摘要
心血管疾病作为人类的第一大疾病,其高发病率及死亡率一直受到世界各国的关注。其生理病理的机制和治疗也一直是心血管领域的研究热点。目前,我国心血管病的发病率和死亡率逐年上升,已接近发达国家水平,严重影响着人民的健康状况,给国民经济带来不可估量的损失。但迄今为止,对心血管疾病的发病机制仍未十分明确。miRNAs(miRNAs)是一种大小约21—26个碱基的单链小分子、非编码RNA,具有高度进化保守性,并在翻译水平调节基因表达。他们广泛参与动植物生长发育、细胞分化、增殖与凋亡、激素分泌、肿瘤形成等各种过程。miRNA在心血管系统的发生、发育及心血管疾病的发生中也发挥了极其重要的作用。前期研究在构建成人正常心脏组织miRNA基因cDNA文库时发现,miR-126约占整个克隆总数的20%—23%,根据以往研究的结果,miRNA的克隆数目往往与该miRNA基因表达量呈正相关。我们由此认为miR-126是一个人类心脏高表达的miRNA。因此,拟选定该miRNA,对其功能进行深入研究。本课题拟从以下几个方面研究:首先我们拟在组织及细胞水平检测miR-126的表达谱,明确miR-126在不同组织细胞的表达水平;其次,拟在293细胞中初步研究miR-126与VEGF 3'-UTR的关系,通过外源表达miR-126和携带VEGF 3'-UTR的报告基因,通过检测报告基因,明确miR-126对靶基因表达的调节。再次,构建miR-126的慢病毒表达载体,并包装重组慢病毒颗粒,检测病毒梯度,建立miR-126过表达的稳转细胞株,利用分子生物学手段RPA、RT-PCR及Western blot明确上调miR-126表达对VEFG mRNA和蛋白表达的影响。另外,将表达miR-126的慢病毒感染内皮细胞及不同的肺癌细胞株在细胞水平检测miR-126对VEGF表达的影响,最后我们用稳转表达lv-mir-126的A549细胞株接种到裸鼠体内,在肿瘤动物模型体内确定miR-126对VEGF表达的调控作用。
本研究的目的是检测miR-126的表达谱,比较其组织及细胞水平的表达差异,并初步探讨其高表达的作用。
第一部分:miR-126的表达谱分析
目的:分析miR-126在人体各组织及部分细胞的表达分布情况。
方法:1、运用RNA酶保护法(RNA Protective Assay,RPA)检测:根据miR-126设计DNA片段(正义链),并添加与mirVana~(TM) miRNA探针制备试剂盒中T7启动子3'末端匹配的序列,合成DNA片段,然后与T7启动子引物片段杂交,并使用Klenow DNA聚合酶补平,然后使用T7 RNA聚合酶转录32P标记的反义探针,加入DNaseⅠ消化DNA模板,使用聚丙烯酰胺凝胶电泳分离探针,洗脱、沉淀并且重悬RNA探针,用于RNA酶保护检测miR-126。2、运用RT-PCR方法检测:根据miR-126的序列设计其特异的颈环状逆转录引物及PCR扩增的前向及逆向引物,提取不同组织和细胞的RNA在反转录酶的作用下先行反转录获取cDNA,cDNA在特异酶及引物的作用下行PCR,并在PCR反应体系中加入荧光探针,利用荧光信号累积实时监测整个PCR进程,最后通过标准曲线对miR-126进行定量分析。
结果:RPA及RT-PCR结果均显示miR-126主要在人的心脏、肺脏及胃组织中高表达,在肾脏、大脑和骨骼肌中仅少量表达或无表达;在脐静脉内皮细胞中miR-126表达量明显高于其他细胞,而在hela细胞、293细胞、A549细胞及Y90细胞等肿瘤细胞中miR-126的表达量极低。
结论:miR-126在人体各组织及细胞中表达有差异,其在心脏、肺及内皮细胞中高表达而在肿瘤细胞中表达量明显降低,提示miR-126可能参与这些组织细胞的病理生理过程。
第二部分:MiR-126靶基因的预测及初步验证
目的:通过生物信息学方法初步预测、并筛选出可能与心血管疾病密切相关的miRNA的靶基因,并通过荧光素酶报告基因系统确认miR-126对靶基因的作用。
方法:运用miRanda、TargetScan、PicTar、MiRanda和BibiServ等靶基因预测软件检索mir-126可能的靶基因,筛选出保守程度高、结合自由能低,并且与心血管病理生理过程相关的基因做为候选靶基因,我们选定VEGF为研究的靶基因,然后构建萤火虫荧光素酶表达载体,在293细胞中验证miR-126与VEGF之间的关系。将包含VEGF的3'-UTR区克隆入pMIR-REPORT~(TM) Luciferase vector(pLuc,Ambion)载体编码荧光素酶基因序列后,构建pLuc-VEGF/miR-126BS重组质粒。将pLuc-VEGF/miR-126BS重组质粒和pLV-miR-126表达质粒通过脂质体转染分方法转染293细胞,转染后48小时,收集细胞,然后进行荧光素酶检测。系统采用双荧光素酶检测系统,以海肾荧光素酶作为参照,检测细胞干预前后荧光素酶的变化趋势。同时采用点突变的方法使VEGF-3'UTR中miR-126的结合位点发生碱基突变,检测其抑制作用是否消失。
结果:(1)通过不同的的预测软件我们发现多种miR-126的可能靶基因,我们选定在心血管病理生理过程中发挥重要作用的VEGF作为我们研究的靶基因。(2)在293细胞中共转染pLV-miR-126,可以抑制pLuc-VEGF/miR-126BS载体表达荧光素酶,而当3'-UTR进行突变修饰后,miR-126不能够抑制其表达。
结论:生物学信息学预测VEGF可能是miR-126的靶基因,miR-126通过作用于VEGF的3'UTR端而起到抑制基因表达的作用。
第三部分:miR-126慢病毒生产
目的:构建miR-126的慢病毒表达载体,包装生产重组慢病毒,慢病毒滴度、活性测定。
方法:根据miR-126序列信息设计PCR引物,两端分别添加酶切位点和保护碱基。从A549细胞中提取人基因组DNA,以基因组DNA为模板,使用设计的引物进行PCR扩增,得到499bp的目的条带,切胶回收PCR产物,然后双酶切消化,琼脂糖凝胶电泳后切胶回收。表达载体质粒(pCDH-CMV-MCS-EF1-copGFP~(TM))也进行双酶切消化,电泳后切胶回收。连接目的片段和表达载体,连接产物转化感受态菌株DH5a,涂布平板,过夜培养,使用PCR的方法进行阳性菌落鉴定。接种阳性菌落于LB培养基,摇床过夜培养,提取质粒,然后使用载体多克隆位点上游的引物进行测序。分析测序结果,选择测序正确的重组质粒,摇床扩增其对应的转化菌株,使用去内毒素质粒抽提试剂盒进行无内毒素质粒DNA的提取。
复苏293TN细胞,经过两三次传代培养,调整细胞至适合转染状态。接种细胞至10cm细胞培养皿。使用脂质体转染的方法进行共转染实验,即将miR-126慢病毒表达载体以及慢病毒包装质粒混合物(Lentivirus Package plasmid mix)共转染293TN细胞。使用慢病毒表达载体上携带的绿色荧光基因copGFP作为荧光标记物,观察转染效率。转染后24小时,荧光显微镜下观察转染效率,并且对细胞进行换液处理。转染后48小时,收取细胞上清液,使用0.45um的微孔膜过滤去掉细胞碎片,冰浴保存。使用梯度稀释法测定病毒滴度。
结果:(1)成功调取miR-126基因序列,构建miR-126的慢病毒表达载体。(2)成功进行了LV-miR-126重组病毒的生产和滴度测定。(3)慢病毒感染293细胞后,能够大大增强目的基因的表达。
结论:通过慢病毒途径可以有效增强miR-126在宿主细胞中的表达。
第四部分:在内皮细胞中分析miR-126与VEGF表达之间的关系
目的:在内皮细胞中分析miR-126与VEGF表达之间的调控关系
方法:(1)使用LV-miR-126慢病毒颗粒感染内皮细胞,感染后72小时,收集细胞,然后分别提取总RNA和总蛋白,通过RPA和West Blot的方法观察miR-126与VEGF之间的关系。(2)使用慢病毒LV-miR-126和对照LV-GFP感染内皮细胞,感染后72小时,取200μl细胞传代到96孔板中,分别孵育24,48,72个小时,然后加入10μl MMT试剂,再孵育4小时,去除上清液,加入DMSO溶解,然后在酶标仪上检测OD570,并使用OD630作为参照。
结果:(1)在内皮细胞高表达miR-126,VEGF的表达水平明显减低。(2)使用慢病毒感染内皮细胞后,细胞可以高表达miR-126,但其增殖受到抑制。
结论:在内皮细胞中过表达miR-126,可以抑制VEGF表达,并且抑制内皮细胞增殖。推测miR-126可能通过调节VEGF参与了内皮相关疾病的病理生理过程。
第五部分:动物模型分析miR-126与VEGF表达之间的关系
目的:采用活体动物模型,分析miR-126与VEGF表达之间的关系
方法:(1)在肺癌细胞株A549、Y-90和SPC-A1中研究过表达miR-126对VEGF及细胞周期的影响(2)使用慢病毒LV-miR-126和对照LV-GFP感染上述三种肺癌细胞株,感染后48小时,收集细胞,然后使用70%乙醇固定1小时,然后用PI染色,进行流式细胞仪检测细胞周期。(3)使用Lv-miR-126和LV-GFP感染A549细胞,摸索适合细胞感染的最佳MOI值。根据最佳MOI值,进行A549细胞的病毒感染实验。感染后每隔24小时进行观察,待绿色荧光蛋白表达稳定后,使用有限稀释法进行单细胞克隆的筛选。得到细胞克隆之后,通过RT-PCR和West Blot的方法对细胞克隆进行性能检测。(4)使用稳定的A549/LV-miR-126和A549/LV-GFP接种到裸鼠体内,每只小鼠在左前足腋下接种2*10~6细胞。在25天后,处死小鼠,剪下肿瘤,然后称重,确定miR-126过表达后对体内肿瘤模型VEGF的抑制作用。
结果:(1)在肺癌细胞株A549、Y-90和SPC-A1高表达miR-126,可抑制VEGF的表达及抑制细胞增殖。(2)在肺癌细胞株A549、Y-90和SPC-A1使用慢病毒感染高表达miR-126,提高了处在G1期的细胞的比例,而S期细胞的比例则发生减少。(3)LV-miR-126稳定转染的A549细胞株在裸鼠模型中的所形成的肿瘤重量明显低于对照细胞所成肿瘤。
结论:在肺癌细胞中过表达miR-126,可以抑制VEGF表达,并且通过调节细胞周期,抑制肿瘤细胞增殖。并且在动物体内进一步证实miR-126对VEGF表达的抑制作用。
Background:Cardiovascular diseases are the most common human diseases,which have the high morbidity and mortality and aroused extensive concern in the international community.Its physiological and pathological mechanisms and treatment have also been hot spots all around the world.At present,the morbidity and mortality of cardiovascular disease are increasing every year,which have seriously affected on the people's health status and bring an immeasurable loss to the national economy.Howerer,the pathogenesis of cardiovascular disease has not yet very clear.
MiRNAs(miRNAs) are single-stranded small molecule non-coding RNA containing about 21-26 bases,which are highly conservative in evolution and regulate the gene expression at the transcriptional level.Posttranscriptional regulation by miRNAs is important for many aspects of development,cell differentiation,proliferation and apoptosis, hormone secretion and tumor formation.MiRNAs also play an extremely important role in the cardiovascular development and cardiovascular diseases.In the past research contructing the miRNA gene cDNA library,we found that miR-126 represents about 20%-23%of the total clone.According to the findings of previous studies,the miRNA gene expression was positively correlated with the miRNA clone number.We thus consider miR-126 is a high expression miRNA in the human heart.Therefore,we select miR-126 for further research and investigate its function.
This study is to study the following aspects:First,we intend to detect the miR-126 expression in different tissues and cells,and we want to make clear wether the miR-126 highly expressed in the heart;Secondly,we predicted the target gene of miR-126 using bioinformatics and preliminary investigated the relationship between miR-126 and VEGF 3'-UTR in 293 cells by exogenous expressing of miR-126 and carrying the VEGF 3'-UTR reporter gene in 293 cells and detecting the reporter gene.Thirdly,we constructed a miR-126 lentiviral expression vector,and packaged the recombinant lentiviral particles, detected the virus titer.Then we set up stable miR-126 over-expressed transgenic cell lines (endothelial cell and lung cancer cell) and use RPA and Westblot to test the VEGF mRNA and protein expression when the the expression of miR-126 has increased.In addition,we used the stable expressed lentiviral miR-126 cells inoculated into nude mice to detect the effect of miR-126 over-expression on the inhibition of tumor models in vivo.
The significance of this study is:To detect the expression of miR-126 and to make clear that miR-126 is cardiac highly expressed.And then we use cell lines to investigate the effect of miR-126 on different potential targets.Accordingly,it provides theoretical gist for studying the heart physiological function,exploring the pathogenesis of cardiovascular disease,as well as the screening of new therapeutic targets.
PartⅠAnalysis of expression pattern of miR-126
Object:Analyze the distribution of miR-126 in various tissues and cells of human.
Methods:(1) The use of RPA method:Designed the DNA fragment according to miR-126,and added the sequence complementary to 3' sequence of the T7 promoter of mirVana miRNA Probe Construction Kit,and synthesized the oligonucleotide. Resuspended the oligonucleotide template to a concentration of 100μmol/L,and hybridized the template with T7 Promoter Primer of the kit,and used Klenow DNA polymerase to fill in.Transcription by T7 RNA polymerase was performed to produce 32P labeled probe.DNase I was added to digest the DNA template.The probes were purified by denaturing polyacrylamide gel,and were eluted from acrylamide gel slice and precipitated and resuspended to detect miR-126 in ribonuclease protection assay(RPA). Total RNA was extracted using Trizol from various tissues,5μg of total RNA was hybridized with the probes at 52℃for 16 h.RNases A/T1 were added and the mixture was incubated at 37℃for 30 min.The remained RNA was precipitated by ethanol and then separated on a 15%denaturing PAGE.The probes were visualized by autoradiography.(2) The use of RT-PCR method:The sequence-specific primers of miR-126 are designed in accordance with miR-126's sequenceir,and then we extracted the total RNA from different tissues and cells.Using the RNA and the RNA reverse transcriptase enzyme to obtain cDNA,cDNA is used as the template of PCR.The PCR reaction system was added with fluorescent probe,so we use the real-time monitoring of fluorescent signal to measure the quantity of miR-126.
Results:RPA and RT-PCR showed that miR-126 was expressed predominantly in lung,heart and stomach,and can be detected in spleen,but was absent in kidney,brain and skeletal muscle.The expression of miR-126 is high in HUVEC and very low in HeLa cell,293 cell,A549cell,and Y90cell.
Conclusion:The expression of miR-126 is different in different kinds of tissues and cells.MiR-126 is mainly expressed in heart,lung,and endothelial cell which might give us the cue that miR-126 probably participate the pathological and physical mechanism.
PartⅡThe prediction of miR-126 target gene and validation
Object:Predict the target genes of miR-126 using bioinformatics and verify its role on target genes using reporter gene system.
Methods:Firefly luciferase expression vector was constructed to verify the relationship between miR-126 and VEGF.3'-Untranslated region(3'-UTR) of VEGF was PCR-amplified from human cDNA library and the product was inserted into pMIR-REPORT~(TM) Luciferase vector(pLuc,Ambion),downstream of the firefly luciferase coding region to construct pLV-miR-126.PLuc-VEGF/miR-126BS and pLV-miR-126 were co-transfected into 293 cells with Lipofectamine reagent.After 48-hour incubation,the cells were harvested and subjected to Luciferase assay.Firefly luciferase activities were measured by double luciferase assays System(Promega),and Renilla luciferase was used as internal control.Point mutation was used to mutate the binding site of miR-126 in VEGF-3'UTR to detect the inhibition effect.
Results:(1) There are several target genes of miR-126,and we choose VEGF which is associated with the cardiovascular disease to be the target of reaseach.(2) Co-transfection of pLV-miR-126 in 293 cells can inhibit the expression of luciferase of pLuc-VEGF/miR-126BS.When the 3'-UTR was mutated,the inhibition was eliminated.
Conclusion:The prediction of bioinformatics suggests that VEGF is the corresponding target gene of miR-126 which probably repress the the mRNA translation by combining with the 3'UTR of VEGF.
PartⅢProduct of miR-126 lentivirus
Object:C,onstruct Lentivirus vector to express miR-126,produce pseudoviral particles and determine its titer.
Methods:PCR primers were designed according to miR-126 sequence,restriction endonuclease digesting sequences and protection bases were added.Genomic DNA was extracted from A549 cells and used as template.The target PCR products were purified by recovery from agarose electrophoresis.The DNA fragment was digested by EcoRⅠand BamHⅠ,and purified.Expression vector,pCDH-CMV-MCS-EF1-copGFP~(TM) was digested by EcoR I and BamH I and recovered.And the fragment was inserted into the vector and transformed competent DH5a.The DH5a was plated to plating medium and were incubated overnight.PCR was conducted to determine positive clones.The positive clones were seeded to LB medium and were incubated overnight,and plasmids were isolated,and sequencing was conducted with the primer from upstream site of vector. The sequencing results were analyzed and the correct plasmid was amplified by corresponding strain culture.The plasmids were prepared by Endotoxin-Free Plasmid Midi Kit.
293TN cells were grown and plated for 2-3 times.The cells were seeded to a fresh 10-cm plate.And Lipofectamine 2000 was used to co-transfect the miR-126 lentiviral expression Vector and Lentivirus Package plasmid mix into 293 TN cells.The transfection efficiency was measured.Medium displacement was performed 24 hours after transfection.The supernatant was harvested,passed through a 0.45μm syringe filter to remove cell debris 48 hours after transfection.Pseudoviral titer was determined by serial dilution method.
Results:(1) miR-126 genomic sequence was amplified and lentiviral expression vector were constructed.(2) LV-miR-126 pseudovirus was packaged and the titer was determined.(3) 293 cell infected by LV-miR-126 can over-express miR-126.
Conclusion:LV-miR-126 infection can enhance the expression level of miR-126 in 293 cell lines.
PartⅣThe relationship between miR-126 and VEGF in endothelial cell
Object:Analyze the relationship between the expression of miR-126 and VEGF in endothelial cells.
Methods:(1)Human Umbilical Vein Endothelial Cell was infected by recombinant lentivirus LV-miR-126.Cells were harvested 72 hours after infection,and total RNA and protein was isolated,and RPA and west blot were used to confirm the relationship of miR-126 and VEGF.(2) EC was infected by recombinant lentivirus LV-miR-126.Cells were harvested 72 hours after infection,and re-seeded to 96-well plate and were incubated for 24,48 and 72 hours.10μl MMT reagent was added and the cells were incubated for another 4 hours.The supernatant was removed and 200μl DMSO was added to dissolve the formazan crystals.Optical density value(OD) of each sample was measured at a wavelength of 570nm(630 nm as a reference) on a microplate reader.
Results:In vitro,infection of LV-miR-126 in HUVEC can inhibit the expression of VEGF and the proliferation of HUVEC
Conclusion:Over-expression of miR-126 in HUVEC can inhibit the expression of VEGF and repress the proliferation of endothelial cells,which indicate that miR-126 may take part in the atherosclerosis by binding to the 3'UTR of VEGE
PartⅤThe relationship between miR-126 and VEGF in vivo
Object:Analyze the relationship between the expression of miR-126 and VEGF in vivo.
Methods:(1) A549,Y-90 and SPC-A1 cell lines were infected by recombinant lentivirus LV-miR-126.RPA and west blot were used to confirm the relationship of miR-126 and VEGF.MMT was used to detect the cell proliferation.(2)A549,Y-90 and SPC-A1 cell lines were infected by recombinant lentivirus LV-miR-126.Cells were harvested 48 hours after infection,typsinized and fixed with 70%ethanol on ice for 1 hour.The cell suspension was stained by propidium idide(PI) and was measured by flow cytometry.(3)A549 cells were infected by Lv-miR-126 and LV-GFP,and optimal MOI were determined.A549 cells were infected with optimal MOI.When the GFP level of infected cells was stable in 24-hour observation,limiting dilution assay was used to screen monoclone.When clones were selected,RT-PCR and west blot were conducted to detect the expression of miR-126 and VEGF.(4)Viable 2×106 of stable A549/LV-miR-126 and A549/LV-GFP were injected in the left foreleg armpit of nude mouse to establish cancer model.25 days after injection,all the mice were euthanized. The tumors were excised and weighted to determine the inhibitory effect of over-expression of miR-126 on cancer in vivo.
Results:(1) Over-expression of miR-126 in lung cancer lines A549,Y-90 and SPC-A1 can inhibit expression of VEGF and the proliferation of A549,Y-90 and SPC-A1 cell lines.(2) Infection of LV-miR-126 can increase the percentage of cells at G1 phase and decrease the percentage of cells at S phase in A549,Y-90 and SPC-A1 cell lines.(3) The weight of tumors formed by A549 cells with stable miR-126 expression in nude mice was lower than control group.
Conclusion:Over-expression of miR-126 in lung cancer cell lines,can inhibit the expression of VEGF and repress the proliferation of cancer cells and modulate cell cycles and decrease its tumorigenicity in nude mice by binding to the 3'UTR of VEGF.
引文
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