Open Access

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

  • Authors:
    • Lele Zhang
    • Longlong Wang
    • Mengyuan Lei
    • Runsheng Ma
    • Fangqin Yu
    • Chenguang Liu
    • Detao Yin
  • View Affiliations

  • Published online on: September 10, 2019     https://doi.org/10.3892/ol.2019.10839
  • Pages: 4495-4502
  • Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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 5.0x108 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.
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November-2019
Volume 18 Issue 5

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Spandidos Publications style
Zhang L, Wang L, Lei M, Ma R, Yu F, Liu C and Yin D: Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes. Oncol Lett 18: 4495-4502, 2019.
APA
Zhang, L., Wang, L., Lei, M., Ma, R., Yu, F., Liu, C., & Yin, D. (2019). Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes. Oncology Letters, 18, 4495-4502. https://doi.org/10.3892/ol.2019.10839
MLA
Zhang, L., Wang, L., Lei, M., Ma, R., Yu, F., Liu, C., Yin, D."Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes". Oncology Letters 18.5 (2019): 4495-4502.
Chicago
Zhang, L., Wang, L., Lei, M., Ma, R., Yu, F., Liu, C., Yin, D."Generation and identification of a thyroid cancer cell line with stable expression of CCDC67 and luciferase reporter genes". Oncology Letters 18, no. 5 (2019): 4495-4502. https://doi.org/10.3892/ol.2019.10839