Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes

Lele Zhang; Longlong Wang; Mengyuan Lei; Runsheng Ma; Fangqin Yu; Chenguang Liu; Detao Yin

doi:10.3892/ol.2019.10839

Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes

Oncol Lett. 2019 Nov;18(5):4495-4502. doi: 10.3892/ol.2019.10839. Epub 2019 Sep 10.

Authors

Lele Zhang^{1

2}, Longlong Wang^{1

2}, Mengyuan Lei^{1

2}, Runsheng Ma^{1

2}, Fangqin Yu^{1

2}, Chenguang Liu^{1

2}, Detao Yin^{1

2}

Affiliations

¹ Department of Thyroid Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.
² Department of Thyroid Surgery, Key Discipline Laboratory of Clinical Medicine of Henan, Zhengzhou, Henan 450050, P.R. China.

Abstract

Coiled-coil domain containing 67 (CCDC67) gene is a tumor suppressor gene that exhibits a significant inhibitory effect on a variety of tumors. Our previous study demonstrated that the upregulation of CCDC67 gene in TPC-1 cells inhibited cell proliferation, migration and invasion, and promoted apoptosis in vitro. However, due to the lack of a suitable cell tool, these results were not validated in vivo. In the present study, a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated and identified. Firstly, cDNA clones of the CCDC67 gene were obtained by reverse transcription using a custom-designed primer. The results of subsequent electrophoresis analysis and sequencing revealed that the cDNA clones of CCDC67 gene were obtained successfully, with a length of 1,862 bp. The lentiviral vectors, containing the CCDC67, luciferase reporter and puromycin acetyltransferase genes, were co-transfected with two plasmids that encode lentiviral structural proteins and envelope proteins into 293T cells. Following ultracentrifugation, the titer of lentivirus was determined by ELISA to be 5.0×10⁸ TU/ml. The constructed lentiviral vector was used to transfect TPC-1 thyroid cancer cells, and stabilization was achieved by puromycin screening. The expression of CCDC67 gene, luciferase activity and tumorigenic ability of the generated cell line were detected. Reverse transcription-qPCR results demonstrated that the expression levels of CCDC67 gene in TPC-1 cells following transfection were increased 194,46.782-fold compared with those in the negative control group (P<0.01). A higher fluorescence intensity was detected in the generated cell line, while no detectable fluorescence was observed in untransfected TPC-1 cells. The tumorigenic ability of TPC-1-Luc-Puromycin-CCDC67 cells was verified by bioluminescence imaging and histopathological analysis using a pulmonary metastasis model. These results demonstrated that a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes was generated successfully. The TPC-1-Luc-Puromycin-CCDC67 cell line may be a helpful tool for further research on CCDC67 in vivo.

Keywords: TPC-1; coiled-coil domain containing 67; luciferase reporter gene; puromycin; thyroid cancer.