ABSTRACT
Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.
ABSTRACT
Objective To study the effect of curcumin on the radiosensitivity of the human papillary thyroid cancer cell line TPC?1, to investigate the signaling pathway probably targeted by curcumin, and to provide new insights for the development of radiosensitizers for thyroid cancer. Methods The human papillary thyroid cancer cell line TPC?1 was treated with curcumin and radioactive iodine. CCK?8 assay, colony formation assay, and flow cytometry were used to evaluate cell proliferation, colony formation ability, and cell apoptosis, respectively. Western blot was used to measure the expression of p50, p65, and apoptosis?related proteins, Bcl?2 and Bax. Cell proliferation, colony formation ability, and cell apoptosis were determined again after the activity of the NF?κB signaling pathway was blocked by a NF?κB signaling pathway inhibitor PDTC. Results After treatment with curcumin and radioactive iodine, the human papillary thyroid cancer TPC?1 cells had reduced cell proliferation and colony formation, an elevated apoptosis rate, downregulated expression of anti?apoptotic Bcl?2, and upregulated expression of pro?apoptotic Bax in a dose?dependent manner. These results indicated that curcumin enhanced the radiosensitivity of TPC?1 cells. Curcumin inhibited the activation of the NF?κB signaling pathway in the TPC?1 cells treated with radioactive iodine. When the activity of the NF?κB pathway was blocked by PDTC, cell proliferation and colony formation were reduced and the apoptosis rate was increased, indicating an enhanced radiosensitivity of TPC?1 cells. Conclusions Curcumin is likely to target the NF?κB signaling pathway. It regulates the radiosensitivity of thyroid cancer cells by inhibiting the activity of the NF?κB signaling pathway.
ABSTRACT
PURPOSE: Response to radioiodine therapy for thyroid cancer is related to the loss of sodium-iodine symporter protein caused by dedifferentiation of thyroid cancer cells. So we aimed to study mRNA expression of CD97, dedifferentiation marker, and sodium-iodine symporter after retinoic acid treatment according to retinoids receptor status. METHODS: Thyroid cancer cell lines; ARO, FRO, NPA, TPO, and FTC133 were prepared. 5µM of all trans retinoic acid were administered to each cell lines and then expression of m RNA for retinoids receptors (RARα, RARβ, RARγ, RXRα, RXRβ, RXRγ), CD97, and Sodium-Iodine symporter by RT-PCR. RESULTS: RARs and RXRs were differently expressed in each cell line. After retinoic acid treatment, relative density of retinoic acid receptor mRNA were increased by time dependently in each cell line except TPO cell line. Expression of CD97 also was decreased in every cell lines (P<0.001). Retinoic acid increased expression of sodium-iodine symporter only in FTC133 cell line (P<0.001), and TSH or forskolin did not enhance NIS expression by retinoic acid. RARβ and RXRγ were expressed only in FTC 133cell line before treatment. Induction of sodium-iodine symporter by retinoids disappeared after RARβ specific antagonist LE135 or pan RXR antagonist PA452 administration. CONCLUSION: Retinoic acid reduced expression of CD97 in five thyroid cancer cell lines. However, retinoic acid could restore sodium-iodine symporter expression in only FTC 133 cell line specifically containing RARβ and RXRγ. Restoration of sodium-iodine symporter expression by retinoic acid is related to RARβ and RXRγ expression.