The Interaction Between Hnrnpk And C-Src And Its Effects On C2C12Myoblasts Proliferation And Differentiation

Hnrnpk, a multifunctional protein, participates in DNA repair, transcription and translation regulation, RNA splicing and other biological processes. Hnrnpk may play an important role in skeletal muscle cells proliferation and differentiation, but how and why it has this effect remains unknown. c-Src, a member of the non-receptor protein tyrosine kinase family, plays a critical role in skeletal muscle formation through Raf/MEK/ERK and p38MAPK signal pathway, but its expression and regulation mechanism are not clear. Previous studies showed that Hnrnpk can interact with c-Src at multi-level. In this study, we set about researching on cloning and spatial and temporal expression analysis of Hnrnpk and c-Src gene, then used C2C12cells as a model, combining with qRT-PCR, immunoblotting, cell transfection, subcellular localization, immunefluorescence, over expression, RNAi, Co-IP, GST pull-down, yeast two hybrid and RIP to study the interaction between Hnrnpk and c-Src and its effects on C2C12cells proliferation and differentiation. It lays a foundation for illustrating the regulatory mechanism of skeletal muscle development and regeneration, and is helpful for increasing the meat production and quality for livestock genetic improvement and controlling the muscle regeneration of human being. The results are as follows:1. Cloning and spatial and temporal expression analysis of Hnrnpk geneWe cloned Hnrnpk cDNA sequence, which length was2056bp containing a1395bp ORF and coding464amino acid; Hnrnpk gene in longissimus dorsi of meishan pig at different developmental stages was differentially expressed. At embryonic period, Hnrnpk gene was expressed at the highest level, then was downregulated with the growth of skeletal muscle; Hnrnpk gene in large white at120days was expressed at a relatively high level in muscle and adipose tissue, a moderate level in the lung and kidney, and a relatively lower level in heart, brain, liver, spleen and ovary. As for the four different types of muscle, Hnrnpk was expressed at the highest level in longissimus dorsi muscle and semitendinosus, a moderate level in masseter muscle, and a low level in biceps femoris; Hnrnpk gene was downregulated during the differentiation of C2C12cells, and c-myc, c-Src and p21gene, which were regulated directly by Hnrnpk, had a similar expression trend.2. Cloning and spatial and temporal expression analysis of c-Src geneWe cloned c-Src cDNA sequence, which length was4126bp containing a1629bp ORF and coding542amino acid; Expression pattern of c-Src gene in large white and meishan was similar. Both the two breeds had the highest expression level at fetal65days, and decreased significantly3days after birth, and then increased at21days, and then decreased with the development of skeletal muscle. At fetal65days, c-Src gene was expressed at a higher level in meishan than large white (P<0.05). However, at3days,21days and6months, c-Src gene was expressed at a significantly higher level in large white than meishan (P<0.01). c-Src gene was expressed extensively in pig tissues. At PT periods, expression level of c-Src gene was the highest in the brain, at a lowest level in the liver; At3days, expression level of c-Src gene was at the highest level in liver, and at a lowest level in the skeletal muscle. At4months, expression level of c-Src gene was the highest in lung, and at a lowest level in the skeletal muscle. c-Src gene expression level increased firstly and then decreased gradually during the differentiation of C2C12cells.3. Effect of Hnrnpk on the proliferation and differentiation of C2C12cellsAfter over expression of Hnrnpk gene, myogenic transcription factor, such as MyoG and Myf6were downregulated, however MyoD was upregulated. Mature skeletal muscle relating molecular markers, such as MLC2, MCK and TAK1were downregulated. c-Src was upregulated significantly. p21was downregulated. After Hnrnpk knocked down by siRNA, MyoG, Myf6, MyoD, MLC2and MCK were upregulated. c-Src was down-regulated, p21was upregulated. This indicated that Hnrnpk may promot C2C12cells proliferation or inhibit C2C12differentiation by upregulating c-Src and downregulating MRFs.4. Effect of c-Src on the proliferation and differentiation of C2C12cellsAfter over expression of c-Src gene, myogenic transcription factor, such as MyoG, Myf6and MyoD were downregulated. Mature skeletal muscle relating molecular markers, such as MLC2, MCK and TAK1were downregulated. p21was downregulated. After treated by PP2inhibitor, MyoG, MyoD, p21and caspase3were upregulated. p53and c-myc were downregulated. c-Src expression was remained unchanged. This indicated that c-Src may play an important role in C2C12cells proliferation and differentiation by regulating MRFs and p21directly or indirectly.5. Analysis of the interaction between Hnrnpk and c-SrcWe found that Hnrnpk can interact with many elements, containing SH3domain of c-Src through ELM online prediction. Then we preliminarily confirmed that Hnrnpk can interact with c-Src in differentiated C2C12cells by Co-IP. In addition, we made a further study on interaction between Hnrnpk and c-Src by yeast two hybrid and GST pull-down.The results of yeast two hybrid showed that Hnrnpk can interact with c-Src full length and SH3domain alone, but cannot interact with other deletions. And the binding activity of c-Src full length was higher than SH3domain alone. The results of GST pull-down showed that Hnrnpk can interact with c-Src and other deletions in vitro. The results of RIP showed that in differentiated C2C12cells, the binding capacity of c-Src mRNA and Hnrnpk was significantly higher than negative control IgG (P<0.01). However, in proliferated C2C12cells, there was no significant difference between the binding activity of c-Src mRNA and the negative control IgG (P>0.05). This indicated that Hnrnpk can interact with c-Src mRNA only in the differentiated C2C12cells. The result was in accord with subcellular location of mouse Hnrnpk in C2C12cells.