Construction of the INTU-siRNA Plasmid and Screening in Human Thyroid Nthy-ori-3-1 Cells
Objective:The target-specific INTU-siRNA plasmids were constructed and transfected into Human thyroid Nthy-ori-3-1 cells to establish the Nthy-ori-3-1 cell model of silenced-INTU.This model provides a suitable cell model for exploring the pathogenesis of Graves'disease.Methods:Four pairs of human INTU sequences(numbered INTU-29,INTU-31,INTU-33,INTU-35)siRNA plasmids and an empty vector plasmid were constructed and individually transfected into Nthy-ori-3-1 cells,which were then cul-tured for 24 hours.The fluorescence expression of each group was observed using a Fluorescent Inverted Microscope,and the fluores-cence intensity was detected by a Multi-function Measuring Instrument.The silencing efficiency of the INTU gene was determined by fluorescence quantitative PCR and Western blotting.Finally,the most effective silenced-INTU plasmid was identified through screening.Results:After the empty vector and the four INTU-siRNA plasmids were transfected into Nthy-ori-3-1 cells for 24 hours,the results of Fluorescent Inverted Microscope and Multi-function Measuring Instrument indicated that the four INTU-siRNA groups showed green flu-orescence expression,and their fluorescence intensity was significantly higher than the normal group(P＜0.01),suggesting that the plas-mids were successfully transfected.The results of fluorescence quantitative PCR demonstrated that the relative mRNA expression of the INTU gene in four INTU-siRNA groups was significantly reduced compared with the normal group and the empty vector group(P＜0.01),with the most significant reduction of the INTU-35 group decreased by 61.02％.The results of Western blotting showed that compared to the normal group and the empty vector group,the INTU-29 and the INTU-31 group did not exhibit a significant decrease,while the IN-TU-33 and INTU-35 groups showed a remarkable decline(P＜0.05,P＜0.01),with the protein expression of the INTU-35 group reduced bv 44.54％.These results suggest that the INTU-35 group achieved the most effective silencing of the INTU gene.Conclusions:In this study,the INTU-siRNA plasmids were successfully constructed and transfected into human thyroid Nthy-ori-3-1 cells.The construction of the INTU-siRNA thyroid cell model lays the experimental foundation for further exploring the pathogenesis of Graves'disease.
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