| Background and Objective Thyroid cancer, one of the most common tumor high-risk endocrine system tumor, showed a trend of rising in people. Pathological types of thyroid carcinoma include: differentiated thyroid cancer, undifferentiated thyroid cancer, and derived from the follicular C cells of medullary thyroid carcinoma. In well differentiated thyroid cancer, it contains thyroid papillary carcinoma and follicular thyroid carcinoma. And well differentiated thyroid cancer, which accounts for more than 90% of all thyroid cancer, is a common endocrine malignancy and diversification. The study found that the majority of differentiated thyroid cancer can be cured, but the disease recurrence rates are relatively high, around 10% ~30%, 5% of the patients on conventional treatment are incurable. For those patients with thyroid cancer, we need to find new treatments. In the past 20 years, thyroid cancer research has made significant development of molecular biology, the study found that a large number of genetic mutations associated with thyroid cancer occurrence and development. Therefore, we need to further understand the mechanism and the influence of these in tumor and clinical relevance. According to a recent study shows that CCDC67 gene is a new candidate tumor suppressor gene, and found its expression in malignant tumor and so on in gastric cancer, breast cancer is absent or reduced, but its research in thyroid carcinoma are rarely reported.We take real-time fluorescent quantitative PCR technology to test the CCDC67 mRNA expression in differentiated thyroid cancer cells. Then we use cell immunofluorescence technique to detect CCDC67 protein expression in differentiated thyroid cancer. And we collected clinical histological specimens thyroid cancer, through immunohistochemical technology to detect CCDC67 gene expression in clinical patients with thyroid cancer, combined with the clinical pathological characteristics analysis of the correlation. Finally, to explore CCDC67 gene in thyroid tumor molecular mechanism, we extracted the DNA in the sample, through DNA sequencing technology to detect CCDC67 thyroid carcinoma with classical genetic BREAF V600 E correlation, with slow virus transfection papillary thyroid cancer cells, then makes the cells of CCDC67 expression, and analyzed before and after transfection of thyroid papillary carcinoma in the changes of the apoptosis related factors. The role of these genes to determine CCDC67 in thyroid tumors, CCDC67 gene is can be a tumor suppressor genes, thyroid cancer and genetics targeted therapy may provide the theory basis.Material and Method(1)Real-Time PCR was used to detect the expression of CCDC67 mRNA in differentiated thyroid cancer cells(TCP-1, FTC-133), the normal thyroid cell lines(Nthy-ori- 3-1) was negative control.(2)Cell immunofluorescence was to detect the expression of CCDC67 protein in differentiated thyroid cancer cells(TCP-1, FTC-133), the normal thyroid cell lines(Nthy-ori- 3-1) was negative control.(3)Immunohistochemical was used to detect the protein of 60 cases of thyroid carcinoma clinical histological specimens and to analyze the correlation with clinical pathology.(4)DNA sequencing technology used was to detect CCDC67 gene in thyroid cancer classic BREAF V600 E relevance.(5)To construct a lentiviral expression vector carrying CCDC67 and to obtain CCDC67 efficient and stable expression in TPC-1. Then Real-Time PCR was used to detecte the expression of Bcl-2, Bax mRNA in TPC-1 in which CCDC67 stable expression.(6)Statistical analyses were performed using SPSS 21.0, t-test, and one-way ANOVA were used to analyze the significance. And P<0.05 was proved statistically significant.Results(1) CCDC67 mRNA was down-regulated in differentiated thyroid cancer cells(TPC-1, FTC- 133, P < 0.05).(2) CCDC67 protein was down-regulated in differentiated thyroid cancer cells(TPC-1, FTC- 133, P < 0.05). CCDC67 protein distributed in cytoplasm and nuclei.(3) There is 52%(31/60) CCDC67 protein expression is missingin the PTC, and it was correlated with TNM stage, pathological grade and lymph node metastasis in patient(P < 0.05)(4) There was a strong association between the down-regulation of CCDC67 expression and the BRAF mutation, r = 0.707, P < 0.05.(5) CCDC67 gene can promote cell apoptosis.Conclusion The expression of CCDC67 mRNA and CCDC67 protein were down-regulation in PTC. And the down-regulation of CCDC67 expression and the BRAF mutation is strong association, and CCDC67 gene can promote cell apoptosis. The results confirmed CCDC67 gene as a papillary carcinoma of thyroid tumor suppressor genes. |
