小鼠肾脏缺血-再灌注损伤miRNAs差异表达谱研究

吴奥; 王水良; 祝玲; 杨顺良

doi:10.3969/j.issn.1674-7445.2017.02.009

小鼠肾脏缺血-再灌注损伤miRNAs差异表达谱研究

doi: 10.3969/j.issn.1674-7445.2017.02.009

350025 福州，解放军福州总医院泌尿外科全军器官移植研究所厦门大学附属东方医院（吴奥、王水良、祝玲、杨顺良）；武汉市第九人民医院泌尿外科（吴奥）

基金项目:

福建省科技厅引导性项目 2015Y5007

详细信息

通讯作者:
杨顺良, Email：ysliang@medmail.com.cn

中图分类号: R617, R-332
计量
- 文章访问数: 295
- HTML全文浏览量: 204
- PDF下载量: 11
- 被引次数: 0
出版历程
- 收稿日期: 2016-12-18
- 网络出版日期: 2021-01-19
- 刊出日期: 2017-03-15

Differential gene expression profile of miRNAs in mouse models with renal ischemia-reperfusion injury

Department of Urology Surgery, Fuzhou General Hospital, Organ Transplantation of Dongfang Hospital Affiliated to Xiamen University, Fuzhou 350025, China

More Information

Corresponding author: Yang Shunliang, Email:ysliang@medmail.com.cn

摘要

摘要: 目的筛选小鼠肾脏缺血-再灌注损伤（IRI）差异性表达的微小核糖核酸（miRNAs），为进一步深入阐明肾脏IRI发生、发展的分子机制奠定基础。方法应用微型动脉夹夹闭小鼠双侧肾动脉制备急性肾缺血损伤模型，将15只小鼠分为IRI组和假手术组（E组），IRI组再分为A组（缺血时间45 min，再灌注时间24 h）、B组（缺血时间25 min，再灌注时间24 h）、C组（缺血时间45 min，再灌注时间4 h）、D组（缺血时间25 min，再灌注时间4 h），每组3只。采用肾脏组织形态学改变和肾功能评估结果来鉴定各组小鼠的IRI程度；采用miRNAs芯片聚类分析对小鼠IRI特定缺血时间（25、45 min）和再灌注时间（4、24 h）下肾脏差异性表达差异表达的miRNAs进行筛选鉴定；采用实时定量逆转录-聚合酶链反应（qRT-PCR）对小鼠IRI后差异表达miRNAs芯片miR-695与miR-145进行验证。结果肾脏组织形态学改变和肾功能评估结果显示成功建立IRI模型。与假手术组相比，肾脏IRI组共检出71种差异性表达显著的miRNAs，其中30种下调表达，41种上调表达。qRT-PCR法检测结果表明，以E组特定miRNAs的表达量标准化为1，miR-695及miR-145在肾脏IRI组的相对表达量分别为11.82和0.31（均为P＜0.05），与芯片结果基本一致。结论肾脏IRI后，miRNAs表达谱发生差异性改变，这些差异性表达的miRNAs将可作为肾脏IRI的分子标志物而具有潜在的临床及科研应用价值。
- 肾组织 /
- 肾动脉夹闭 /
- 缺血-再灌注损伤 /
- 微小核糖核酸（miRNAs） /
- 芯片 /
- miR-695 /
- miR-145 /
- 基因差异表达谱 /
- 实时定量逆转录-聚合酶链反应
Abstract: Objective To screen the differentially-expressed microRNAs (miRNAs) in mouse models with renal ischemia-reperfusion injury (IRI), aiming to offer foundation for unraveling the molecular mechanism of the incidence and progression of IRI. Methods The mouse models with acute IRI were established by renal artery clamping. Fifteen mice were divided into the IRI group and sham surgery group (group E). The animals in the IRI group were subdivided into the A group (45 min ischemia followed by 24 h reperfusion), B group (25 min ischemia followed by 24 h reperfusion), C group (45 min ischemia followed by 4 h reperfusion) and D group (25 min ischemia followed by 4 h reperfusion) (n=3 for each group). The severity of IRI was evaluated by histological changes and renal function. The differentially-expressed miRNAs in the IRI mouse models at different ischemia time (25 and 45 min) and reperfusion time (4 and 24 h) were screened by using cluster analysis of miRNAs microarray data. The differential expression of miR-695 and miR-145 was validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Results Both histological changes and renal function confirmed that the IRI mouse models were successfully established. Compared with the sham surgery group, 71 differentially-expressed miRNAs were detected in the IRI group including 30 down-regulated miRNAs and 40 up-regulated miRNAs. The results of qRT-PCR demonstrated that if the standardized expression level of miRNAs in the E group was 1, the relative expression levels of miR-695 and miR-145 were 11.82 and 0.31 in the IRI group (both P < 0.05), which were consistent with the chip results. Conclusions After renal IRI, different changes occur in the gene expression profile of miRNAs. These differentially-expressed miRNAs act as molecular biomarkers for renal IRI with potential clinical and scientific research values.
- Renal tissue /
- Renal artery clamping /
- Ischemia-reperfusion injury /
- Micro ribonucleic acid (miRNAs) /
- Chip /
- miR-695 /
- miR-145 /
- Differential gene expression profile /
- Quantitative real-time reverse transcription polymerase chain reaction

HTML全文

图 1 各组小鼠的肾脏组织形态学改变（HE，×100）

A~E图分别为A~E组的肾脏组织

Figure 1. Morphology of kidney tissues of mice in each group

下载: 全尺寸图片幻灯片

图 2 各组小鼠肾脏IRI差异表达的miRNAs（聚类分析图）

Figure 2. Cluster analysis of differentially expressed miRNAs in mouse kidney of IRI in each group

下载: 全尺寸图片幻灯片

图 3 miR-695、miR-145及内参U6的PCR扩增曲线

Figure 3. PCR amplification curves of miR-695, miR-145 and internal control U6

下载: 全尺寸图片幻灯片

图 4 miR-695与miR-145在假手术组与IRI组小鼠肾脏组织中相对表达量的比较

Figure 4. Comparison of relative expression levels of miR-695 andmiR-145 in kidney tissues of mice between IRI group and sham operation group

下载: 全尺寸图片幻灯片

表 1 各组小鼠IRI后肾功能的变化

Table 1. Changes of renal function of mice in each group (x±s)

组别	n	Scr (滋mol/L)	BUN (mmol/L)
A组	3	142.7±13.6^a	31.7±2.5^a
B组	3	105.3±9.4^a	19.0±1.0^a
C组	3	98.7±2.9^a	15.7±1.1^a
D组	3	67.0±7.0^a	13.7±0.6^a
E组	3	34.0±9.8	9.0±2.0
与E组比较，^aP＜0.05

下载: 导出CSV

参考文献(17)

[1]	Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay[J]. Nat Rev Genet, 2010, 11(9):597-610. DOI: 10.1038/nrg2843.
[2]	Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease[J]. Cell, 2012, 148(6):1172-1187. DOI: 10.1016/j.cell.2012.02.005.
[3]	Bellini MH, Coutinho EL, Filgueiras TC, et al. Endostatin expression in the murine model of ischaemia/reperfusion-induced acute renal failure[J]. Nephrology (Carlton), 2007, 12(5):459-465. DOI: 10.1111/j.1440-1797.2007.00850.x.
[4]	Lawrie CH, Gal S, Dunlop HM, et al.Detection of elevated levels of tumor-associated microRNAs in serum of patients with diffuse large B-cell lymphoma[J].Br J Haematol, 2008, 141(5):672-675.DOI: 10.1111/j.1365-2141.2008.07077.x.
[5]	Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection[J]. Proc Natl Acad Sci U S A, 2008, 105(30):10513-10518. DOI: 10.1073/pnas.0804549105.
[6]	Cortez MA, Bueso-Ramos C, Ferdin J, et al. MicroRNAs in body fluids-the mix of hormones and biomarkers[J]. Nat Rev Clin Oncol, 2011, 8(8):467-477. DOI: 10.1038/nrclinonc.2011.76.
[7]	Wang JF, Zha YF, Li HW, et al. Screening plasma miRNAs as biomakers for renal ischemia-reperfusion injury in rats[J].Med Sci Monit, 2014, 20:283-289. DOI: 10.12659/MSM.889937.
[8]	Wang N, Zhou Y, Jiang L, et al. Urinary MicroRNA-10a and MicroRNA-30d serve as novel, sensitive and specific biomarkers for kidney injury[J]. PLoS One, 2012, 5(12):e51140. DOI: 10.1371/journal.pone.0051140.
[9]	王博, 徐达, 王锡智, 等.青藤碱减轻小鼠肾脏缺血再灌注损伤[J].中华器官移植杂志, 2011, 32(2):73-77. DOI: 10.3760/cma.j.issn.0254-1785.2011.02.002. Wang B, Xu D, Wang XZ, et al. Protective effects of sinomenine on renal ischemia/reperfusion injury in mice[J]. Chin J Organ Transplant, 2011, 32(2):73-77.DOI: 10.3760/cma.j.issn.0254-1785.2011.02.002.
[10]	赵海红, 乔晞, 李荣山, 等.肾上腺髓质素对大鼠肾缺血再灌注损伤中肾小管上皮细胞凋亡的影响及机制研究[J].中华泌尿外科杂志, 2011, 7(32):449-453. DOI: 10.3760/cma.j.issn.1000-6702.2011.07.005. Zhao HH, Qiao X, Li RS, et al. Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury[J]. Chin J Urol, 2011, 7(32):449-453. DOI: 10.3760/cma.j.issn.1000-6702.2011.07.005.
[11]	Liangos O, Tiouart H, Perianayagam MC, et al. Comparative analysis of urinary biomakers for early detection of acute kidney injury following cardiopulmonary bypass[J].Biomakers, 2009, 14(6):423-431. DOI: 10.1080/13547500903067744.
[12]	Godwin JG, Ge X, Stephan K, et al. Identification of a microRNA signature of renal ischemia reperfusion injury[J]. Proc Natl Acad Sci U S A, 2010, 107(32):14339-14344. DOI: 10.1073/pnas.0912701107.
[13]	Park KM, Kramers C, Vayssier-Taussat M, et al. Prevention of kidney ischemia/reperfusion induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction[J]. J Biol Chem, 2002, 277(3):2040-2049. DOI: 10.1074/jbc.M107525200.
[14]	查宜凤, 王嘉锋, 李和文, 等.缺血再灌注损伤大鼠肾脏miRNA表达谱的筛查与分析[J].第二军医大学学报, 2014, 35(5):465-470. DOI: 10.3724/SP.J.1008.2014.00465. Zha YF, Wang JF, Li HW, et al. miRNA expression profile in rat kidney during renal ischemia/reperfusion injury: screening and analysis[J]. Acad J Sec Milit Med Univ, 2014, 35(5):465-470. DOI: 10.3724/SP.J.1008.2014.00465.
[15]	王春阳, 叶冬波, 倪少滨, 等.缺血后处理对大鼠肾脏miRNAs表达谱的影响[J].现代生物医学进展, 2015, 15(28):5408-5413. DOI: 10.13241/j.cnki.pmb.2015.28.003. Wang CY, Ye DB, Ni SB, et al. Expression profiling of miRNAs in rat renal ischemic post-conditioning[J]. Prog Mod Biomed, 2015, 15(28):5408-5413. DOI: 10.13241/j.cnki.pmb.2015.28.003.
[16]	Jeyaseelan K, Lim KY, Armugam A. Micro RNA 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. DOI: 10.1161/STROKEAHA.107.500736.
[17]	郑平, 曾志涌. MicroRNA-134在缺氧预处理新生大鼠缺氧缺血脑组织中的表达及意义[J].广州医学院学报, 2011, 39(2):201-205. DOI: 10.3969/j.issn.1008-1836.2011.02.005. Zheng P, Zeng ZY. Expression of microRNA-134 in brain tissue of hypoxia-preconditioned neonatal rat hypoxic-ischemic models[J]. Acad J Guangzhou Med Col, 2011, 39(2):201-205. DOI: 10.3969/j.issn.1008-1836.2011.02.005.