Effect Of HDAC4 On The Protein Of P27kip1 And Its Phosphorylation In Cells

HDAC4 belongs to class IIa histone deacetylase,which is mainly expressed in the heart,brain and skeletal muscle,and it plays an important role in the growth and development of the body,as well as the generation and development of the disease.It has been reported that HDAC4 can affect cell senescence by interacting with transcription factors or other proteins,but the relationship between HDAC4 and p27kip1 or phosphorylation of p27kip1 needs to be clarified.p27kip1 is a kind of CDKIs,which can hinder the cell cycle arrest in the G1 phase,thereby affecting cell growth and development.In this study,we detected the change of p27kip1 after CR treatment,ensuring the relationship between CR and p27kip1.In addition,we further investigate the effect of HDAC4 on the distribution of p27kip1 and p27kip1 phosphorylation and clarify the regulatory role of HDAC4 in the growth and development,providing an effective target for future research on the treatment and drug development of diseases related to aging.In this study,we first detected the expression of p27kip1 and its nuclear distribution in HEK293 cells by semi-quantitative PCR,real-time quantitative PCR and Western blot,at the same time,we detected the cell viability by cck-8 assay.In addition,to determine the expression and nuclear distribution of p27kip1 under the actin of HDAC4 overexpression,we over-expressed HDAC4 in HEK293 and SH-SY5 Y cells,and then we detected the expression of p27kip1 and p27kip1(T198,S10,T187,157)protein by semi-quantitative analysis,real-time quantitative PCR and Western blot.we also detected the cell viability by cck-8 assay.Results:(1)CR treatment affected the mRNA and protein level of p27kip1.However,the effect of p27kip1 protein mainly on reducing the expression of protein in the cytoplasm,but not the nucleus;Although we detected cell viability,it was not statistically significant(p=0.7599);(2)Overexpression of HDAC4 could enhance the expression of p27kip1 mRNA;(3)Western Blot showed that HDAC4 overexpression in HEK293 cells could downregulate the expression of p27kip1 in the cytoplasm(p<0.05),upregulate the protein expression in the nucleus(p<0.001),and upregulate the protein expression of p27kip1 in total(p<0.05);In SH-SY5 Y cells,overexpression of HDAC4 up-regulated the expression of p27kip1 protein in the cytoplasm and nuclear(p<0.001,p<0.01);up-regulated the expression of p27kip1 protein in total(p<0.01);(4)Western Blot results showed that HDAC4 overexpression could regulate the nuclear distribution of p27kip1 phosphorylation.The expression of p27kip1(T198)in nucleus increased in HEK293 cells after HDAC4 overexpression(p<0.01),but not in cytoplasm(p=0.6607).The expression of p27kip1(S10)increased in the nucleus(p<0.01),and the expression of p27kip1(T187)increased in the nuclear(p<0.001),but not in the cytoplasm(p=0.4202),p27kip1(T157)increased in the nuclear(p<0.001),but not in the cytoplasm(p=0.807).In SH-SY5 Y cells,the expression of p27kip1(T198)in cytoplasm and nucleus decreased after HDAC4 overexpression(p<0.05,p<0.001).There was no significant difference in p27kip1(S10)(p=0.5265),p27kip1(T157)and no significant difference in total protein expression(p=0.8307),but its expression in cytoplasm is down(p<0.001),and in the nucleus is increasing(p<0.01).(5)Cell viability was detected by cck-8.The results showed that HDAC4 overexpression attenuated cell viability.The results showed that CR could downregulate the expression of p27kip1 in HEK293 cells.The expression of p27kip1 mRNA and protein decreased with the decrease of glucose concentration in medium.In both HEK293 and SH-SY5 Y cells,there was a positive correlation between HDAC4 and p27kip1.After HDAC4 overexpression,the correlation mainly reflected in the up-regulation of p27kip1 mRNA and protein expression,and affected the phosphorylation of p27kip1(T198,S10,T187,T157)nuclear distribution.This experiment provides a theoretical basis for future research on aging-related diseases and provides an effective target for disease treatment.