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

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

doi:10.3892/ol.2019.10839

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
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Affiliations: Department of Thyroid Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China
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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1	Yu Y, Yu X, Fan C, Wang H, Wang R, Feng C and Guan H: Targeting glutaminase-mediated glutamine dependence in papillary thyroid cancer. J Mol Med (Berl). 96:777–790. 2018. View Article : Google Scholar : PubMed/NCBI
2	Hardin H, Helein H, Meyer K, Robertson S, Zhang R, Zhong W and Lloyd RV: Thyroid cancer stem-like cell exosomes: Regulation of EMT via transfer of lncRNAs. Lab Invest. 98:1133–1142. 2018. View Article : Google Scholar : PubMed/NCBI
3	Raue F and Frank-Raue K: Thyroid cancer: Risk-stratified management and individualized therapy. Clin Cancer Res. 22:5012–5021. 2016. View Article : Google Scholar : PubMed/NCBI
4	Nikitski AV, Rominski SL, Wankhede M, Kelly LM, Panebianco F, Barila G, Altschuler DL and Nikiforov YE: Mouse model of poorly differentiated thyroid carcinoma driven by STRN-ALK fusion. Am J Pathol. 188:2653–2661. 2018. View Article : Google Scholar : PubMed/NCBI
5	Yang J, Li LF, Zhang XM, Xu Q, Zhang J, Weng WW and Dong MJ: Unusual synchronous skeletal muscle and lung metastasis in papillary thyroid cancer: A case report and review of the literature. Oncol Lett. 9:727–730. 2015. View Article : Google Scholar : PubMed/NCBI
6	Bruglia M, Palmonella G, Silvetti F, Rutigliano P, Criante P, Marmorale C, Boscaro M and Taccaliti A: Skin and thigh muscle metastasis from papillary thyroid cancer. Singapore Med J. 50:e61–e64. 2009.PubMed/NCBI
7	Luo Q, Luo QY, Sheng SW, Chen LB, Yu YL, Lu HK and Zhu RS: Localization of concomitant metastases to kidney and erector spinae from papillary thyroid carcinoma using (131)I-SPECT and CT. Thyroid. 18:663–664. 2008. View Article : Google Scholar : PubMed/NCBI
8	American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, ; Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, Mazzaferri EL, McIver B, Pacini F, et al: Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 19:1167–1214. 2009. View Article : Google Scholar : PubMed/NCBI
9	Park SJ, Jang HR, Kim M, Kim JH, Kwon OH, Park JL, Noh SM, Song KS, Kim SY, Kim YH and Kim YS: Epigenetic alteration of CCDC67 and its tumor suppressor function in gastric cancer. Carcinogenesis. 33:1494–1501. 2012. View Article : Google Scholar : PubMed/NCBI
10	Yu YP, Ding Y, Chen Z, Liu S, Michalopoulos A, Chen R, Gulzar ZG, Yang B, Cieply KM, Luvison A, et al: Novel fusion transcripts associate with progressive prostate cancer. Am J Pathol. 184:2840–2849. 2014. View Article : Google Scholar : PubMed/NCBI
11	Chen ZH, Yu YP, Zuo ZH, Nelson JB, Michalopoulos GK, Monga S, Liu S, Tseng G and Luo JH: Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene. Nat Biotechnol. 35:543–550. 2017. View Article : Google Scholar : PubMed/NCBI
12	Yin DT, Xu J, Lei M, Li H, Wang Y, Liu Z, Zhou Y and Xing M: Characterization of the novel tumor-suppressor gene CCDC67 in papillary thyroid carcinoma. Oncotarget. 7:5830–5841. 2016.PubMed/NCBI
13	Sekiguchi Y, Owada J, Oishi H, Katsumata T, Ikeda K, Kudo T and Takahashi S: Noninvasive monitoring of β-cell mass and fetal β-cell genesis in mice using bioluminescence imaging. Exp Anim. 61:445–451. 2012. View Article : Google Scholar : PubMed/NCBI
14	Tu SH, Hsieh YC, Huang LC, Lin CY, Hsu KW, Hsieh WS, Chi WM and Lee CH: A rapid and quantitative method to detect human circulating tumor cells in a preclinical animal model. BMC Cancer. 17:4402017. View Article : Google Scholar : PubMed/NCBI
15	Feng L, Jia X, Zhu M, Chen Y and Shi F: Chemoprevention by Prunella vulgaris L. extract of non-small cell lung cancer via promoting apoptosis and regulating the cell cycle. Asian Pac J Cancer Prev. 11:1355–1358. 2010.PubMed/NCBI
16	Kato H, Wakabayashi H, Naito Y, Kato S, Nakagawa T, Matsumine A and Sudo A: Anti-tumor necrosis factor therapy inhibits lung metastasis in an osteosarcoma cell line. Oncology. 88:139–146. 2015. View Article : Google Scholar : PubMed/NCBI
17	Boivin GP, Bottomley MA, Schiml PA, Goss L and Grobe N: Physiologic, behavioral, and histologic responses to various euthanasia methods in C57BL/6NTac male mice. J Am Assoc Lab Anim Sci. 56:69–78. 2017.PubMed/NCBI
18	Randle RW, Balentine CJ, Leverson GE, Havlena JA, Sippel RS, Schneider DF and Pitt SC: Trends in the presentation, treatment, and survival of patients with medullary thyroid cancer over the past 30 years. Surgery. 161:137–146. 2017. View Article : Google Scholar : PubMed/NCBI
19	Pstrag N, Ziemnicka K, Bluyssen H and Wesoły J: Thyroid cancers of follicular origin in a genomic light: In-depth overview of common and unique molecular marker candidates. Mol Cancer. 17:1162018. View Article : Google Scholar : PubMed/NCBI
20	Bath SC, Pop VJ, Furmidge-Owen VL, Broeren MA and Rayman MP: Thyroglobulin as a functional biomarker of iodine status in a cohort study of pregnant women in the United Kingdom. Thyroid. 27:426–433. 2017. View Article : Google Scholar : PubMed/NCBI
21	Xing M: BRAF mutation in thyroid cancer. Endocr Relat Cancer. 12:245–262. 2005. View Article : Google Scholar : PubMed/NCBI
22	Liu X, Bishop J, Shan Y, Pai S, Liu D, Murugan AK, Sun H, El-Naggar AK and Xing M: Highly prevalent TERT promoter mutations in aggressive thyroid cancers. Endocr Relat Cancer. 20:603–610. 2013. View Article : Google Scholar : PubMed/NCBI
23	Basolo F, Pisaturo F, Pollina LE, Fontanini G, Elisei R, Molinaro E, Iacconi P, Miccoli P and Pacini F: N-ras mutation in poorly differentiated thyroid carcinomas: Correlation with bone metastases and inverse correlation to thyroglobulin expression. Thyroid. 10:19–23. 2000. View Article : Google Scholar : PubMed/NCBI
24	Dwight T, Thoppe SR, Foukakis T, Lui WO, Wallin G, Höög A, Frisk T, Larsson C and Zedenius J: Involvement of the PAX8/peroxisome proliferator-activated receptor gamma rearrangement in follicular thyroid tumors. J Clin Endocrinol Metab. 88:4440–4445. 2003. View Article : Google Scholar : PubMed/NCBI
25	Marini F, Falchetti A, Del Monte F, Carbonell Sala S, Tognarini I, Luzi E and Brandi ML: Multiple endocrine neoplasia type 2. Orphanet J Rare Dis. 1:452006. View Article : Google Scholar : PubMed/NCBI
26	Zhang Y, Wang S, Hu B, Zhao F, Xiang P, Ji D, Chen F, Liu X, Yang F, Wu Y, et al: Direct detection of Helicobacter pylori in biopsy specimens using a high-throughput multiple genetic detection system. Future Microbiol. 11:1521–1534. 2016. View Article : Google Scholar : PubMed/NCBI
27	Cheng L, Du C, Murray D, Tong X, Zhang YA, Chen BP and Hawley RG: A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells. Gene Ther. 4:1013–1022. 1997. View Article : Google Scholar : PubMed/NCBI
28	Fischer M, Haase I, Simmeth E, Gerisch G and Müller-Taubenberger A: A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium. FEBS Lett. 577:227–232. 2004. View Article : Google Scholar : PubMed/NCBI
29	Kao TH, Chen Y, Pai CH, Chang MC and Wang AH: Structure of a NADPH-dependent blue fluorescent protein revealed the unique role of Gly176 on the fluorescence enhancement. J Struct Biol. 174:485–493. 2011. View Article : Google Scholar : PubMed/NCBI
30	Cissell KA, Rahimi Y, Shrestha S, Hunt EA and Deo SK: Bioluminescence-based detection of microRNA, miR21 in breast cancer cells. Anal Chem. 80:2319–2325. 2008. View Article : Google Scholar : PubMed/NCBI