Rab5a, to detect these two genes at the mRNA and protein level in the group of benign cervical tissues (hysterectomy were performed in patients suffering from myoma of uterus), the group of cervical squamous cell carcinoma without lymph node metastasis (operable patients with stageⅠa～Ⅱb squamous cell cervical cancer), the group of cervical squamous cell carcinoma with lymph node metastasis (operable patients with stageⅠa～Ⅱb squamous cell cervical cancer). It's purpose is to understand the impact of S100A6, Rab5a to cervical squamous cell carcinoma and it's clinical significance.Methods Collections of cervical tissues are divided into three groups, group A (control):cervical benign lesions (hysterectomy were performed in patients suffering from myoma of uterus); group B:cervical squamous cell carcinoma without lymph node metastasis (operable patients with stageⅠa～Ⅱb squamous cell cervical cancer); group C:cervical squamous cell carcinoma with lymph node metastasis (operable patients with stageⅠa～Ⅱb squamous cell cervical cancer).1. Using trizol to extract total RNA from cervical tissues of three groups in one step, reverse transcription to cDNA; 2. Design primer of S100A6 and Rab5a specific fragment, after PCR, amplification, purification, recycling and a series of steps, specific fragment was linked with DH-5a to obtain plasmid of S100A6 and Rab5a specific fragment;3. Using real-time fluorescence quantitative PCR to detect the expressions of S100A6 and Rab5a mRNA in three groups;4. Detecting S100A6 and Rab5a protein of three groups by immunohistochemical technique, simultaneous to detect these two proteins of primary tumor and metastatic lymph node of group C;5. Record and collate data, using SPSS 13.0 software to analyze the Results.Results1. Successfully extracted the total RNA of cervical tissues,there are clearly strips of 28S,18S and 5S by 1% agarose electrophoresis. Using Takara's RT-kit, RNA reverse transcription to cDNA successfully, results to observe the effect of clear and bright electrophoresis strip;2. Results of the specific sequence alignment and sequencing showed that we successfully built a S100A6-specific fragment plasmid;3. Results of the specific sequence alignment and sequencing showed that we successfully built a Rab5a-specific fragment plasmid;4. Using real-time fluorescence quantitative PCR to detect expression levels of S100A6 mRNA in cervical tissues of three groups. In 13 cases of group A,10 cases had the positive expression, positive rate was 76.9%; In 25 cases of group B,22 cases had the positive expression, positive rate was 88%; In 25 cases of group C,23 cases had the positive expression, positive rate was 92%. No statistic differences existed among the rates of the three groups CP>0.05). But there were significant statistical differences in the quantity of mRNA expression in three groups (P<0.05);5. Using real-time fluorescence quantitative PCR to detect expression levels of Rab5a mRNA in cervical tissues of three groups. In 13 cases of group A,11 cases had the positive expression, positive rate was 84.6%; In 25 cases of group B,22 cases had the positive expression, positive rate was 88%; In 25 cases of group, C,23 cases had the positive expression, positive rate was 92%. No statistic difference existed among the rates of the three groups (P>0.05). But there were significant statistical differences in the quantity of mRNA expression in three groups (P<0.05);6. Immunohistochemical techniques were used to detect the gene expression of S100A6 among the three groups. In 10 cases of group A,1 case had the positive expression, positive rate was 10%; In 30 cases of group B,7 cases had the positive expression, positive rate was 23.3%; In 30 cases of group C,16 primary tumors had the positive expression, positive rate was 53.3%,11 metastatic lymph nodes had the positive expression, positive rate was 36.7%. The differences of S100A6 protein expression between primary tumors and metastatic lymph nodes in group C was not significant (P>0.05). The difference between group A and B was not significant (P>0.05) and differences between group A and C, group B and C were significant (P<0.05);7. Immunohistochemical techniques were used to detect the gene expression of Rab5a among the three groups. In 10 cases of group A, no case had the positive expression, positive rate was 0%; In 30 cases of group B,4 cases had the positive expression, positive rate was 13.3%; In 30 cases of group C,11 primary tumors had the positive expression, positive rate was 36.7%,1 metastatic lymph node had the positive expression, positive rate was 3.3%.The difference of Rab5a protein expression between primary tumors and metastatic lymph nodes in group C was significant (P<0.05). The difference between group A and B was not significant (P>0.05) and differences between group A and C, group B and C were significant (P<0.05).Conclusions1. The up-regulation of S100A6 mRNA was positively corelated with malignant degree, development and metastasis of cervical squamous cell carcinoma;2. The up-regulation of Rab5a mRNA was positively corelated with the pathogenesis, development and metastasis of cervical squamous cell carcinoma;3. The expression of S100A6 protein in cervical squamous cell carcinoma was increased,had the positive relationship with the malignant degree and lymph node metastasis of cervical squamous cell carcinoma;4. The expression of Rab5a protein had the positive relationship with the malignant degree and lymph node metastasis of cervical squamous cell carcinoma;5. The overexpression of S100A6 and Rab5a may be linked to the pathogenesis, development and metastasis of cervical squamous cell carcinoma. They could be taken as an index for evaluating the infiltration, metastasis and prognosis of cervical squamous cell carcinoma.6. The expression of S100A6 and Rab5a proteins had the positive relationship with the malignant degree and lymph node metastasis of cervical squamous cell carcinoma. They could be taken as an index for evaluating the infiltration, metastasis and prognosis of cervical squamous cell carcinoma.