KLF5 Functions In Proliferation,Differentiation And Apoptosis Of Chicken Satellite Cells

KLF5(Krüppel like factor 5)is a member of the Krüppel like factors subfamily of zinc finger transcription proteins,it is a basic transcription factor which regulates genes transcription by binding to GC boxes of gene promoters.Lots of studies have shown that KLF5 is an important regulator of cell proliferation,differentiation and apoptosis in mammals,but there was no relevant studies have been reported in poultry.Hence,a series of experiments were designed to explore the role of KLF5 gene in chicken cells.First,the expression level of KLF5 gene in different tissues of chicken was detected by RT-PCR.Then we knocked down KLF5 by KLF5-specific small interfering RNA(siRNA)to identify the function of KLF5 in chicken skeletal muscle satellite cells(SMSCs)proliferation,differentiation,and apoptosis.Finaly the RNA-seq was used to further explore its potential regulation mechanisms.The main results obtained are as follows:(1)KLF5 gene is observed to express in heart,liver,spleen,lung,kidney,breast muscle,leg muscle,intestine,muscle stomach,glandular stomach,fat and brain of chicken,and it has the highest expression level in breast muscle and leg muscle.(2)The proliferation rate of SMSCs were detected after transfected with KLF5 siRNA and negative siRNA.There was no significant difference between KLF5 siRNA group and negative siRNA group during the 72 h period.The mRNA expression levels of CCND1 and PCNA which closely related with cells proliferation were no significant difference between KLF5 siRNA group and negative siRNA group during the 72 h period.Knockdown of KLF5 expression had no significant effect on the proliferation of SMSCs.(3)SMSCs were transfected with negative siRNA and KLF5 siRNA respectively at the SMSCs differentiation stage.Morphologically,there was no obvious myotube formation was observed in KLF5 siRNA group within 72 h after interfering,but in the negative siRNA group,the SMSCs gradually differentiated within 72 hrs and formed a large number of myotubes within 72 h.The mRNA and protein expression levels of MYHC and MyoG which related to muscle differentiation in SMSCs with KLF5 siRNA were significantly lower than negative siRNA groups in 24 h,48h and 72 h after interfering(P<0.05).Immunofluorescence images also showed that the fluorescence activity of MYHC decreased in KLF5 siRNA group,the protein of MYHC in siRNA group was lower than negative siRNA group.Knockdown of KLF5 expression inhibited the differentiation of SMSCs.(4)The apoptosis rate of SMSCs were detected after transfected with KLF5 siRNA and negative siRNA,the apoptotic rate of KLF5 siRNA group significantly higher than negative siRNA group(P<0.05).The mRNA levels of caspase-3 which is a pro-apoptotic factor was significantly upregulated(P<0.05),and mRNA levels of survivin which can make cells resistant to apoptosis was significantly downregulated in KLF5 siRNA group(P<0.05).Knockdown of KLF5 expression promoted apoptosis of SMSCs.(5)The KLF5 siRNA group and the negative siRNA group were detected by RNA-seq,a total of 1195 differentially expressed genes were obtained,of which 729 genes were significantly down-regulated and 466 genes were significantly up-regulated(P<0.05).Differentially expressed genes were mainly concentrated in GO terms such as skeletal muscle tissue development,muscle fiber development,myofilament,muscle alpha-actinin binding and ribosome.KEGG analysis revealed that differentially expressed genes were mainly concentrated in Wnt signaling pathway,Ribosome,ECM-receptor interaction and Hypertrophic cardiomyopathy.(6)The expression of related genes in Wnt signaling pathway was detected after knockdown of KLF5 gene in SMSCs,The mRNA expression levels of Wnt3 a,Wnt5a,Wnt5 b,Wnt7a,Wnt10 a and β-catenin were significantly decreased in the KLF5 siRNA group except C-Myc gene(P<0.05).In summary,KLF5 is an important regulator in the differentiation and apoptosis of chicken SMSCs,it regulates the development of chicken skeletal muscle through Wnt signaling pathways and other related signaling pathways.Our results provide a theoretical basis for studying the development of chicken skeletal muscle after birth.