Wound healing response is characterized by progression from clot formation to an inflammatory phase, a repair phase, and finally remodeling. MicroRNAs (miRNAs) are 20-24 nucleotide noncoding RNAs that can affect protein expression at the posttranscriptional level by modulating the stability and translation of corresponding messenger RNAs (mRNAs). The roles that miRNAs play in various physiological and pathological processes have received increasingly widespread attention. However, the differentially expressed miRNAs involved in different stages of wound healing and their roles remain to be further explored. In this study, we established wound model on the shaved dorsum using a punch biopsy tool in C57BL/6J mice. Biopsies were harvested on postoperative days 0, 1, 3, and 7. Then, we identified the differentially expressed miRNAs in different stages of wound healing through Illumina sequencing and miRDeep2 analysis. This study help to find out the expression profile of microRNAs during normal wound healing in mice.

Animals

C57BL/6J mice were purchased from Weitonglihua Co., Ltd. (Beijing, China). All animals were maintained in an animal facility at 22°C ± 2°C and 55% ± 10% humidity under a 12 h light-dark cycle. Food and water were freely available.

Wound healing model

Male mice aged 2 months were anesthetized with pentobarbital (i.p., 80 mg/kg). The dorsal surface was then shaved and cleaned with 75% ethanol. Two round wounds with a diameter of 5 mm were made on each side of the midline of the shaved dorsum using a punch biopsy tool (Miltex, USA). The wounds were left undressed, and all mice were housed separately thereafter. The wounds were imaged using a high-resolution camera, the sizes of which were calculated using ImageJ software (National Institutes of Health, USA). Biopsies were harvested on unharmed skin tissues (D0) and during inflammation (postinjury day 1, D1), tissue formation (postinjury day 3, D3) and tissue reconstruction (postinjury day 7, D7) phases during wound healing. Experiments were conducted in three biological replicates (n = 3) for each group.

RNA isolation and library preparation

During the inflammation, tissue formation, and tissue reconstruction phases of skin wound healing in control mice, namely, at 0, 1, 3 and 7 days postoperation, the wounds and a 5 mm unwounded skin border were harvested and stored at −80℃ until further processing. Total RNA was isolated using the miRNeasy Serum/Plasma Kit (Qiagen, USA). The A (260/280) absorbance ratio of isolated RNA was 1.8–2.0, while the A (260/230) absorbance ratio was greater than 1.6. Integrity of total RNA was determined using formaldehyde denaturing gel electrophoresis. The libraries were constructed from total RNA using the Illumina TruSeq Small RNA Sample Preparation kit (Illumina, USA). Single-stranded cDNA was created by reverse transcription reactions with the ligation products. The cDNA was amplified by PCR. Finally, Illumina sequencing technology was used to sequence these prepared samples.

Illumina sequencing

Illumina miRNA-sequencing was performed on the equally mixed samples of three replicates for each time point. Raw sequences were processed using the Illumina pipeline program. Clean reads were filtered, and contaminated reads were removed. Secondary structure prediction of individual miRNAs was performed using Mfold software. Clean sequence reads were analyzed by miRDeep2 (version, 2.0.0.5).

miRDeep2 is a popular algorithm for miRNA identification. For details, please refer to the following literature: Friedlander, M. R., Mackowiak, S. D., Li, N., Chen, W. & Rajewsky, N. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res. 40, 37–52. https://doi.org/10.1093/nar/gkr688 (2012).