Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing

Etich, Julia; Bergmeier, Vera; Pitzler, Lena; Brachvogel, Bent

doi:10.1080/03008207.2016.1210606

Cited by 19 publications

(16 citation statements)

References 48 publications

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“…Wound healing is a complex process in which tissue homeostasis and the protective role of the skin are restored (1). Skin is a complex organ that is composed of the epidermis, dermis, and skin appendages, while wound healing in adult mammals leads to scar formation without skin appendages (2).…”

Section: Introductionmentioning

confidence: 99%

Adipose extracellular matrix promotes skin wound healing by inducing the differentiation of adipose‑derived stem cells into fibroblasts

et al. 2018

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Fibroblasts are the major effector cells of skin wound healing. Adipose-derived stem cells can differentiate into fibroblasts under certain conditions. In the present study, it was hypothesized that adipose-derived stem cells (ADSCs) could be induced by the adipose extracellular matrix (ECM) to differentiate into fibroblasts in order to promote skin wound healing. First, flow cytometry was used to detect the ratio of fibroblasts and relative expression of the fibroblast markers cytokeratin 19 (CK19) and vimentin in ADSCs. Then, the effect of the adipose ECM during the differentiation of ADSCs into fibroblasts was investigated by detecting the total amount of collagen fibers and degree of fibrosis, and the proliferation and cell cycle of differentiated fibroblasts, using the MTT assay and flow cytometry analysis respectively. Finally, a mouse skin wound model was established and treated with PBS, ADSC suspension or ECM + ADSCs to compare wound healing rate and expression of collagen I and collagen III by immunohistochemistry. Following induction of ADSCs with the adipose ECM, more fibroblasts were found, expression of CK19 and vimentin increased, and a greater degree of fibrosis occurred, which revealed the positive effect of the adipose ECM on the differentiation of ADSCs into fibroblasts. In addition, the induced fibroblasts had enhanced proliferation activity, with more cells in the S phase and fewer in the G2/M phase. The in vivo experiment indicated that the ECM produced by the ADSCs had a faster wound healing rate and increased expression of collagen I and collagen III compared with mice injected with PBS or ADSCs alone, which verified that ADSCs induced by the adipose ECM had a positive effect on skin wound healing. The present study demonstrated that the adipose ECM in combination with ADSCs may be a novel therapeutic target for the repair of skin injury, due to the ability of the adipose ECM to induce the differentiation of ADSCs into fibroblasts and to facilitate the wound healing process.

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Section: Introductionmentioning

confidence: 99%

Adipose extracellular matrix promotes skin wound healing by inducing the differentiation of adipose‑derived stem cells into fibroblasts

et al. 2018

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“…: 4.16.003, approval date: 18 February 2016). Full thickness wounds were inflicted on the back of C57BL/6N mice as described earlier [92]. Three and five days post injury wounds were embedded in tissue tek (Sakura Finetek Europe, Staufen im Breisgau, Germany), sectioned (Leica Cryotome CM3050, Wetzlar, Germany) and analyzed by immunofluorescence microscopy (Nikon Europe Eclipse TE2000-U Microscope, Tokyo, Japan) or the wound area was cropped using a fresh 6mm biopsy punch, underlying fat tissue was removed and the tissue was stored at −80 °C for up to two years.…”

Section: Methodsmentioning

confidence: 99%

Gene Expression Profiling of the Extracellular Matrix Signature in Macrophages of Different Activation Status: Relevance for Skin Wound Healing

Etich

Koch

Wagener

et al. 2019

IJMS

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The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFβ)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.

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“…The following are some of the criteria we applied for selecting candidate reference genes: to minimize the effect of co-regulation, the nucleic acid sequence should encode a protein that plays various roles in cellular metabolism with different molecular functions; sequences that have previously been examined for stability in, to a certain extent, similar biological contexts; for reliability, the sequence should be tested specifically to verify the accuracy of the data (Die et al., 2017). The previously published candidate reference genes (Purohit et al, 2015; Altmann et al, 2015; Etich et al, 2016) are cyclophilin ( CYC ), beta actin ( ACTB ), elongation factor-1 alpha ( EF1a ), ribosomal protein L28 ( RPL28S ), β-tubulin ( TUBB ), ubiquitin ( Ubiq ) and 18S ribosomal RNA ( 18S RNA ). To select corresponding sequences, all candidate genes selected from the transcriptome were analysed in the NCBI database using BLAST, and sequences were uploaded to the NCBI database to obtain the GenBank accession number (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury inOnchidium reevesii

Yang

et al. 2019

PeerJ

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Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions.

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Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing

Abstract: RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.

Cited by 19 publications

References 48 publications

Adipose extracellular matrix promotes skin wound healing by inducing the differentiation of adipose‑derived stem cells into fibroblasts

Adipose extracellular matrix promotes skin wound healing by inducing the differentiation of adipose‑derived stem cells into fibroblasts

Gene Expression Profiling of the Extracellular Matrix Signature in Macrophages of Different Activation Status: Relevance for Skin Wound Healing

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury inOnchidium reevesii

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