Identification On The Changes Of PTC Cell’s Biological Properties By Lentivirus Vector-mediated CCDC67 Transfection

Background and ObjectiveThyroid cancer, a kind of head and neck malignant tumor, is the most common malignant tumor within endocrine system. It accounts for about 3% of malignant tumors and the incidence is higher among women. Thyroid papillary carcinoma, as the most common type of pathology in Thyroid cancer, accounts for about 90% of thyroid cancer with an ever-increasing incidence in recent years. Previous research on Thyroid cancer has made remarkable progress, but the specific mechanism of carcinogenesis is yet to be explored in depth. Tumor occurrence and development is a pathological process involving in multi-factors, multi-phases, alterations of multi genes. As gene mutation, gene recombination, the activation of oncogene, epigenetic changes and the inactivation of tumor suppressor genes play an important role in the cancerization, tumor suppressor genes in the genome and transcription of abnormal expression turns out to be one of the common causes of cancer. Research shows that if the inactivated tumor suppressor genes can be reexpressed and their functions be restored, they will inhibit the growth of tumor cells. Recent studies have shown that CCDC67 is a new candidate tumor suppressor gene which is associated with the cell signal transduction of tumor formation. The study also found CCDC67 m RNA expression was found missing or reduced in lung cancer, cervical cancer, breast cancer and other malignancies. However, expression and clinical data about CCDC67 gene in thyroid cancer have been reported rarely. Therefore, we used RT-PCR to detect the expression of CCDC67 m RNA, Western blot to detect the CCDC67 protein expression in PTC, and analyzed CCDC67’s association with clinical pathology. Then we constructed a lentiviral expression vector carrying CCDC67 to obtain efficient and stable expression of CCDC67 in TPC-1 and investigated the changes in biological properties of papillary thyroid carcinoma cell, in order to determine whether the CCDC67 gene is a PTC tumor suppressor gene, which can help us to seek a new target for cancer treatment. Material and Method:1、 RT-PCR and Western blot were used to detect the expression of CCDC67m RNA and protein in 56 cases of PTC and their matched adjacent non-carceiousepithelium(NCE).2、 RT-PCR and Western blot were used to detect the expression of CCDC67m RNA and protein in TPC- 1, SW579.3、 Construction and identification of CCDC67 gene lentiviral vector.4、 To construct a lentiviral expression vector carrying CCDC67 and obtain theefficient and stable expression of CCDC67 in TPC-1. CCK-8, Transwell cellmigratory assay, Adhesion-dependent colony-formation assy, FCM, Woundscratch assy were used to detect the function of CCDC67.5、 Statistical analyses were performed using SPSS software version 17.0, χ2 test,t-test, and one-way ANOVA were used to analyze the significance. P<0.05 wasconsidered statistically significant. Results:1、 Only 41.4%(23/56) in 56 cases of thyroid carcinoma appeared CCDC67m RNA expression,it was correlated with the tumor TNM stage, pathologicalgrade and lymph node metastasis(χ2=11.368,4.861,6.321, P<0.05). Theexpression of CCDC67 protein was down-regulated significantly, only 46.4%(26/56) in 56 cases of thyroid carcinoma appeared CCDC67 protein expression, it was correlated with the tumor TNM stage, pathological grade and lymph node metastasis(χ2=4.229, 4.313,5.540, P<0.05). Furthermore, there was a distinct correlation between the m RNA and protein expression of CCDC67 gene(χ2=25.775, P<0.01) 2、 In thyroid carcinoma cell lines, there is no expression of CCDC67 in SW579 and TPC-1. 3、 The lentiviral expression vector carrying CCDC67 was successfully constructed and CCDC67 protein expression was detected in 293 T cells. 4、 CCDC67 can suppress cell proliferation, colony formation and migration of TPC-1. Conclusions: 1、 The expression m RNA and protein of CCDC67 were down-regulated significantly in papillary thyroid carcinoma and negative in PTC cell line. It confirmed initially CCDC67 is a candidate tumor suppressor gene in PTC. 2、 Lentiviral expression vector can transfect and stable expression in TPC-1. 3、 GV208-GFP-CCDC67 can significantly promote the reversal of TPC-1 cell lines of malignant phenotype, further confirmed CCDC67 as a candidate tumor suppressor gene of PTC.