| Objectives ZNF703 is a newly discovered member of the NET family.It is believed that ZNF703 mainly acts as a negative transcription factor,activates cell cycle related gene transcription and promotes cell proliferation.In this study,RNA interference was used to silence thyroid carcinoma K1 cells.The expression of ZNF703 gene was detected by detecting the expression of ZNF703 gene in thyroid papillary carcinoma K1 cells and its effect on K1 cell proliferation and apoptosis.Thyroid cancer gene targeting therapy provides new research directions.Methods 1 RNAi technique down-regulates the expression of ZNF703 to screen out the most effective ZNF703-siRNA sequence and the optimal conditions for cell transfection:K1 cells were divided into 5 groups: blank control group,negative control group,transfection group,(ZNF703-siRNA-2,ZNF703-siRNA-3)targeting the human ZNF703 gene was transfected into the papillary thyroid papilla Cancer K1 cells to determine the optimal viral transfection sequence as well as MOI values.Real-time PCR technique was applied to explore the utterance of ZNF703 m RNA in each group,then filter the effective siRNA for the following experiment.2 Effects of ZNF703 gene silencing on protein expression of K1 cells in papillary thyroid carcinoma: K1 cells were divided into 3 groups.The expression level of protein in blank control group,negative control group and transfection group was detected by immunoblotting.3 Effects of ZNF703 gene silencing on proliferation,apoptosis and invasion of thyroid papillary carcinoma K1 cells: MTT,flow cytometry,Hoechst 33342 staining and Transwell invasion assay were used to detect the control group,transfected negative carrier control group,transfected positive vector group apoptosis and invasion.4 Statistical analysis: all the experiments in this study were repeated 3 times.The experimental data which belongs to normal distribution were analyzed by SPSS 17.0 statistical software,and the single factor analysis of variance between the two groups.The results were expressed as mean ± standard deviation((?)±s).The test level is set to ? = 0.05,P<0.05 indicates that the difference is statistically significant.Results 1 Optimization of transfection efficiency and optimal siRNA screening:Immunofluorescence results showed that transfection efficiency was the best when transfected with K1 cells with different infection complexes in lentivirus transfection,respectively.The transfection efficiency was the best at transfection efficiency > 80%.The relative expression levels of ZNF703-siRNA-1,ZNF703-siRNA-2 and ZNF703-siRNA-3m RNA were 0.14±0.13,0.49±0.08 and 0.55±0.07 P<0.05)after transfection of KN cells with KNF703-siRNA for 72 h,indicating that all three ZNF703-siRNAs had their effects,especially ZNF703-siRNA1 was the best,and there was no significant difference between the blank group and the negative carrier group(P>0.05),the difference has obvious statistically implication.The expression of ZNF703 m RNA in ZNF703-siRNA1 group was lower than that in blank control group(81.60±2.03)%(P<0.05),while there are no significant difference for above indice between control group and negative vector group(P>0.05).Follow-up experiments were carried out to use this interference sequence.2Protein expression level were detected by Western-Blot:We compare the blank control group and transfected negative carrier control group simultaneously,ZNF703 expression was significantly decreased after the infection for 48 h,and the quantitive analysis in three groups above showed 0.295±0.015,0.843±0.054 and 0.815±0.034 respectively(P<0.05),the difference is statistically significant,however,the one between blank control group and negative control group got no statistically significance(P> 0.05).3 MTT assay showed that MTT assay was performed at 24 h,48 h and 72 h after transfection.Compared with the control group,the growth inhibition was observed at 24 h after transfection,and the inhibitory effect was 48.3%(P<0.05).But there are no significant difference for above indice between control group and negative vector group(P>0.05).4 FCM and Hoechst33342 staining.After 48 hours of transfection,FCM results showed that the apoptotic rate of the transfected group was remarkable higher than blank control group and transfected negative carrier control group(15.32 ± 0.18(P<0.05).There was no significant difference in the apoptotic rate between the blank group and the negative control group(P> 0.05).The results of Hoechst 33342 staining showed that the difference was not significant(P<0.05)(3.49±0.07)%,(4.84±0.19)%,(34.50±0.55)%(P<0.05),and the apoptotic rates of the two groups were significantly higher than those in the control group Consistency.5Transwell detection of cell invasion: Compared with the blank control group,negative control group,transferred into the ZNF703-sh RNA K1 cell invasion number decreased significantly,the blank control group,negative control group,transfection group,the number of transmembrane cells were respectively 138.33±2.08?133.33±3.21?62.67±2.51(P<0.05).There was no evident difference between the blank control group and transfected negative carrier control group(P> 0.05).Conclusions In vitro experiments confirmed that transfection of ZNF703-siRNA significantly down-regulated the expression of ZNF703 m RNA in thyroid carcinoma K1 cells by lentivirus interference,considerable restrained the invasion and diffusion of K1 cells and could significantly increase the apoptotic rate.Suggesting that ZNF703-siRNA is involved in the development of thyroid cancer. |
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