造血干细胞发育相关miRNAs的筛选及其功能初步研究

发布时间：2018-02-27 17:01

  本文关键词： 造血干细胞 microRNA 非编码RNA 基因芯片 干细胞功能 基因表达调控 生物信息学 发育生物学　出处：《第三军医大学》2007年博士论文　论文类型：学位论文

【摘要】： 造血干细胞(hematopoietic stem cell,HSCs)是目前研究最为深入的成体干细胞,其具有向髓系、淋巴系、巨核系祖细胞的多向分化的功能,是极富应用前景的研究领域,很可能给人类带来革命性的变化。然而,造血干细胞研究尚存在许多亟待解决的困惑,比如,造血干细胞特异性分子标志、多向分化潜能分子机制、造血干细胞在未分化状态下长期增殖的关键因子、造血干细胞受损相关疾病(白血病、再生障碍性贫血、骨髓增生异常综合征)发生机制等,既是造血干细胞研究的热点,也是造血干细胞研究的难点。近年来,一类叫做microRNAs(miRNAs)的非编码小RNA分子,在监控个体发育时相转变、调控特定细胞增殖、分化进程等方面起着重要作用,其在维持干细胞定向分化和自我更新功能维持中的作用,逐渐被科学家们发现,目前已经掀起miRNAs在干细胞研究中的热潮。HSCs中miRNAs表达状况尚未见报道,miRNAs在HSCs发育中的作用尚知之甚少。建立高通量筛选miRNAs的技术平台,获得并鉴定造血干细胞发育相关的miRNAs,是研究造血干细胞相关miRNAs的起点。因此,本课题拟研制新型哺乳动物miRNAs检测基因芯片,筛选造血干细胞发育相关miRNAs,生物信息学分析及验证其作用靶点,并探讨其在促进造血干细胞发育中的作用,研究结果为探明miRNAs调控造血干细胞发育的分子机制、探索诱导造血干细胞定向分化的新手段提供理论指导和实验依据。 研究目的 1、研制新型哺乳动物miRNAs检测芯片。 2、通过miRNAs芯片杂交,获取CD34+CD38-造血干细胞发育相关miRNAs表达谱。 3、生物信息学分析预测并验证miRNAs作用靶点。 4、探索miRNAs在造血干细胞发育中的作用。 研究内容和方法 1、研制新型哺乳动物miRNAs检测芯片 以已知哺乳动物基因组miRNAs序列为依据,合成相应DNA探针,制作新型哺乳动物miRNAs检测基因芯片。 2、CD34+CD38-造血干细胞发育相关miRNAs表达谱的筛选 流式细胞仪细胞分选人脐血CD34+CD38-造血干细胞及CD34+造血祖细胞,提取其总RNA,分离纯化18-100nt小片段RNA,经扩增及荧光标记后,与基因芯片进行分子杂交,获得CD34+CD38-造血干细胞miRNAs表达谱。 3、实时荧光定量RT-PCR(Q-RT-PCR)对miRNAs芯片结果的验证 选取6个在CD34+CD38-造血干细胞中差异表达的miRNAs,以Q-RT-PCR加以验证,对miRNAs芯片结果进行初步验证,对结果一致的miRNAs行3次重复实验以确证Q-RT-PCR结果的可靠性。 4、生物信息学分析及miRNAs作用靶点的验证 生物信息学分析miR-129、miR-526b*和miR-520h的染色体定位、保守性分析等,利用网络共享软件picTar、miRanda 3.0和targetscan3.1,分析其可能作用的靶基因。选择在CD34+CD38-差异较为显著的miR-129、miR-520h的进行作用靶基因的验证。首先合成与miRNAs结合的靶位点DNA序列及互补序列,并分别在两条寡核苷酸5'端悬垂添加Hind III和Spe I酶切位点;退火形成形成带酶切位点的双链插入序列,与线性化pMIR-REPORT荧光素酶(Luc)报告基因载体(pMIR-Luc)连接构建重组载体(pMIR-Luc-miR),并进行酶切及测序验证。 重组载体(pMIR-Luc-miR)、标准化对照载体pMIR-REPORT-β-gal(pMIR-β-gal)与miRNAs前体(Pre-miRNAs)共转染HeLa细胞(实验组),72h后分别检测HeLa细胞的荧光素酶(Luc)和β半乳糖苷酶(β-gal)的活性,计算二者的比值(Luc/β-gal)。同时设立三个对照组(no miRNA空白对照组、Pre-miR-negative阴性对照组和Anti-miRNAs反证对照组),比较各组的Luc/β-gal比值,验证miRNAs作用靶点的真实性。 5、miR-520h促进造血发育功能研究 选择在CD34+CD38-造血干细胞中高表达的miR-520h,研究其在促进造血发育中的作用。流式细胞仪分选的CD34+细胞,miR-520h转染CD34+细胞,转染后24h行CFC实验检测CD34+细胞集落形成能力改变,转染后72h行FACS检测CD34+、CD34+CD38-细胞比例变化,同时设立三个对照组(no miRNA空白对照组、Pre-miR-129阴性对照组和Anti-miR-520h反证对照组),分析miR-520h在促进造血发育中的作用。 研究结果 1、成功研制新型哺乳动物miRNAs检测芯片 整个点阵分成4个亚阵,每个亚阵有23行,21列,点间距为185μm,点的直径约为130μm。每条探针重复三次。为保证实验的可靠性,在芯片的不同部位多次设定各种对照,经点样后芯片共有1754个位点,包含469个miRNAs序列,总共588个基因(每个基因重复3个位点)。 2、CD34+CD38-造血干细胞发育相关miRNAs的筛选 与CD34+细胞miRNAs表达微阵列比较,HSC细胞共筛选出31个miRNAs,其中4倍低于CD34+细胞的miRNAs为22个(包括miR-129等),4倍高于CD34+细胞的miRNAs为9个(包括miR-526b*和miR-520h等) ,其中预测的miRNAs (PREDICTED_MIR)为4个。 3、实时荧光定量RT-PCR(Q-RT-PCR)对miRNAs芯片结果的验证 选取6个CD34+CD38-造血干细胞中差异表达的miRNAs,以Q-RT-PCR加以验证,与芯片结果的吻合率达到50%,两种实验结果相符的miRNAs为:CD34+CD38-造血干细胞低表达的miR-129和CD34+CD38-造血干细胞中高表达的miR-526b*和miR-520h,相对于CD34+细胞的相对表达率分别为0.160±0.005、2.276±0.058和5.596±0.861。 4、生物信息学分析及miRNAs作用靶点的验证 利用在线软件Pictar、miRanda 3.0和targetscan3.1分别预测miR-129、miR-526b*和miR-520h的作用靶基因。结果发现,①miR-129在人、鼠、狗、猩猩、鸡等动物中保守存在33个可能的靶基因,包括EIF2C3、CAMTA1、SH3KBP1、TGIF2、ING3、DLGAP2等与miRNA加工、转录因子、信号转导等相关的基因。②miR-526b*仅在人类存在,其可能的作用靶点有771个,包括ABCG2、EIF2C3、CAMK4、CSF2RA等关于信号转导、转录因子、造血发育、凋亡调控、干细胞功能维持、miRNAs加工成熟的基因。③miR-520h共有226个可能的作用靶点,在人类、小鼠和类人猿中保守的靶基因共有30个,包括ABCG2、ID 1 (DNA结合抑制剂1)、SRP19 (一种信号识别蛋白)以及SMAD6 (一种转录调控因子)等与干细胞功能维持、转录、信号转导等相关的调控蛋白。 选取miR-129可能的作用靶点EIF2C3和CAMTA1、miR-520h可能的作用靶点ABCG2和SMAD6进行验证。利用pMIR-REPORT荧光素酶报告基因载体,经酶切鉴定和测序验证,分别成功构建四种重组载体: pMIR-Luc-miR129-EIF2C3、pMIR-Luc-miR129-CAMTA1、pMIR-Luc-miR520h-ABCG2和pMIR-Luc-miR520h-SMAD6。 pMIR-REPORT荧光素酶报告基因系统进行作用靶点验证实验表明,三个对照组(no miRNAs空白对照组、Pre-miR-negative阴性对照组和Anti-miRNAs反证对照组),Luc/β-gal比值接近。实验组,miR-129对于EIF2C3和CAMTA1,荧光素酶活性分别下降41%和47%,表明EIF2C3和CAMTA1都是miR-129的真实作用靶点;miR520h对于ABCG2和SMAD6,荧光素酶活性分别下降68%和27%,表明ABCG2是miR-520h的真实作用靶点,而miR-520h对SMAD6的抑制能力作用较弱。 5、miR-520h促进造血干细胞发育功能研究 CFC实验:三个对照组(no miRNAs空白对照组、miR-129阴性对照组和Anti-miR-520h反证对照组),各种祖细胞集落和集落总数均相近,其中空白对照组CFU-E、BFU-E、CFU-GM和CFU-GEMM和集落总数分别为28.50±4.32、37.83±5.60、57.67±5.16、1.50±0.55和145.50±14.35,实验组分别为37.83±7.70、46.5±4.85、68.17±7.73、1.67±0.52和174.33±17.82。经方差分析表明,CFU-E、BFU-E、CFU-GM和集落总数有显著统计学差异(P0.05,n=6)。 FACS分析:三个对照组CD34+和CD34+CD38-细胞比例相近,其中空白对照组CD34+和CD34+CD38-细胞比例为22.91%±2.03和12.57%±0.81,而实验组则为30.43%±2.34和13.08%±1.08。经方差分析表明,实验组CD34+细胞比例较高,有统计学差异(P0.05,n=6),而CD34+CD38-比例相差不大,无统计学差异(P0.05,n=6)。 实验表明miR-520h具有促进造血干细胞发育和分化的功能。 结论 1、成功研制新型哺乳动物miRNAs检测芯片,表明利用miRNAs基因芯片来获取CD34+CD38-造血干细胞相关miRNAs是可行的,也为研究其它成体干细胞miRNAs表达谱建立了技术平台。 2、筛选到CD34+CD38-造血干细胞发育相关miRNAs表达谱,并部分经实时定量RT-PCR验证,为探讨miRNAs参与CD34+CD38-造血干细胞发育分子机制和生物功能研究奠定了基础。 3、验证了miR-129和miR-520h部分作用靶点,表明miRNAs通过调控EIF2C3、CAMTA1、ABCG2等在miRNA加工、转录因子、信号转导、干细胞功能维持等方面的靶基因,从而参与造血干细胞的发育,并形成miRNAs基因网络,在造血干细胞发育进程中起着协同或者拮抗作用。 4、miR-520h具有显著的促进造血干细胞发育和向祖细胞分化的作用。研究结果不仅对miRNAs作为促进造血发育的分子靶点的探索具有重要的应用前景,而且对于发育生物学、发展组织工程和再生医学都具有重要的理论意义。
[Abstract]:Hematopoietic stem cells (hematopoietic stem cell, HSCs) is currently the most in-depth study of adult stem cells, which has to myeloid, lymphoid, megakaryocyte progenitor cell differentiation, is a field with significant application prospect, is likely to bring revolutionary changes to human life. However, such as hematopoietic there are still many problems of stem cell research, confusion, hematopoietic stem cell specific molecular markers and multilineage potential molecular mechanisms of hematopoietic stem cells in an undifferentiated state under the key factor of the long-term proliferation of hematopoietic stem cell damage related diseases (leukemia, aplastic anemia, myelodysplastic syndrome) occurrence mechanism etc. hematopoietic stem cell research is the hotspot, also is the difficulty of hematopoietic stem cell research. In recent years, a class called microRNAs (miRNAs) encoding non small RNA molecules, the phase transition in the monitoring of individual development, regulation of specific cells The proliferation plays an important role in the differentiation process and other aspects, in the maintenance of stem cell differentiation and self-renewal function in maintenance, gradually by scientists found that currently there has been miRNAs in stem cell research in the upsurge of.HSCs expression of miRNAs has not been reported. The role of miRNAs in the development of HSCs is still poorly understood. Establish a technology platform for high-throughput screening of miRNAs, and identification of hematopoietic stem cell development related to miRNAs, is the starting point of hematopoietic stem cell related miRNAs. Therefore, this paper intends to design a new gene chip for the detection of mammalian miRNAs, screening of hematopoietic stem cell development related miRNAs, bioinformatics analysis and validation of its target, and on the promotion of hematopoietic stem cells during the development of the role of research results to explore the molecular mechanism of miRNAs regulation of hematopoietic stem cell development, explore the induction of hematopoietic stem cell division The novice section provides theoretical guidance and experimental basis.
research objective
1, develop a new mammal miRNAs detection chip.
2, CD34+CD38- hematopoietic stem cell development related miRNAs expression profiles were obtained by miRNAs chip hybridization.
3, bioinformatics analysis predicts and validates miRNAs targets.
4, explore the role of miRNAs in the development of hematopoietic stem cells.
Research contents and methods
1, develop a new mammal miRNAs detection chip
Based on the known miRNAs sequence of the mammalian genome, the corresponding DNA probe was synthesized to produce a novel mammalian miRNAs detection gene chip.
2, screening of miRNAs expression profiles related to CD34+CD38- hematopoietic stem cell development
Flow cytometry cells were selected to select cord blood CD34+CD38- hematopoietic stem cells and CD34+ hematopoietic progenitor cells, extract their total RNA, and isolate and purify 18-100nt small fragment RNA. After amplification and fluorescence labeling, hybridized with gene chip to obtain miRNAs expression profile of CD34+CD38- hematopoietic stem cells.
3, verification of miRNAs chip results by real time fluorescence quantitative RT-PCR (Q-RT-PCR)
6 differentially expressed miRNAs in CD34+CD38- hematopoietic stem cells were selected and verified by Q-RT-PCR. The results of miRNAs chip were preliminarily verified, and 3 times repeated miRNAs experiments were performed to confirm the reliability of Q-RT-PCR results.
4, bioinformatics analysis and verification of the target of miRNAs
Analysis of miR-129 biological information, chromosomal location of miR-526b* and miR-520h, conservative analysis, sharing network by using the software picTar, miRanda 3 and targetscan3.1, and analyze the possible role of the target gene. Selection in CD34+CD38- is significant difference in miR-129, verify the miR-520h target gene. The first synthesis of target sites of DNA sequence and complementary sequence combined with miRNAs, and respectively in two oligonucleotide 5'end adding Hind III and Spe I dangling endonuclease site; annealed to form double chains are formed with restriction sites of insertion sequence, and linear pMIR-REPORT luciferase (Luc) reporter gene vector (pMIR-Luc). The recombinant vector (pMIR-Luc-miR), and enzyme digestion and sequencing.
The recombinant vector (pMIR-Luc-miR), the standard control vector pMIR-REPORT- -gal beta (pMIR- beta -gal) and precursor miRNAs (Pre-miRNAs) were transfected into HeLa cells (experimental group), 72h after HeLa cells were detected by luciferase (Luc) and beta galactosidase (beta -gal) activity, calculate the ratio of the two (Luc/ beta -gal). Three control groups were set up (no miRNA control group, Pre-miR-negative negative control group and Anti-miRNAs control group, Luc/ beta disproof) -gal ratio were compared, to verify the authenticity of the miRNAs target.
5, miR-520h promotes the function of hematopoietic development
In the selection of CD34+CD38- high expression of hematopoietic stem cells in miR-520h and study its role in promoting development of hematopoiesis. Flow cytometry sorting of CD34+ cells, miR-520h cells transfected with CD34+, 24h after transfection by CFC assay, CD34+ cell colony formation ability to change 72h after transfection were detected with FACS CD34+, changes in the proportion of CD34+CD38- cells. At the same time, the establishment of the three control groups (no miRNA control group, Pre-miR-129 negative control group and Anti-miR-520h control group, miR-520h analysis of disproof) role in promoting the development of hematopoiesis.
Research results
1, a successful development of a new mammal miRNAs detection chip
The matrix is divided into 4 sub arrays, each sub array with 23 rows, 21 rows, spacing of 185 m, the diameter is about 130 M. for each probe was repeated three times. In order to ensure the reliability of the experiment, repeatedly set various in different parts of the control chip, the chip samples had a total of 1754 site contains 469 miRNAs sequences, a total of 588 genes (gene duplication each 3 loci).
2, screening of CD34+CD38- hematopoietic stem cell development related miRNAs
Compared with CD34+ cells miRNAs expression microarray, HSC cells were screened in 31 miRNAs, which is 4 times lower than that of CD34+ cells for 22 miRNAs (including miR-129), 4 times higher than that of CD34+ cells of 9 miRNAs (including miR-526b* and miR-520h), the prediction of the miRNAs (PREDICTED_MIR) 4.
3, verification of miRNAs chip results by real time fluorescence quantitative RT-PCR (Q-RT-PCR)
We selected 6 differentially expressed in CD34+CD38- HSCs were miRNAs, Q-RT-PCR and verified with the microarray data rate reached 50%, two kinds of experimental results with miRNAs: CD34+CD38- low expression of hematopoietic stem cells miR-129 and CD34+CD38- hematopoietic stem cells have high expression of miR-526b* and miR-520h, the relative expression rate relative to CD34+ cells were 0.160 + 0.005,2.276 + 0.058 and 5.596 + 0.861.
4, bioinformatics analysis and verification of the target of miRNAs
Using online software Pictar, miRanda 3 and targetscan3.1 respectively predicted miR-129 target genes of miR-526b* and miR-520h. The results showed that the miR-129 in human, rat, dog, gorilla, 33 possible target genes are conserved, chicken and other animal including EIF2C3, CAMTA1, SH3KBP1, TGIF2, ING3, DLGAP2 and miRNA processing gene, transcription factor, signal transduction related. The miR-526b* only exists in human, the possible targets of 771, including ABCG2, EIF2C3, CAMK4, CSF2RA and so on signal transduction, transcription factor, hematopoietic development, apoptosis regulation, maintaining of stem cell function, gene of mature miRNAs. The processing of miR-520h there are 226 potential targets, in humans, mice and apes in the target gene for a total of 30, including ABCG2 1, ID (inhibitor of DNA binding 1), SRP19 (a signal recognition protein) and SMAD6 (a transcription factor) and stem cells The regulatory proteins related to cellular function maintenance, transcription, signal transduction and so on.
Select the potential targets of miR-129, EIF2C3 and CAMTA1, potential targets of miR-520h ABCG2 and SMAD6 to verify. By using the pMIR-REPORT luciferase reporter gene vector and identified by enzyme digestion and sequencing, the recombinant vector was successfully constructed respectively four kinds: pMIR-Luc-miR129-EIF2C3, pMIR-Luc-miR129-CAMTA1, pMIR-Luc-miR520h-ABCG2 and pMIR-Luc-miR520h-SMAD6.
The pMIR-REPORT luciferase reporter system showed that the target verification experiment, three control group (no miRNAs Pre-miR-negative blank control group, negative control group and Anti-miRNAs control group, Luc/ disproof) beta -gal ratio is close to the experimental group, miR-129 for EIF2C3 and CAMTA1, luciferase activity were 41% and 47%, indicating that EIF2C3 and CAMTA1 are the real target of miR-129; miR520h for ABCG2 and SMAD6, luciferase activity were 68% and 27%, indicating that ABCG2 is the real target of miR-520h, and the inhibition effect of miR-520h on SMAD6 is weak.
5, miR-520h to promote the development of hematopoietic stem cell development
CFC experiment: three control group (no miRNAs miR-129 blank control group, negative control group and Anti-miR-520h control group, all kinds of disproof) progenitor cell colony and colony count were similar to that of the control group CFU-E, BFU-E, CFU-GM and CFU-GEMM and the total number of colonies were 28.50 + 4.32,37.83 + 5.60,57.67 + 5.16,1.50 + 0.55 and 145.50 + 14.35, the experimental group were 37.83 + 7.70,46.5 + 4.85,68.17 + 7.73,1.67 + 0.52 and 174.33 + 17.82. by variance analysis showed that CFU-E, BFU-E, CFU-GM and the total number of colonies with significant difference (P0.05, n=6).
FACS analysis: three CD34+ control group and CD34+CD38- cells was similar to that of the control group CD34+ and the percentage of CD34+CD38- cells was 22.91% + 2.03 and 12.57% + 0.81, while the experimental group was 30.43% + 2.34 and 13.08% + 1.08. by the analysis of variance showed that the proportion of CD34+ cells in experimental group was higher, there was significant difference (P0.05, n=6), while the proportion of CD34+CD38- had little difference, no statistical difference (P0.05, n=6).
The experimental results show that miR-520h has the function of promoting the development and differentiation of hematopoietic stem cells.
conclusion
1, we successfully developed a new mammalian miRNAs detection chip. It showed that it is feasible to obtain CD34+CD38- hematopoietic stem cell related miRNAs by miRNAs gene chip, and also set up a technological platform for studying miRNAs expression profiles of other adult stem cells.
2, we screened the miRNAs expression profiles of CD34+CD38- hematopoietic stem cell development, and partially verified by real-time quantitative RT-PCR, which laid a foundation for exploring miRNAs involved in the molecular mechanism and biological function of CD34+CD38- hematopoietic stem cell development.
3, to verify the miR-129 and miR-520h part of the target, indicated that miRNAs regulated by EIF2C3, CAMTA1, ABCG2 in miRNA processing, transcription factor, signal transduction, function of stem cells to maintain target genes, and thus participate in hematopoietic stem cell development, and the formation of the miRNAs gene network plays a synergistic or antagonistic effects on hematopoiesis stem cell development process.
4, miR-520h has a significant role in promoting the development of hematopoietic stem cells and progenitor cells to differentiation. The results of miRNAs as not only has important application prospect to promote the exploration of molecular target of hematopoietic development, but also for the development of biology, has the important theory significance and development of tissue engineering and regenerative medicine.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

【引证文献】

相关硕士学位论文 前1条

1 潘长福;低温环境下microRNA对小鼠脑外伤后海马区神经干细胞增殖分化的调控作用[D];南昌大学;2011年

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