Gallo Phillip H, Satish Latha, Johnson Sandra, Kathju Sandeep
Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA 15212, USA.
BMC Res Notes. 2011 Jun 6;4:175. doi: 10.1186/1756-0500-4-175.
Adult mammalian tissues heal injury to the skin with formation of scar; this process quickly seals an injured area, however, excessive scar formation can become a source of persistent pathology, interfering with multiple vital functions. In contrast, mammalian fetal tissue can heal without scar formation. We previously sought to model scarless healing in a rabbit fetal skin wound and identified gene products differentially expressed during fetal wound healing through PCR suppression subtractive hybridization (PCR SSH). One of these transcripts, previously identified simply as clone 11, showed putative increased expression in wounded fetal skin. This study establishes its identity as Ero1L-alpha and confirms its elevated expression in healing fetal wounds.
After obtaining further sequence by 5' rapid amplification of cloned ends (RACE) we find that clone 11 is Ero1L-alpha. We determined that clone 11, a differentially expressed transcript in fetal wound healing, comprises the 3' untranslated region (UTR) of an approximately 4 kb transcript in rabbit tissues that corresponds to Ero1L-alpha. We showed that Ero1L-alpha is expressed predominantly as two transcripts in rabbit skin, namely a 1.6 kb transcript and the 4.0 kb transcript recovered in our PCR SSH screen via its 3' UTR sequence. However, a third transcript of 2.9 kb was also detected in Northern blots and was subsequently cloned and confirmed by 3' RACE. Knockdown of the clone 11 sequence in rabbit adult fibroblasts via siRNA resulted in significantly decreased Ero1L-alpha message expression. Increased expression of clone 11 (Ero1L-alpha) in a variety of cell types during the wound healing response was also confirmed by in situ hybridization.
Ero1L-alpha is one of the previously unknown clones identified in a PCR SSH screen for genes differentially expressed in fetal wounded tissue. In situ hybridization confirms that Ero1L-alpha shows increased expression in multiple cell types after wounding of the fetal integument.
成年哺乳动物组织通过形成瘢痕来愈合皮肤损伤；这一过程能迅速封闭受伤区域，然而，过度的瘢痕形成会成为持续病理状态的根源，干扰多种重要功能。相比之下，哺乳动物胎儿组织能够无瘢痕愈合。我们之前试图在兔胎儿皮肤伤口中模拟无瘢痕愈合，并通过PCR抑制性消减杂交（PCR SSH）鉴定了胎儿伤口愈合过程中差异表达的基因产物。这些转录本之一，之前简单地鉴定为克隆11，在受伤的胎儿皮肤中显示出假定的表达增加。本研究确定其身份为Ero1L-α，并证实其在愈合的胎儿伤口中表达升高。
通过5'克隆末端快速扩增（RACE）获得进一步序列后，我们发现克隆11是Ero1L-α。我们确定克隆11，即胎儿伤口愈合中差异表达的转录本，包含兔组织中一个约4 kb转录本的3'非翻译区（UTR），该转录本与Ero1L-α相对应。我们表明，Ero1L-α在兔皮肤中主要以两种转录本形式表达，即1.6 kb转录本和我们通过PCR SSH筛选通过其3'UTR序列回收的4.0 kb转录本。然而，在Northern印迹中也检测到了第三种2.9 kb的转录本，随后通过3'RACE进行克隆和确认。通过siRNA敲低兔成年成纤维细胞中的克隆11序列导致Ero1L-α信息表达显著降低。原位杂交也证实了在伤口愈合反应期间，多种细胞类型中克隆11（Ero1L-α）的表达增加。
Ero1L-α是在PCR SSH筛选中鉴定出的在胎儿受伤组织中差异表达的基因中先前未知的克隆之一。原位杂交证实，Ero1L-α在胎儿体表受伤后在多种细胞类型中表达增加。
