Hnrnpk与c-Src互作及对C2C12细胞增殖分化的影响

发布时间：2018-04-12 08:49

本文选题：猪 + Hnrnpk　；参考：《信阳师范学院》2015年硕士论文

【摘要】：核不均一核糖核蛋白k (heterogeneous nuclear ribonucleoprotein k, Hnrnpk)是一个多功能蛋白,参与DNA修复、转录和翻译调控、RNA的剪切加工等生物学过程,能通过多种途径对成肌细胞增殖与分化起重要调控作用,但其作用机制及信号通路尚不清楚。c-Src作为非受体蛋白酪氨酸激酶家族的一员,可以通过Rat/MEK/ERK和p38MAPK信号通路对骨骼肌肌生成过程起着重要的调节作用,但其在成肌细胞中的表达调控机制却不清楚。以往研究表明Hnrnpk与c-Src存在多层次的互作并发挥重要生物学功能。本研究从猪Hnrnpk和c-Src基因的克隆及表达着手,以小鼠C2C12成肌细胞为模型,结合qRT-PCR、免疫印迹、细胞转染、亚细胞定位、免疫荧光、过表达、RNA干涉、Co-IP、GST pull-down、酵母双杂交、RIP等方法,深入探讨Hnrnpk与c-Src的互作及其在C2C12成肌细胞增殖分化中的作用,这将为阐明骨骼肌发育和再生的复杂网络调节机制奠定基础,而且还可望为畜牧业提高动物的产肉量、肉品质及医学上控制肌肉再生提供理论依据。本研究得到以下结果： 1. Hnrnpk基因的克隆及时空表达分析克隆得到2056bp猪Hnrnpk基因的cDNA序列,包含1395bp的开放阅读框,编码464个氨基酸；Hnrnpk基因在梅山猪不同发育时期的背最长肌中均差异表达,胚胎期表达量最高,并且随着生长发育呈下调表达。Hnrnpk基因在120日龄的大白猪肌肉和脂肪组织中相对高表达,肾、肺中次之,心、大脑、肝、脾和卵巢中的表达相对较低。而就四种不同类型肌肉组织而言,半腱肌和背最长肌中表达量最高,咬肌次之,股二头肌中最低；Hnrnpk基因在C2C12细胞分化过程中表达下调,其直接调控的c-myc、c-Src、p21等基因表达趋势与之相似。 2. c-Src基因的克隆及时空表达分析克隆得到4126bp猪c-Src基因的cDNA序列,包含1629bp的开放阅读框,编码542个氨基酸；c-Src基因在大白猪和梅山猪肌肉中的表达模式相似,均在胚胎65日龄高表达,出生后3天表达量极显著减少,而在21日龄又表达上调,此后随着发育逐渐下调；两品种相比,胚胎65日龄,c-Src基因在梅山猪中的表达量显著高于大白猪(P0.05),而出生后3天、21天及6月龄大白猪中的表达量均极显著高于梅山猪(P0.01); c-Src基因在猪不同组织中广泛表达,在PT期,大脑中c-Src基因的表达量最高,肝脏中表达量最低。在3天,肝脏中c-Src基因的表达量最高,骨骼肌中表达量最低。在4月龄,肺中c-Src基因的表达量最高,骨骼肌中表达量最低；在C2C12细胞诱导分化过程中,c-Src基因随着诱导分化表达先上调后逐渐下调。 3. Hnrnpk对C2C12细胞增殖分化的影响超表达Hnrnpk基因后,成肌转录因子MyoG, Myf6的表达量下调,而MyoD却上调,成熟骨骼肌相关分子标记物MLC2、MCK、TAK1下调,c-Src表达量上调,p21下调；siRNA干涉Hnrnpk后,成肌转录因子MyoG、Myf6和MyoD以及成熟骨骼肌相关分子标记物MLC2、 MCK表达量上调,c-Src表达量下调,p21上调。表明Hnrnpk可能通过上调c-Src和下调成肌转录因子MRFs家族的表达从而促进C2C12细胞增殖或抑制分化。 4. c-Src对C2C12细胞增殖分化的影响超表达c-Src基因后,成肌转录因子MyoG、 Myf6、 MyoD的表达量下调,而且成熟骨骼肌相关分子标记物MLC2、 MCK、 TAK1表达量下调,p21表达量下调；PP2抑制剂处理C2C12细胞后,MyoG和MyoD分化标记基因、p21、凋亡基因Caspase3表达量均上调,p53和c-myc基因下调表达,而c-Src表达没有变化。表明c-Src对C2C12细胞增殖分化发挥作用可能是通过直接或间接调节成肌转录因子MRFs家族和p21的表达来实现的。 5.Hnrnpk与c-Src的互作分析通过ELM在线分析发现与Hnrnpk相互作用的众多元件包括了c-Src蛋白的SH3结构域,进而采用Co-IP初步证实内源性的Hnrnpk和c-Src在分化的C2C12细胞中相互作用；此外,又用酵母双杂交和GST pull-down方法对Hnrnpk与c-Src的互作进一步分析,酵母双杂交结果表明Hnrnpk与c-Src的全长以及单独的SH3结构域互作,与其他缺失体不互作,与c-Src全长的结合活性要强于单独的SH3结构域；GST pull-down结果表明Hnrnpk能够在体外与c-Src的全长及所有缺失体相互作用。RIP实验结果表明分化的C2C12细胞中c-Src mRNA与Hnrnpk的结合能力要极显著高于阴性对照IgG抗体(P0.01),而增殖的C2C12细胞中c-Src mRNA和Hnrnpk的结合能力与阴性对照IgG抗体相比差异不显著(P0.05),表明Hnrnpk只在分化的C2C12细胞中与c-Src mRNA互作,这与鼠Hnrnpk在C2C12细胞中的亚细胞分布是一致的。
[Abstract]:The effects of Hnrnpk on the proliferation and differentiation of myoblasts were investigated by means of rat / MEK / ERK and p38MAPK signaling pathways .

Cloning and Expression Analysis of 1 . Hnrnpk Gene

The cDNA sequence of the 2056bp porcine Hnrnpk gene was cloned , and the cDNA sequence was 1395bp open reading frame , and 464 amino acids were encoded .
The expression of Hnrnpk gene in muscle and adipose tissue of 120 - day - old pig muscle and adipose tissue was the highest , and the expression of Hnrnpk gene in muscle and adipose tissue was relatively low .
The expression of Hnrnpk gene was down regulated in C2C12 cell differentiation , and its direct regulation of c - myc , c - myc , p21 and other genes were similar .

Cloning and Expression Analysis of the 2 . c - src Gene

the cDNA sequence of the 4126 bp porcine c - src gene was cloned , and a 1629 bp open reading frame was obtained , and 542 amino acids were encoded ;
the expression pattern of c - src gene in muscle of pig and meishan pig was similar , which was expressed at the age of 65 days of embryo , and the expression level was significantly decreased at the third day after birth , while the expression rate was up - regulated at the age of 21 , and then gradually decreased with development .
Compared with the two breeds , the expression of c - src gene was significantly higher in the pigs than that in Mishan ( P0.05 ) , while the expression of c - src gene was the highest in the three days after birth . The c - src gene expression was the highest in the liver and the lowest in skeletal muscle .
During the induction and differentiation of C2C12 cells , the c - src gene was down - regulated with the increase of induction differentiation .

3 . Effect of Hnrnpk on proliferation and differentiation of C2C12 cells

After overexpression of Hnrnpk gene , myogenic transcription factor MyoG , Myf6 expression was down - regulated , while MyoD was up - regulated , and the mature skeletal muscle - related molecular markers MLC2 , MCK , TAK1 were down - regulated , and the expression of c - src was up - regulated and p21 down - regulated ;
After interfering with Hnrnpk by siRNA , myogenic transcription factors , MyoG , Myf6 and MyoD , and mature skeletal muscle - related molecular markers MLC2 , MCK expression were up - regulated , and the expression of c - src was down - regulated , and p21 was up - regulated . It was suggested that Hnrnpk might promote the proliferation of C2C12 cells or inhibit differentiation by up - regulating the expression of c - src and down - regulating the MRFs family .

4 . Effects of c - src on the proliferation and differentiation of C2C12 cells

The expression of myogenic transcription factor MyoG , Myf6 , MyoD was down regulated after overexpression of c - src gene , and the expression of related molecular markers MLC2 , MCK , TAK1 of mature skeletal muscle was down regulated , and the expression level of p21 was down regulated .
After treatment of C2C12 cells , the expression of MyoG and MyoD differentiation marker gene , p21 and apoptosis gene Caspase3 were up - regulated , and the expression of p53 and c - myc was downregulated .

5 . Interaction between Hnrnpk and c - src

Many elements interacting with Hnrnpk were found by ELM on - line analysis , including the SH3 domain of c - src protein , and then Co - IP was used to demonstrate the interaction of endogenous Hnrnpk and c - src in differentiated C2C12 cells .
In addition , the interaction of Hnrnpk and c - src was further analyzed by yeast two - hybrid and GST pull - down methods . The results showed that Hnrnpk was the full - length of c - src and that of the isolated SH3 domains , which did not interact with other deletions , and the binding activity of the full length of c - src was stronger than that of the single SH3 domain ;
The results of RIP showed that Hnrnpk was significantly higher than that of negative control IgG antibody ( P0.01 ) , and the binding ability of c - src mRNA and Hnrnpk in C2C12 cells was significantly higher than that of negative control IgG antibody ( P0.05 ) .

【学位授予单位】：信阳师范学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

【共引文献】