缺血再灌注肾损伤后MiRNA-210、MiRNA-320和MiRNA-92a的变化及其与血管新生关系的探讨
|
摘要
目的:
肾缺血再灌注(Ischemia/Reperfusion,I/R)损伤是在缺血基础上恢复血流,导致组织损伤反而加重的现象,常见于肾部分切除术后和肾移植等过程中,是导致急性肾功能损伤的主要原因之一。肾缺血后造成肾脏血管受阻血流量减少,导致肾脏微血管损伤,改变小管代谢甚至坏死,影响肾脏血流。因而尽快恢复缺血区的血供是修复肾缺血损伤的关键之一。动物实验和临床研究均表明肾脏缺血后可诱导反应性血管新生现象,且伴随有多种血管生长因子的表达。研究发现,VEGF信号通路对血管的发生发展,包括细胞增殖、迁移、分化及血管生成等多个方面有重要调控功能,也是介导肾缺血后血管新生的主要信号通路。近年来,研究报道一些特异的miRNA可调控组织血管新生,缺血后,组织中一系列的miRNA表达发生显著改变。研究提示miRNAs这一作用可能是通过调控其靶标基因参与血管生成的信号通路如VEGF信号通路,影响血管生成。
本研究通过制作肾缺血/再灌注小鼠模型,观察缺血再灌注肾损伤后肾组织血管新生及miR-210、miR- 92a和miR-320的表达变化并探讨其靶向VEGF信号通路的关系,以期为缺血性肾损伤提供新的思路。积极探讨缺血性肾损伤后血管新生的分子机制也对于修复缺血性肾损伤具有重要意义。
方法:
1、制作缺血性肾损伤模型,采用微型动脉夹夹闭小鼠双侧肾蒂制备急性肾缺血/再灌注损伤模型。于I/R后4h、24h、48h、72h分批处死各组小鼠,取肾组织标本待检。通过观察血清肌酐(Scr)和尿素氮(BUN)及肾组织病理改变判断模型成功。
2、采用实时荧光定量PCR方法,观察组织缺血诱导的microRNAs( miRNA210、miRNA-92a、miRNA-320)的表达变化。
3、免疫组织化学染色检测CD31表达,判断缺血组织微血管内皮细胞增生状况;
4、采用Western blot和Real-time PCR方法观察与VEGF信号通路相关分子血管内皮生长因子及其受体(VEGF、Flk-1)各分子蛋白和基因表达水平的变化。
结果:
1、肾缺血再灌注模型判断:肾脏I/R损伤后24h血清Scr和BUN水平升高明显,与假手术组(sham)比较有统计学意义(P<0.05),HE染色结果显示肾缺血组织病理改变明显,判断肾缺血再灌注模型制作成功。
2、缺血性肾损伤后miRNA210、miRNA-92a和miRNA-320的表达变化:肾脏I/R4h和24h后,miR-210的表达明显上调,其上调倍数分别为2.02±0.29,5.58±0.16;miR-92a的表达上调,其上调倍数分别为3.23±0.74,1.53±0.33;而I/R4h和24h后,miR-320其上调倍数分别为2.13±0.95,3.43±1.05。
3、缺血性肾损伤后组织微血管密度变化:CD31免疫组化检测微血管密度结果显示,肾缺血后缺血组织中新生血管增生明显,与假手术组比较有统计学意义(P<0.05)。
4、缺血性肾损伤后VEGF信号通路相关分子的动态变化:RT-PCR结果显示,肾I/R损伤4h、24h后,缺血组织VEGF和Flk-1mRNA表达水平显著上调,其上调倍数分别为4.75±0.35,3.09±0.86和1.90±0.05,2.46±0.36(P<0.05)。Western Blot结果显示,肾I/R损伤24h组、72h组Flk-1蛋白条带均较假手术组蛋白条带深,蛋白表达量较高(P <0.05),提示I/R损伤后Flk-1蛋白的表达水平上升。
结论:
实验结果表明,肾缺血后组织反应性新生血管增生明显,miRNA210、miRNA-92a和miRNA-320的表达变化显著上调,且伴随着VEGF信号通路相关分子VEGF和Flk-1蛋白和基因表达水平上调。研究结果提示,肾缺血后miRNA210、miRNA-92a和miRNA-320可能通过靶向VEGF信号通路参与肾缺血后血管再生的调控过程。
Renal ischemia injury is in relation with renal transplantation and renal operation. As an important pathological process, the timely reconstruction of renal blood flow in ischemic region is a key treatment for renal ischemic injury. Animal experiments and clinical studies showed that compensatory angiogenesis was increased after issues ischemia. Most studies have confirmed that the VEGF pathway signalling has the critical regulatory role in endothelial cell proliferation, differentiation, migration and angiogenesis and involved in the regulation of ischemia-induced angiogenesis in tissue ischemia, which are perhaps the most important mechanism in regulation of angiogenesis in renal ischemia. Recently, studies have showed that microRNAs (miRNAs) are small noncoding RNAs and regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA that regulate physiological and pathological processes. And a few specific miRNAs targeting endothelial cell function and angiogenesis in tissue ischemia-induced have been identified. Our results showed that some of miRNAs expression changes after renal ischemia may be involved in targeting VEGF pathway signaling to regulate angiogenesis.
Methods: Mice were subjected to a standard renal I/R to induce acute kidney injury after 45 min of bilateral renal artery clamping. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. MiRNAs expression changes used Quantitative Real-time RT-PCR analysis at 4h and 24 h following I/R. VEGF and Flk-1 mRNA expression changes used Quantitative Real-time RT-PCR analysis at 4h and 24h following I/R. Flk-1 protein expression changes used western Blotting analysis at 24h and 72h following I/R.
Results: 1.Blood samples were obtained at 24 h and at the time of animal death to evaluate the degree of AKI by serum creatinine (SCr) and urea nitrogen(BUN)(P﹤0.05), and the renal tissue were obtained to evaluate histological changes after I/R at 24h.
2. Quantitative Real-time RT-PCR analysis showed that ischemic kidney injury significantly increased miRNA-210, miRNA-320 and miRNA-92a expression compared to the sham controls, with prominent changes at 4h and 24h after recovery (P< 0.05).
3. The immunohistochemistry staining results of CD31 showed a significant increase of microvessels in ischemic region in renal ischemia compared with control group.
4. VEGF and Flk-1 mRNA expression were increased in I/R injury at 4h and 24h compared to the sham controls (P<0.05). And Flk-1 protein expression was increased in I/R injury at 24h and 72h compared to the sham controls (P< 0.05).
Conclusion: Renal I/R to induce acute kidney injury (AKI) significantly increased miRNA-210, miRNA-320 and miRNA-92a expression with prominent changes at 4h and 24h after recovery.And compensatory angiogenesis was increased significantly after renal ischemia.Meanwhile,VEGF and Flk-1 mRNA expression were increased in I/R injury compared to the sham controls. And Flk-1 protein expression were also increased in renal I/R injury compared to the sham controls .These results provided evidence that miR-210,miRNA-320 and miRNA-92a may be somehow involved in the mechanisms of renal ischemic injury disease through targeting VEGF pathway to regulate angiogenesis.
肾缺血再灌注(Ischemia/Reperfusion,I/R)损伤是在缺血基础上恢复血流,导致组织损伤反而加重的现象,常见于肾部分切除术后和肾移植等过程中,是导致急性肾功能损伤的主要原因之一。肾缺血后造成肾脏血管受阻血流量减少,导致肾脏微血管损伤,改变小管代谢甚至坏死,影响肾脏血流。因而尽快恢复缺血区的血供是修复肾缺血损伤的关键之一。动物实验和临床研究均表明肾脏缺血后可诱导反应性血管新生现象,且伴随有多种血管生长因子的表达。研究发现,VEGF信号通路对血管的发生发展,包括细胞增殖、迁移、分化及血管生成等多个方面有重要调控功能,也是介导肾缺血后血管新生的主要信号通路。近年来,研究报道一些特异的miRNA可调控组织血管新生,缺血后,组织中一系列的miRNA表达发生显著改变。研究提示miRNAs这一作用可能是通过调控其靶标基因参与血管生成的信号通路如VEGF信号通路,影响血管生成。
本研究通过制作肾缺血/再灌注小鼠模型,观察缺血再灌注肾损伤后肾组织血管新生及miR-210、miR- 92a和miR-320的表达变化并探讨其靶向VEGF信号通路的关系,以期为缺血性肾损伤提供新的思路。积极探讨缺血性肾损伤后血管新生的分子机制也对于修复缺血性肾损伤具有重要意义。
方法:
1、制作缺血性肾损伤模型,采用微型动脉夹夹闭小鼠双侧肾蒂制备急性肾缺血/再灌注损伤模型。于I/R后4h、24h、48h、72h分批处死各组小鼠,取肾组织标本待检。通过观察血清肌酐(Scr)和尿素氮(BUN)及肾组织病理改变判断模型成功。
2、采用实时荧光定量PCR方法,观察组织缺血诱导的microRNAs( miRNA210、miRNA-92a、miRNA-320)的表达变化。
3、免疫组织化学染色检测CD31表达,判断缺血组织微血管内皮细胞增生状况;
4、采用Western blot和Real-time PCR方法观察与VEGF信号通路相关分子血管内皮生长因子及其受体(VEGF、Flk-1)各分子蛋白和基因表达水平的变化。
结果:
1、肾缺血再灌注模型判断:肾脏I/R损伤后24h血清Scr和BUN水平升高明显,与假手术组(sham)比较有统计学意义(P<0.05),HE染色结果显示肾缺血组织病理改变明显,判断肾缺血再灌注模型制作成功。
2、缺血性肾损伤后miRNA210、miRNA-92a和miRNA-320的表达变化:肾脏I/R4h和24h后,miR-210的表达明显上调,其上调倍数分别为2.02±0.29,5.58±0.16;miR-92a的表达上调,其上调倍数分别为3.23±0.74,1.53±0.33;而I/R4h和24h后,miR-320其上调倍数分别为2.13±0.95,3.43±1.05。
3、缺血性肾损伤后组织微血管密度变化:CD31免疫组化检测微血管密度结果显示,肾缺血后缺血组织中新生血管增生明显,与假手术组比较有统计学意义(P<0.05)。
4、缺血性肾损伤后VEGF信号通路相关分子的动态变化:RT-PCR结果显示,肾I/R损伤4h、24h后,缺血组织VEGF和Flk-1mRNA表达水平显著上调,其上调倍数分别为4.75±0.35,3.09±0.86和1.90±0.05,2.46±0.36(P<0.05)。Western Blot结果显示,肾I/R损伤24h组、72h组Flk-1蛋白条带均较假手术组蛋白条带深,蛋白表达量较高(P <0.05),提示I/R损伤后Flk-1蛋白的表达水平上升。
结论:
实验结果表明,肾缺血后组织反应性新生血管增生明显,miRNA210、miRNA-92a和miRNA-320的表达变化显著上调,且伴随着VEGF信号通路相关分子VEGF和Flk-1蛋白和基因表达水平上调。研究结果提示,肾缺血后miRNA210、miRNA-92a和miRNA-320可能通过靶向VEGF信号通路参与肾缺血后血管再生的调控过程。
Renal ischemia injury is in relation with renal transplantation and renal operation. As an important pathological process, the timely reconstruction of renal blood flow in ischemic region is a key treatment for renal ischemic injury. Animal experiments and clinical studies showed that compensatory angiogenesis was increased after issues ischemia. Most studies have confirmed that the VEGF pathway signalling has the critical regulatory role in endothelial cell proliferation, differentiation, migration and angiogenesis and involved in the regulation of ischemia-induced angiogenesis in tissue ischemia, which are perhaps the most important mechanism in regulation of angiogenesis in renal ischemia. Recently, studies have showed that microRNAs (miRNAs) are small noncoding RNAs and regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA that regulate physiological and pathological processes. And a few specific miRNAs targeting endothelial cell function and angiogenesis in tissue ischemia-induced have been identified. Our results showed that some of miRNAs expression changes after renal ischemia may be involved in targeting VEGF pathway signaling to regulate angiogenesis.
Methods: Mice were subjected to a standard renal I/R to induce acute kidney injury after 45 min of bilateral renal artery clamping. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. MiRNAs expression changes used Quantitative Real-time RT-PCR analysis at 4h and 24 h following I/R. VEGF and Flk-1 mRNA expression changes used Quantitative Real-time RT-PCR analysis at 4h and 24h following I/R. Flk-1 protein expression changes used western Blotting analysis at 24h and 72h following I/R.
Results: 1.Blood samples were obtained at 24 h and at the time of animal death to evaluate the degree of AKI by serum creatinine (SCr) and urea nitrogen(BUN)(P﹤0.05), and the renal tissue were obtained to evaluate histological changes after I/R at 24h.
2. Quantitative Real-time RT-PCR analysis showed that ischemic kidney injury significantly increased miRNA-210, miRNA-320 and miRNA-92a expression compared to the sham controls, with prominent changes at 4h and 24h after recovery (P< 0.05).
3. The immunohistochemistry staining results of CD31 showed a significant increase of microvessels in ischemic region in renal ischemia compared with control group.
4. VEGF and Flk-1 mRNA expression were increased in I/R injury at 4h and 24h compared to the sham controls (P<0.05). And Flk-1 protein expression was increased in I/R injury at 24h and 72h compared to the sham controls (P< 0.05).
Conclusion: Renal I/R to induce acute kidney injury (AKI) significantly increased miRNA-210, miRNA-320 and miRNA-92a expression with prominent changes at 4h and 24h after recovery.And compensatory angiogenesis was increased significantly after renal ischemia.Meanwhile,VEGF and Flk-1 mRNA expression were increased in I/R injury compared to the sham controls. And Flk-1 protein expression were also increased in renal I/R injury compared to the sham controls .These results provided evidence that miR-210,miRNA-320 and miRNA-92a may be somehow involved in the mechanisms of renal ischemic injury disease through targeting VEGF pathway to regulate angiogenesis.
引文
[1] Basile DP. The endothelial cell in ischemic acute kidney injury: implications for acute and chronic function [J]. Kidney Int, 2007,72(2): 151-156.
[2] Velazquez O, Snyder R, Liu Z J, et al. Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like 3-dimensional networks [J]. FASEB J, 2002,16 (10): 1316-1318.
[3]Yancopoulos GD.Vascular-specific growth factors and blood vessel formation [J].Nature, 2000, 407(6801):242-248.
[4] Mi H, Haeberle H, Barres B A. Induction of astrocyte differentiation by endothelial cells [J]. JNeurosci, 2001, 21 (5): 1538-1547.
[5] Larrivee B, A. Karsan. Signaling pathways induced by vascular endothelial growth factor (review) [J]. IntJ Mol Med,2000,5(5): 447-456.
[6] Hicklin DJ, LM Ellis.Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis[J]. JClin Oncol, 2005. 23(5): 1011-1027.
[7] Rogers SA, Hammermann MR, et al. Transplantation of rat metanephroi into mice [J].Am J Physiol 2001; 280(6):1865-1869.
[8] Kroll, J. , J. Waltenberger.The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells [J] . Biol Chem, 1997. 272(51): 32521-32527.
[9] Waltenberger J, et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor [J] .Biol Chem, 1994. 269(43): 26988-26995.
[10] Filip, A. MiRNA--new mechanisms of gene expression control [J]. Postepy Biochem, 2007. 53(4): 413-419.
[11] Yang WJ, Yang DD, Na S, et al. Dicer is required for embryonic angiogenesis during mouse development [J] .J Biol Chem 2005; 280(10):9330-9335.
[12] Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human miRNA [J] .Nature Genet 2005; 37(7):766-770.
[13] Hua Z, et al.MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia [J]. PLoS One, 2006,1: e116.
[14] Fasanaro, P., et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. [J]J Biol Chem, 2008. 283(23): 15878-15883.
[15] Pulkkinen K, Malm T, Turunen M, et al. Hypoxia induces microRNA miR-210 in vitro and in vivo ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210. [J]FEBS Lett, 2008, 582(16): 2397-2401.
[16] Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions[J]. Trends Cardiovasc Med, 2007, 17(5): 145-151.
[17] Ren XP, Wu J,Wang X, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20[J]. Circulation, 2009, 119(17): 2357-2366.
[18] Wang XH, Qian RZ, Zhang W,et al. MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats[J]. Clin Exp Pharmacol Physiol, 2009, 36(2):181-188.
[19] Bonauer A, Carmona G ,Iwasaki M ,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J]. Science 2009; 324(5935): 1710-1713.
[20] Garmy, Susini B, JA Varner.Roles of integrins in tumor angiogenesis and lymphangiogenesis. [J] Lymphat Res Biol, 2008, 6(3-4): 155-163.
[21]Weidner N, Semple J P, Welch W R, et al. Tumor angiogenesis and metastasis correlation in invasive breast carcinoma [J]. N Engle J Med, 1991, 324 (1): 1-8.
[22] Chen C,Ridzon DA,Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR [J]. Nucleic Acids Res, 2005, 33(20):e179.
[23] Lee RC, Feinbaum RL, AmbrosV. The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75 (5): 843-854.
[24] Reinhart BJ, Slack F, J.Basson M, et al.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J].Nature,2000,403(6772): 901-906.
[25] Ambros V. microRNAs: tiny regulators with great potential [J].Cell, 2001,107 (7): 823-826.
[26] Krol J, D Kaczynska, WJ Krzyzosiak. MicroRNA--members of non-coding RNA family [J]. Postepy Biochem, 2003, 49(4): 214-228.
[27] Li CM, Zheng JG, and Du GS.miRNA: a new regulator of gene expression [J]. Yi Chuan, 2004, 26(1): 133-1366.
[28] Liang RQ, Li W, Li Y, et al.An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantumdot and nanogold probe [J]. Nucleic Acids Res, 2005, 33(2):e17
[29] Thomson JM,Parker J,Perou CM, et al. A custom microarray platformfor analysis of microRNA gene expression [J].Nat Methods,2004,1 (1): 47253
[30] Nelson PT, Baldwin DA, Scearce LM et al .Microarray-based, High-throughput gene expression profiling of microRNAs [J]. Nat Methods, 2004,1(2) : 155-161
[31] Miska EA , AlvarezSaavedra E , Townsend M, et al. Microarray analysis of microRNA expression in the developing mammalian brain[J].Genome Biol,2004,5 (9) : R68.
[32] Sun Y, Koo S,White N,et al. Development of a microarray to detect human and mouse microRNAs and characterization of expression in human organs[J]. Nucleic Acids Res, 2004, 32(22):e188.
[33]Baskerville S, Bartel D P. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes [J]. RNA, 2005, 11(3): 241-247.
[34] J.萨姆布鲁克,D.W.拉塞尔,等著,黄培堂,等译[M].分子克隆实验指南.北京:科学出版社, 2002(16):532.
[35] He HL, Jazdewski K, Li W, et al.The role of microRNA genes in papillary thyroid carcinoma [J]. PNAS, 2005, 102 (5):19075-19080.
[36] Hwang H.W, Wentzel E.A, Mendell JT.A hexamucleotide element directs microRNA nuclear import [J].Science, 2007, 315(1):97-100.
[37] Lee EJ, Gusev Y, Jiang J, et al [J].Expression profiling identifies microRNA signature in pancreatic cancer [J]. Int J Cancer, 2007, 120 (5):1046-1054.
[38] Schmittgen TD, Jiang J, Liu Q, et al.A high-throughput method to monitor the expression of microRNA precursors [J]. Nucleic Acids Res, 2004, 32(4):e43.
[39] JiangJ, Lee EJ, Gusev Y, et al. Real-time expression profiling of microRNA precursors in human cancer cell lines [J].Nucleic Acids Res , 2005 , 33(17) 5394-5403.
[40] Chen C, Ridzon DA, Broomer AJ,et al. Real-time quantification of microRNAs by stem-loop RT-PCR [J].Nucleic Acids Res, 2005, 33(20):e179.
[1] Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell, 1993,75(5): 843-854.
[2] Reinhart BJ, Slack F, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J].Nature, 2000, 403(6772): 901-906.
[3] Du T, Zamore PD. microPrimer: the biogenesis and function of microRNA [J].Development, 2005,132 (21): 4645-4652.
[4]FilipowiczW, Bhattacharyya SN, SonenbergN. Mechanisms of post-transcriptional regulationby microRNAs: are the answers in sight? [J] Nat Rev Genet 2008, 9(2): 102-114.
[5] Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation [J]. Science 2007,318 (5858): 1931-1934.
[6] Fish JE, Santoro MM, Morton SU, et al. miR-126 regulates angiogenic signaling and vascular integrity [J]. Dev Cell, 2008, 15(2): 272-284.
[7] van Solingen C, Seghers L, Bijkerk R, et al. Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis [J]. J Cell Mol Med, 2009, 13(8A): 1577-1585.
[8] Bonauer A ,Carmona G, Iwasaki M, et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J].Science ,2009, 324(5935): 1710-1713.
[9] Pulkkinen K, Malm T, Turunen M, et al. Hypoxia induces microRNA miR-210 in vitro and in vivo ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210 [J]. FEBS Lett, 2008, 582(16): 2397-2401.
[10] Fasanaro P, D'Alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3 [J].J Biol Chem ,2008,283(23): 15878-15883.
[11] Xiao-ping Ren, Jing-haiWu, Xiao-hong Wang, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20[J].Circulation ,2009, 119(17): 2357-2366.
[12] Wang Xin-hong, Qian Rui-zhe, Zhang Wei, et al. MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats [J].Clin Exp Pharmacol Physiol, 2009, 36(2): 181-188.
[13] Shen Ji-kui, Yang Xiao-ru, Xie Bing et al. MicroRNAs regulate ocular neovascularization [J].Mol Ther, 2008, 16(7): 1208-1216.
[14] Jeyaseelan K, Lim KY, Armugam A. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion [J].Stroke, 2008,39 (3): 959-966.
[15] Dharap A, BowenK, Place R, et al. Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome [J].J Cereb Blood Flow Metab, 2009, 29(4): 675-687.
[16] Roy SS,Khanna S, Hussain SR. et al. MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue [J].Cardiovasc Res , 2009, 82(1): 21-29.
[17] Yin Chang, Wang Xiao-yin, Kukreja RC. Endogenous microRNAs induced by heat-shock reduce myocardial infarction following ischemia-reperfusion in mice [J]. FEBS Lett, 2008, 582(30): 4137-4142.
[2] Velazquez O, Snyder R, Liu Z J, et al. Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like 3-dimensional networks [J]. FASEB J, 2002,16 (10): 1316-1318.
[3]Yancopoulos GD.Vascular-specific growth factors and blood vessel formation [J].Nature, 2000, 407(6801):242-248.
[4] Mi H, Haeberle H, Barres B A. Induction of astrocyte differentiation by endothelial cells [J]. JNeurosci, 2001, 21 (5): 1538-1547.
[5] Larrivee B, A. Karsan. Signaling pathways induced by vascular endothelial growth factor (review) [J]. IntJ Mol Med,2000,5(5): 447-456.
[6] Hicklin DJ, LM Ellis.Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis[J]. JClin Oncol, 2005. 23(5): 1011-1027.
[7] Rogers SA, Hammermann MR, et al. Transplantation of rat metanephroi into mice [J].Am J Physiol 2001; 280(6):1865-1869.
[8] Kroll, J. , J. Waltenberger.The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells [J] . Biol Chem, 1997. 272(51): 32521-32527.
[9] Waltenberger J, et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor [J] .Biol Chem, 1994. 269(43): 26988-26995.
[10] Filip, A. MiRNA--new mechanisms of gene expression control [J]. Postepy Biochem, 2007. 53(4): 413-419.
[11] Yang WJ, Yang DD, Na S, et al. Dicer is required for embryonic angiogenesis during mouse development [J] .J Biol Chem 2005; 280(10):9330-9335.
[12] Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human miRNA [J] .Nature Genet 2005; 37(7):766-770.
[13] Hua Z, et al.MiRNA-directed regulation of VEGF and other angiogenic factors under hypoxia [J]. PLoS One, 2006,1: e116.
[14] Fasanaro, P., et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. [J]J Biol Chem, 2008. 283(23): 15878-15883.
[15] Pulkkinen K, Malm T, Turunen M, et al. Hypoxia induces microRNA miR-210 in vitro and in vivo ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210. [J]FEBS Lett, 2008, 582(16): 2397-2401.
[16] Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions[J]. Trends Cardiovasc Med, 2007, 17(5): 145-151.
[17] Ren XP, Wu J,Wang X, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20[J]. Circulation, 2009, 119(17): 2357-2366.
[18] Wang XH, Qian RZ, Zhang W,et al. MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats[J]. Clin Exp Pharmacol Physiol, 2009, 36(2):181-188.
[19] Bonauer A, Carmona G ,Iwasaki M ,et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J]. Science 2009; 324(5935): 1710-1713.
[20] Garmy, Susini B, JA Varner.Roles of integrins in tumor angiogenesis and lymphangiogenesis. [J] Lymphat Res Biol, 2008, 6(3-4): 155-163.
[21]Weidner N, Semple J P, Welch W R, et al. Tumor angiogenesis and metastasis correlation in invasive breast carcinoma [J]. N Engle J Med, 1991, 324 (1): 1-8.
[22] Chen C,Ridzon DA,Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR [J]. Nucleic Acids Res, 2005, 33(20):e179.
[23] Lee RC, Feinbaum RL, AmbrosV. The C.elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75 (5): 843-854.
[24] Reinhart BJ, Slack F, J.Basson M, et al.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J].Nature,2000,403(6772): 901-906.
[25] Ambros V. microRNAs: tiny regulators with great potential [J].Cell, 2001,107 (7): 823-826.
[26] Krol J, D Kaczynska, WJ Krzyzosiak. MicroRNA--members of non-coding RNA family [J]. Postepy Biochem, 2003, 49(4): 214-228.
[27] Li CM, Zheng JG, and Du GS.miRNA: a new regulator of gene expression [J]. Yi Chuan, 2004, 26(1): 133-1366.
[28] Liang RQ, Li W, Li Y, et al.An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantumdot and nanogold probe [J]. Nucleic Acids Res, 2005, 33(2):e17
[29] Thomson JM,Parker J,Perou CM, et al. A custom microarray platformfor analysis of microRNA gene expression [J].Nat Methods,2004,1 (1): 47253
[30] Nelson PT, Baldwin DA, Scearce LM et al .Microarray-based, High-throughput gene expression profiling of microRNAs [J]. Nat Methods, 2004,1(2) : 155-161
[31] Miska EA , AlvarezSaavedra E , Townsend M, et al. Microarray analysis of microRNA expression in the developing mammalian brain[J].Genome Biol,2004,5 (9) : R68.
[32] Sun Y, Koo S,White N,et al. Development of a microarray to detect human and mouse microRNAs and characterization of expression in human organs[J]. Nucleic Acids Res, 2004, 32(22):e188.
[33]Baskerville S, Bartel D P. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes [J]. RNA, 2005, 11(3): 241-247.
[34] J.萨姆布鲁克,D.W.拉塞尔,等著,黄培堂,等译[M].分子克隆实验指南.北京:科学出版社, 2002(16):532.
[35] He HL, Jazdewski K, Li W, et al.The role of microRNA genes in papillary thyroid carcinoma [J]. PNAS, 2005, 102 (5):19075-19080.
[36] Hwang H.W, Wentzel E.A, Mendell JT.A hexamucleotide element directs microRNA nuclear import [J].Science, 2007, 315(1):97-100.
[37] Lee EJ, Gusev Y, Jiang J, et al [J].Expression profiling identifies microRNA signature in pancreatic cancer [J]. Int J Cancer, 2007, 120 (5):1046-1054.
[38] Schmittgen TD, Jiang J, Liu Q, et al.A high-throughput method to monitor the expression of microRNA precursors [J]. Nucleic Acids Res, 2004, 32(4):e43.
[39] JiangJ, Lee EJ, Gusev Y, et al. Real-time expression profiling of microRNA precursors in human cancer cell lines [J].Nucleic Acids Res , 2005 , 33(17) 5394-5403.
[40] Chen C, Ridzon DA, Broomer AJ,et al. Real-time quantification of microRNAs by stem-loop RT-PCR [J].Nucleic Acids Res, 2005, 33(20):e179.
[1] Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell, 1993,75(5): 843-854.
[2] Reinhart BJ, Slack F, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans [J].Nature, 2000, 403(6772): 901-906.
[3] Du T, Zamore PD. microPrimer: the biogenesis and function of microRNA [J].Development, 2005,132 (21): 4645-4652.
[4]FilipowiczW, Bhattacharyya SN, SonenbergN. Mechanisms of post-transcriptional regulationby microRNAs: are the answers in sight? [J] Nat Rev Genet 2008, 9(2): 102-114.
[5] Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate translation [J]. Science 2007,318 (5858): 1931-1934.
[6] Fish JE, Santoro MM, Morton SU, et al. miR-126 regulates angiogenic signaling and vascular integrity [J]. Dev Cell, 2008, 15(2): 272-284.
[7] van Solingen C, Seghers L, Bijkerk R, et al. Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis [J]. J Cell Mol Med, 2009, 13(8A): 1577-1585.
[8] Bonauer A ,Carmona G, Iwasaki M, et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice[J].Science ,2009, 324(5935): 1710-1713.
[9] Pulkkinen K, Malm T, Turunen M, et al. Hypoxia induces microRNA miR-210 in vitro and in vivo ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210 [J]. FEBS Lett, 2008, 582(16): 2397-2401.
[10] Fasanaro P, D'Alessandra Y, Di Stefano V, et al. MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3 [J].J Biol Chem ,2008,283(23): 15878-15883.
[11] Xiao-ping Ren, Jing-haiWu, Xiao-hong Wang, et al. MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20[J].Circulation ,2009, 119(17): 2357-2366.
[12] Wang Xin-hong, Qian Rui-zhe, Zhang Wei, et al. MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats [J].Clin Exp Pharmacol Physiol, 2009, 36(2): 181-188.
[13] Shen Ji-kui, Yang Xiao-ru, Xie Bing et al. MicroRNAs regulate ocular neovascularization [J].Mol Ther, 2008, 16(7): 1208-1216.
[14] Jeyaseelan K, Lim KY, Armugam A. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion [J].Stroke, 2008,39 (3): 959-966.
[15] Dharap A, BowenK, Place R, et al. Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome [J].J Cereb Blood Flow Metab, 2009, 29(4): 675-687.
[16] Roy SS,Khanna S, Hussain SR. et al. MicroRNA expression in response to murine myocardial infarction: miR-21 regulates fibroblast metalloprotease-2 via phosphatase and tensin homologue [J].Cardiovasc Res , 2009, 82(1): 21-29.
[17] Yin Chang, Wang Xiao-yin, Kukreja RC. Endogenous microRNAs induced by heat-shock reduce myocardial infarction following ischemia-reperfusion in mice [J]. FEBS Lett, 2008, 582(30): 4137-4142.