摘要
利用成体干细胞(adult stem cells,ASCs)进行细胞替代治疗或联合基因治疗是近几年提出的修复组织损伤的新思路。基于成体干细胞的治疗策略能否安全、有效的达到目的在很大程度上取决于我们对成体干细胞增殖与分化的分子机制的认识。细胞分化的核心问题是基因表达在时间和空间上的精确调控,一种新的细胞表型的建立需要相关调控基因的打开和关闭以适应新的功能。近年来发现,一类叫做microRNAs(miRNAs)的非编码小RNA分子,在调控特定细胞增殖、分化进程等方面起着重要作用,特别是其在维持干细胞定向分化和自我更新功能中的作用逐渐被揭示,成为目前干细胞研究中的一个新的亮点和热点。某些miRNAs具有组织表达分布的特异性,提示它们与该组织的分化和特定功能的维持有关。早期的miRNAs克隆结果和基于高通量芯片的组织表达谱分析显示,miR-1,miR-133和miR-206在小鼠的肌肉组织呈特异性表达,而它们在肌肉发育、增殖和分化中的作用尚知之不多。明确这些miRNAs在成体干细胞增殖分化中的作用,将为我们利用成体干细胞进行相关肌损伤修复治疗提供新的线索。
因此,本课题拟利用肌母细胞C2C12体外成肌分化模型,检测肌肉组织特异性表达的miRNAs在成肌分化中的表达变化,通过gain-of-function观察部分肌肉组织特异性表达的miRNAs对成肌分化的影响,并利用生物信息学和相关实验手段预测、分析和验证它们可能的靶基因,同时检测这些miRNAs在某些肌肉损伤过程中的表达变化,并构建相应的治疗性载体,初步探讨其在促进成肌分化中的意义,为进一步探明miRNAs调控骨骼肌增殖与分化的分子机制、探索成体干细胞定向分化的新策略提供理论指导和实验依据。
本研究内容主要包括三部分:
一、肌肉组织特异性miRNAs在C2C12细胞体外成肌分化过程中的表达改变建立C2C12细胞体外成肌分化和以TNF-α为干预因素的抑制分化模型,通过形态学改变,肌球蛋白重链免疫荧光组织化学和肌分化相关基因的RT-PCR对上述模型进行评价,采用Northern blot检测肌肉组织特异性的miR-1、miR-133和miR-206在上述模型的表达变化情况。
二、miR-1和miR-206促进成肌分化的作用和机制研究
根据上述研究结果并结合最新进展,确定进一步深入研究miR-1和miR-206在成肌分化过程中的功能。化学合成miR-1和miR-206,将其以200nM的浓度分别转染至C2C12细胞,形成gain-of-function状态后进行诱导分化,采用形态学、肌球蛋白重链免疫荧光组织化学和肌肉功能蛋白skeletal-α-actin的Western blot对其分化效应评价。
采用生物信息学方法,利用软件PiTar、miRanda和TargetScan4.2分析miR-1和miR-206可能作用的候选靶基因。根据上述研究结果并结合最新进展,分析候选靶基因在成肌分化过程中及过表达miR-1和miR-206后的变化特点,进一步筛选与miR-1和miR-206表达相关联的基因。
利用荧光素酶报告系统验证候选靶基因。为了明确荧光素酶报告系统的敏感性和特异性,构建阳性对照质粒pmiR-206-Luc reporter。将位于候选靶基因3′UTR含有预测miR-1和206作用位点约500bp区域克隆至pMIR-Luc内荧光素酶3′UTR,并进行酶切和测序验证。通过上述pMIR-REPORT荧光素酶报告系统,验证miR-1和miR-206是否与靶基因发生相互作用。
三、检测肌肉组织特异性miRNAs在骨骼肌失神经萎缩中的表达变化并构建miR-1的腺病毒表达载体进行促成肌分化的初步研究。
建立小鼠腓肠肌去神经支配萎缩模型,Northern blot检测miR-1、miR-133和miR-206在该模型的表达变化。着眼于以后的治疗应用,利用PCR从小鼠基因组扩增含有miR-1-1的DNA片段,连接至pAdTrack-CMV中后转化含pAdEasy-1的感受态BJ5183,通过同源重组获得腺病毒载体质粒,转染293包装病毒颗粒,Northern blot验证成熟miR-1表达情况。利用miR-1重组腺病毒感染C2C12细胞,诱导分化72h通过形态学、肌球蛋白重链免疫荧光组织化学和肌肉功能蛋白skeletal-α-actin的Western blot等指标评价其对分化的影响。
通过以上三部分的实验研究与分析,获得以下主要结果:
1、成功建立C2C12细胞体外成肌分化和以TNF-α为干预因素的抑制分化模型。C2C12细胞以2%马血清进行成肌分化诱导,3-5d可见多核肌管, MHC免疫荧光呈胞浆阳性,RT-PCR检测myoD、myoG和skeletal-α-actin分化相关基因诱导分化后表达明显上调,证实我们所建立的C2C12细胞成肌分化模型是成功的。另一方面,观察到TNF-α是C2C12细胞成肌分化的负向调控因子。TNF-α作用后上述成肌分化相关的标志分子均不同程度受到抑制,建立了应用TNF-α抑制成肌分化的模型,用于后续研究。
2、明确了肌肉组织特异性miRNAs miR-1,miR-133和miR-206在上述模型的表达变化。在正常成肌分化模型中三者随着诱导时间的延长,在分化过程中表达量升高明显;在诱导分化培养基中加入20ng/mlTNF-α后成肌分化受抑,三者表达也受到明显抑制。
3、揭示miR-1和206能够促进成肌分化。在C2C12细胞转染化学合成miR-1和206后24h进行诱导分化,诱导48h后进行效应评价。结果显示,在TNF-α(-)组,转染miR-1和206后与阴性对照比较, MHC阳性细胞数目明显较多,skeletal-α-actin水平分别为对照的1.37倍和1.23倍。在TNF-α(+)组,各组MHC阳性细胞数目明显少于TNF-α(-)组,转染miR-1和206后与阴性对照比较, MHC阳性细胞数目明较多,skeletal-α-actin表达水平分别为对照的2.08倍和1.69倍,加入TNF-α这一干预因素后,凸显了miR-1和206的促分化效应。
4、发现CCND1和FOXP1是miR-1和miR-206作用的靶基因。利用在线生物信息学软件PiTar、miRanda和TargetScan4.2预测miR-1和miR-206可能作用的靶基因,并结合其在成肌分化过程中的表达变化特点确定CCND1和FOXP1两个基因做进一步深入研究。进一步通过gain-of-function研究证实CCND1和FOXP1符合靶基因的表达变化特点。进而,将CCND1和FOXP1的3′UTR含有预测作用位点约500bp的片段常规方法连接到pMIR-Luc的荧光素酶3′UTR,成功构建pmiR-Luc-CCND1-3′UTR、pmiR-Luc-FOXP1-3′UTR-1和pmiR-Luc -FOXP1-3′UTR-2等3个重组质粒。分别与pMIR-β-gal及合成之miRNAs共转染293细胞,计算分析Luc/β-gal值,结果显示,对pmiR- Luc -CCND1-3′UTR miR-1和miR-206可使其荧光素酶活性分别下降47.7%和39.3%;对pmiR- Luc -FOXP1-3′UTR-2 miR-1和miR-206可使其荧光素酶活性分别下降46.9%和40.7%;miR-1和miR-206未对pmiR- Luc -FOXP1-3′UTR-1的荧光素酶活性造成影响。以上结果表明,CCND1和FOXP1是miR-1/206的靶基因。
5、明确在小鼠腓肠肌去神经支配所致骨骼肌萎缩过程中肌肉组织特异性miRNAs的表达变化情况。与对照组相比,miR-206随着失神经支配时间的延长其在肌肉组织中的表达明显上调,第28d其强度可达到对照的5倍以上;miR-1和miR-133随着失神经支配时间的延长其表达先迅速下调,而后逐渐回升,但至失神经支配后第28d仍明显低于正常水平。
6、成功构建miR-1的腺病毒表达载体,并初步观察利用其促进C2C12分化的作用,为以后利用其进行实验治疗打下基础。利用PCR在小鼠基因组中扩增含有miR-1-1的DNA片段,正确连接至穿梭质粒pAdTrack-CMV中,然后转化含有骨架质粒pAdEasy的感受态大肠杆菌BJ5183 ,通过同源重组产生腺病毒载体质粒pAdEasy/miR-1-1,转染293细胞后包装出重组腺病毒pAd-miR-1-1,感染293后行Northern blot验证成熟miR-1分子能够高效表达。制备高滴度病毒颗粒感染C2C12,诱导成肌分化72 h进行评价,结果表明,感染Ad-miR-1后MHC阳性细胞数目增多,skeletal-α-actin水平为对照的1.21倍;加入TNF-α后,Ad-miR-1组细胞MHC阳性细胞数目明显增多,α-actin接近对照不加入TNF-α的水平,表明Ad-miR-1可以明显促进成肌分化。
研究结论如下:
1、成功建立C2C12细胞体外成肌分化和TNF-α干预分化的模型,明确了肌肉组织特异性miRNAs在上述模型的表达变化特点。
2、明确miR-1和206是促进成肌分化的正向调控因素,CCND1和FOXP1是它们的两个靶基因。
3、揭示肌肉组织特异性miRNAs在小鼠腓肠肌去神经支配所致骨骼肌萎缩过程中的表达变化情况,为从miRNAs角度研究去神经支配所致骨骼肌萎缩的机制提供了新的思路。
4、成功构建miR-1的腺病毒表达载体并初步研究了其促进成肌分化的作用,为应用miRNAs基因治疗促进肌损伤的修复打下了基础。
How to repair tissue injury effectively has been considered a hot and difficult issue for a long time. In recent years, cell replacement therapy or gene therapy with adult stem cells(ASCs) has been studied in the treatment of tissue injury. Whether therapy strategies based on ASCs could obtain safe and effective purpose, to some degree, depends on our understanding of the molecular mechanisms that regulate proliferation and differentiation. Regulation of gene expression is the central theme of cell differentiation. Establishing a new cellular phenotype requires suppression of features associated with the prior phenotype or other related phenotypes, which needs switching on and off certain genes. So the results of cell differentiation are characterized by differences between cells in diverse spaces and time.MicroRNAs, a group of newly-discovered non-coding small RNAs involved in organism development by regulating cell proliferation and differentiation, have been found to play a role in committed differentiation and self-renewal of stem cells. Now research on stem cell-related miRNAs is becoming a hot field. Some miRNAs have tissue-specific expression pattern, which hints they may have a close relationship with development and maintenance of that tissue. Results from pristine miRNAs clone and microarray indicate muscle-specific expression pattern of miR-1, miR-133 and miR-206. However, to date, little is known about their functions in muscle development, proliferation and differentiation. Undoubtedly identifing their roles in ASCs proliferation and differentiation will give clues to treatments of muscular injury based on ASCs.
In this study, the in vitro model of C2C12 myogenic differentiation was employed to imitate myogenesis process, and expression changes of muscle-specific miRNAs were detected in this model. Then, we evaluated the influence of miR-1 and miR-206 overexpression on the myogenic differentiation, and found their functional targets by bioinformatics and experiments. At the same time, we detected the expression changes of muscle-specific miRNAs during muscle atrophy induced by denervation, and constructed therapeutic vector for further application.Our results provide theoretical support and expremental basis for further research on mechanism of miRNAs regulating skeletal muscle proliferation and committed differentiation.
Our study consisted of three parts:
1. Detecting expression changes of muscle-specific miRNAs in myogenic differentiation.
We established the in vitro model of C2C12 myogenic differentiation and its related TNF-αinhibition model evaluated by morphology、RT-PCR of myogenic differentiation related genes and myosin heavy chain(MHC) immunofluorescence histochemistry. Expressions of miR-1, miR-206 and miR-133 in above mentioned models were detected by Northern blot.
2. Studying the roles of miR-1 and miR-206 in the process of myogenic differentiation and exploring their molecular mechanisms.
Functional analysis of miR-1 and miR-206 was evaluated by gain-of-function. C2C12 transfected with 200 nm synthesized miRNAs were induced to myogenic differentiation for 48 h and the effects were evaluated by morphology, IF for MHC and western blot for skeletal-α-actin.
For analyzing target genes of miR-1 and miR-206, a series of work were done. Firstly, three online softwares for bioinformatics analysis, i.e. PicTar, miRanda and TargetScan4.2, were used for miRNA target prediction. Based on both literatures and biologic function of miR-1/206, we selected several genes as candidate target genes for further study.
Secondly, we detected the expression of candidate target genes in the process of myogenic differentiation at protein or mRNA level as well as in the conditions of overexpression miR-1/206. Thus we gave up the genes whose expressions have no correlations with the level of miR-1/206.
Finaly, the candidate target genes were verified by luciferace reporter system. To ensure sensitivity and specificity of the system, we construct a positive control vector named pmiR-206- Luc reporter in which the 3′UTR of luciferace gene has a complementary sequence to mature miR-206 sequence. The reconstitute vector, pmiR-206- Luc reporter, the control vector for normalization, pMIR-β-gal and miRNAs were co-transfected to 293 cells. 24 h after transfection,β-Galactosidase and luciferase activities were determined .All transfection data were expressed as luciferase activity normalized byβ-galactosidase activity. To analyze candidate target genes, similar vectors were constructed by cloning the fragments containing presumed target site in 3′UTR of candidate target genes to the 3′UTR of luciferace gene in the vector. The reliability of miRNAs targets was verified by above-mentioned means.
3. Exploring expression changes of muscle-specific miRNAs in denervated skeletal muscle and using miR-1 recombinant adenovirus to promot myogenic differentiation in vitro.
To explore the expression changes of muscle-specific miRNAs in the process of skeletal muscle atrophy induced by denervation. The mouse model of sciatic nerve resection was prepared. Northern blot was used to examine the levels of muscle-specific miRNAs expression in the gastrocnemius muscle at different time points after denervation.
To construct the miR-1 adenovirus vector, genomic fragment containing miR-1-1 was amplified by PCR and firstly constructed into pAdTrack-CMV. Subsequently miR-1 was constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence BJ5183. The positive recombinant pAdEasy-1 containing miR-1 gene was linearized by PacⅠand transfected into 293 cells to pachage recombinant adenovirus. The production of mature miR-1 was confirmed by northern blot. The influence of myogenic differentiation by miR-1 recombinant adenovirus was evaluated in C2C12 cells infected by the adenovirus by morphology, IF for MHC and Western blot for skeletal-α-actin.
By analysis the three researches above, the following results can be obtained:
1. Establishment of the in vitro model of C2C12 myogenic differentiation and its related TNF-αinhibition model. C2C12 cells were induced to myogenic differentiation by DMEM containing 2% horse serum for 3-5 d. The enlarged polynucleation myotubes formed accompanied with strong positive signal of MHC IF in cytoplasm and upregulation of myoD, myoG as well as skeletal-α-actin detected by RT-PCR. These results indicated the in vitro model of C2C12 myogenic differentiation was successful. In the TNF-αinhibition model, the above-mentioned index were all inhibited to different degree, which indicate the TNF-αinhibition model was successful too.
2. Identifing expression changes of muscle-specific miRNAs in above-mentioned models. In the model of C2C12 myogenic differentiation,all three miRNAs upregulate during muscle differentiation. While the expression level of all three were inhibited with the existence of TNF-αin the differentiation medium.
3. miR-1 and miR-206 can promot myogenic differentiation. Cells were continuously cultured in growth medium for 24 h after transfection and then transferred to differentiation medium for 48 h before immunostaining for MHC and Western blot for skeletal-α-actin. The results from MHC and skeletal-α-actin indicate synthesized miR-1 and miR-206 promot the differentiation.
4. CCND1 and FOXP1 are target genes of miR-1/206. Three online softwares for bioinformatics analysis, i.e. PicTar, miRanda and TargetScan 4.2, were used for predicting targets of miR-1 and miR-206. We selected four genes including CCND1, FOXP1, PFTK1 and EDN1 as potential targets of miR-1/206. The further detection of their expression changes in the process of myogenic differentiation and miR-1/206 overexpression eliminate PFTK1 and EDN1 out of the list. Then we construct vectors named pmiR-Luc -CCND1-3′UTR、pmiR-Luc-FOXP1-3′UTR-1 and pmiR-Luc-FOXP1-3′UTR-2, which have the potential target sites of miR-1/206 from 3′UTR of CCND1 or FOXP1. The results of experiments demonstrated that: the pmiR-Luc-CCND1-3′UTR luciferase activity for miR-1 and miR-206 decreased 47.4% and 49.3%, respectively; the pmiR- Luc-FOXP1-3′UTR-2 luciferase activity for miR-1 and miR-206 decreased 46.9% and 40.7%, respectively. However, the pmiR-Luc-FOXP1-3′UTR-1 luciferase activity was not affected by miR-1 and miR-206. The results of target verification experiments demonstrated that CCND1 and FOXP1 are targets of miR-1/206.
5. Identifying the expression changes of muscle-specific miRNAs in denervated skeletal muscle. The mouse model of sciatic nerve resection was successfully established. The expression of miR-206 was obviously up-regulated with the time denervation, however, those miR-1 and miR-133 was decreased firstly and then gradually recovered with the time elapse.
6. Construction of miR-1 recombinant adenovirus and observation of its effect on promoting myogenic differentiation. After evaluation and sequencing, we constructed the miR-1-1 recombinant adenovirus successfully which could produce high level mature miR-1 in infected 293 cells. C2C12 infected miR-1 recombinant adenovirus were induced for myogenic differentiation for 72 h, and the index of morphology, immunostaining for MHC, western blot for skeletal-α-actin indicate the miR-1 recombinant adenovirus could promot myogenic differentiation.
Conclusion:
1. The in vitro model of C2C12 myogenic differentiation and its related TNF-αinhibition model were established successfully.In addition, we found the expression features of the muscle-specific miRNAs in the two models.
2. miR-1 and miR-206 are positive regulators of myogenic differentiation, and CCND1 and FOXP1 are two target genes.
3. The expression changes of muscle-specific miRNAs were revealed in denervated skeletal muscle, which offered new view to explore the mechanisms of skeletal muscle atrophy in the level of miRNAs.
4. The miR-1 recombinant adenovirus was constructed successfully and proved to promot myogenic differentiation in vitro in primary experiment, providing basis for repairing muscular injuries with miRNAs.
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