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 detect the expression of long non-coding RNA (lncRNA) CCAT1 in human papillary thyroid cancer, and to observe the effect of CCAT1 down-regulation on the invasion and migration of human papillary thyroid cancer.Methods The expression of CCAT1 was detected in human normal thyroid Nthy-ori 3-1 cells and human papillary thyroid cancer TPC-1 cells.CCAT1 siRNA plasmid was transfected into TPC-1 cells.The effect of CCAT1 down-regulation on cell invasion and migration was observed by Transwell chamber assay and scratch test, and the expressions of BRAF, MUC15 and RKIP proteins were detected by Western blot.Results The level of CCAT1 in human papillary thyroid cancer TPC-1 cells was significantly higher than that in human normal thyroid Nthy-ori 3-1 cells.CCAT1 down-regulation significantly inhibited the invasion and migration of TPC-1 cells.The Transwell invasion assay revealed that the number of migrated TPC-1 cells in the CCAT1 down-regulation group was significantly lower than that in the control group.The scratch test showed an increased distance between cells in the CCAT1 down-regulation group compared to the control group, suggesting a reduced cell motility.The expressions of BRAF and MUC15 proteins were decreased in the CCAT1 down-regulation group, while that of RKIP protein was increased.Conclusions The expression of CCAT1 in papillary thyroid cancer cells is significantly higher than that in normal human thyroid cells.Down-regulation of CCAT1 in papillary thyroid cancer cells may inhibit the cell invasion and migration by regulating the expression of BRAF, MUC15 and RKIP proteins.