Differentially expressed wound healing-related microRNAs in the human diabetic cornea.
Differentially expressed wound healing-related microRNAs in the human diabetic cornea.
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DOI：
10.1371/journal.pone.0084425
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Saghizadeh M
中科院分区：
文献类型：
--
作者：
Funari VA;Winkler M;Brown J;Dimitrijevich SD;Ljubimov AV;Saghizadeh M
microRNA是一种强大的基因表达调控因子，但其在角膜疾病中的作用和潜在变化尚不清楚。我们的目的是通过微阵列分析来鉴定人糖尿病角膜中改变的miRNAs，并在体外培养的端粒酶永生化人角膜上皮细胞（HCEC）中检测它们对伤口愈合的影响。从年龄匹配的人尸检正常（n=6）和糖尿病（n=6）中央角膜提取总RNA，Flash Tag末端标记，并与Affyphy ® GeneChip® miRNA阵列杂交。通过定量RT-PCR（Q-PCR）和原位杂交（ISH）在独立样本中验证与糖尿病角膜相关的选择性miRNA。使用Lipofectamine 2000用人pre-miRTMmiRNA前体（h-miR）或其抑制剂（miR）转染HCEC。用P200移液器吸头刮伤融合的转染培养物。通过数码摄影监测伤口闭合。免疫组化染色和Western blot检测信号蛋白的表达。使用微阵列，29个miRNAs被鉴定为在糖尿病样本中差异表达。通过Q-PCR证实并进一步表征了在糖尿病角膜中表达增加倍数最高的两种miRNA候选物。用h-miR-146 a或h-miR-424转染HCEC显著延迟伤口闭合，但与对照相比，它们各自的miR显著增强伤口愈合。用h-miR-146 a或h-miR-424处理的细胞具有降低的p-p38和p-EGFR染色，但在Escheromir处理后，这些在接近伤口边缘的对照水平上增加。总之，在人糖尿病角膜中发现了几种表达增加的miRNAs，其中两种miRNAs抑制培养的角膜上皮细胞伤口愈合。人糖尿病角膜中miRNA表达的失调可能是伤口愈合异常的重要介质。
MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miRTMmiRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.
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影响因子：
64.8
作者：
Cordes KR;Sheehy NT;White MP;Berry EC;Morton SU;Muth AN;Lee TH;Miano JM;Ivey KN;Srivastava D
通讯作者：
Srivastava D
影响因子：
--
作者：
Chen, Gang;Shen, Zhi-Li;Zhou, Rong-Ping
通讯作者：
Zhou, Rong-Ping
DOI：
10.1007/978-1-61779-207-6_2
发表时间：
2011-01-01
期刊：
CELL MIGRATION: DEVELOPMENTAL METHODS AND PROTOCOLS, SECOND EDITION
影响因子：
--
作者：
Cory, Giles
通讯作者：
Cory, Giles
影响因子：
64.8
作者：
Hatfield, SD;Shcherbata, HR;Ruohola-Baker, H
通讯作者：
Ruohola-Baker, H
影响因子：
4.6
作者：
Deacon, Dekker C.;Nevis, Kathleen R.;Burns, Caroline E.
通讯作者：
Burns, Caroline E.