HOXA11-AS Regulates NIPAL3 via Hsa-miRNA-19a-3p, Hsa-miR-141-3p and Hsa-miR-140-5p in Keloid Fibroblast: Bioinformatic Analysis and In Vitro Validation

Background: The long noncoding RNA HOXA11-AS is significantly increased in keloids with an unknown mechanism.Methods: Keloid-derived fibroblasts were primarily cultured from keloid explants (Sample 0). Small inference RNAs (siRNAs) against HOXA11-AS were transinfected in two keloid fibroblasts (Sample 1 and Sample 2). Nonspecific siRNA was transinfected into one keloid fibroblast (Sample 3). The blank plasmid was transinfected into one keloid fibroblast (Sample 4). Five keloid fibroblast samples were sequenced for lncRNAs, mRNAs and miRNAs. Differentially expressed (DE) mRNAs or lncRNAs were obtained in HOXA11-AS-knockdown keloid fibroblasts via DE RNAs from sample 1 intersected from sample 2, removing overlapping nonspecific interfered DE RNAs from sample 3 combined with sample 4. Using stepwise bioinformatics, dominantly functioning mRNAs downstream of HOXA11-AS were scored based on a first-step lncRNA-mRNA-protein (LMP) network in keloid fibroblasts, removing overlapping mRNAs from the nonspecific group. Screened genes were further validated by real-time PCR and western blotting. Validated mRNAs were overtaken to predict a sequence-matched competing endogenous ceRNA network involving the HOXA11-AS (down)- miRNA (up)- validated mRNA (down) pathway in keloid fibroblasts.Results: Six DE profiles (lncRNA, mRNA, miRNA) were obtained in the HOXA11-AS-knockdown group or in the nonspecific interference group. Fourteen dominantly functional genes were enriched in three biological pathways. SNED1, NIPAL3 and VTN were validated in keloid fibroblasts by knocking down HOXA11-AS via real-time PCR. Only NIPAL3 was predicted as a competing endogenous gene downstream of the HOXA11-AS pathway via three sponging miRNAs (hsa-miRNA-19a-3p, hsa-miR-141-3p, hsa-miR-140-5p).Conclusions: HOXA11-AS regulated NIPAL3 via three miRNAs in keloid fibroblasts.

Exosomale microRNA-21 Promotes Keloid Fibroblast Proliferation and Collagen Production by inhibiting Smad7

In keloid fibroblasts, microRNA-21 (miR-21) enhances activation of the TGF-β–Smad-signaling pathway by downregulating Smad7 expression, thereby promoting keloid fibroblast proliferation and collagen production. However, it is unclear whether miR-21 performs the above-mentioned functions through exosomal transport. Here, we extracted exosomes from the culture supernatants of keloid and normal skin fibroblasts, and observed that exosomes from both cell types secreted exosomes; however, keloid fibroblasts secreted significantly more exosomal miR-21 than normal skin fibroblasts (P < 0.001). Interestingly, we also observed that exosomal miR-21 could enter target keloid fibroblasts. In addition, inhibiting exosomal miR-21 upregulated Smad7 protein expression and reduced Smad2 and Smad3 protein levels in target keloid fibroblasts. Furthermore, inhibiting exosomal miR-21 downregulated collagen I and collagen III expression in target keloid fibroblasts, increased the proportion of apoptotic cells, and reduced cell proliferation. Taken together, these results show that exosomal miR-21 promoted proliferation and collagen production in keloid fibroblasts by inhibiting Smad7. Thus, we identified regulatory roles for miR-21 in promoting keloid fibroblast proliferation and participating in keloid formation and development. These findings imply that miR-21 may serve as a novel target for controlling the development of keloids.

Therapeutic candidates for keloid scars identified by qualitative review of scratch assay research for wound healing

The scratch assay is an in vitro technique used to analyze cell migration, proliferation, and cell-to-cell interaction. In the assay, cells are grown to confluence and then ‘scratched’ with a sterile instrument. For the cells in the leading edge, the resulting polarity induces migration and proliferation in attempt to ‘heal’ the modeled wound. Keloid scars are known to have an accelerated wound closure phenotype in the scratch assay, representing an overactivation of wound healing. We performed a qualitative review of the recent literature searching for inhibitors of scratch assay activity that were already available in topical formulations under the hypothesis that such compounds may offer therapeutic potential in keloid treatment. Although several shortcomings in the scratch assay literature were identified, caffeine and allicin successfully inhibited the scratch assay closure and inflammatory abnormalities in the commercially available keloid fibroblast cell line. Caffeine and allicin also impacted ATP production in keloid cells, most notably with inhibition of non-mitochondrial oxygen consumption. The traditional Chinese medicine, shikonin, was also successful in inhibiting scratch closure but displayed less dramatic impacts on metabolism. Together, our results partially summarize the strengths and limitations of current scratch assay literature and suggest clinical assessment of the therapeutic potential for these identified compounds against keloid scars may be warranted.

Is the Mitochondrial Function of Keloid Fibroblasts Affected by Cytoglobin?

Background: A keloid is a benign skin tumour characterised by excessive proliferation of fibroblasts, a process that requires a sufficient amount of energy. The energy needs are associated with adequate oxygen (O2) flow and well-functioning mitochondria. It is known that cytoglobin (CYGB) has a function in O2 distribution. The aim of the present study was to explore whether the inhibition of CYGB expression caused impaired mitochondrial function of keloid fibroblasts. Methods: An in vitro study was conducted on a keloid fibroblast derived from our previous study. The study was carried out in the laboratory of the Biochemistry & Molecular Biology Department, Faculty of Medicine, Universitas Indonesia (FMUI), from July to December 2018. CYGB expression was inhibited by small interfering ribonucleic acid (siRNA) and CYGB. Analysis of mitochondrial function was observed through peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), a mitochondrial biogenesis marker and the activity of the succinate dehydrogenase (SDH) enzyme in mitochondria. Results: The CYGB gene and protein were downregulated after treatment with CYGB siRNA. Inhibition of CYGB expression with siRNA also tended to decrease the levels of PGC-1α messenger ribonucleic acid (mRNA) and protein, as well as SDH enzyme activity. Conclusion: Inhibition of CYGB expression with siRNA tended to decrease mitochondrial biogenesis and function. This may be useful for understanding the excessive proliferation of fibroblasts in keloids and for development of treatment for keloids.

A selective small-molecule inhibitor of c-Met suppresses keloid fibroblast growth in vitro and in a mouse model

Keloids, tumor-like lesions that result from excessive scar formation, have no definitive treatment modality. Activation of c-mesenchymal-epithelial transition factor (c-Met) promotes cell proliferation and survival. Selective c-Met inhibitors, such as PHA-665752, may attenuate the activity of keloid fibroblasts and reduce keloid formation. Here, we aimed to evaluate the effect of PHA-665752, a second-generation selective small-molecule inhibitor of c-Met, on human keloid fibroblasts in vitro and in a mouse model. We performed in vitro cytotoxicity assays, scratch tests, western blotting, and immunofluorescence on human keloid fibroblasts. We also injected human fibroblasts into severe combined immunodeficient mice and measured the degree of nodule formation and skin histologic characteristics. We found that keloid fibroblast migration was inhibited by PHA-665752. Inhibitor treatment was also associated with lower expression of members of the hepatocyte growth factor/c-Met pathway, and lower fibroblast activity and collagen synthesis. In the in vivo experiments, PHA-665752—treated mice had lower nodule volumes and weights, accompanied by less inflammatory cell infiltration and collagen deposition, than those in control mice. These findings showed that although an in vivo model may not accurately represent the pathophysiology of human keloid development, PHA-665752 suppressed keloid fibroblast activity by inhibiting the c-Met—related tyrosine kinase pathway.

Differential responses to folic acid in an established keloid fibroblast cell line are mediated by JAK1/2 and STAT3

Keloids are a type of disordered scar formation which not only show heterogeneity between individuals and within the scar itself, but also share common features of hyperproliferation, abnormal extra-cellular matrix deposition and degradation, as well as altered expression of the molecular markers of wound healing. Numerous reports have established that cells from keloid scars display Warburg metabolism—a form of JAK2/STAT3-induced metabolic adaptation typical of rapidly dividing cells in which glycolysis becomes the predominant source of ATP over oxidative phosphorylation (OxPhos). Using the JAK1/2 inhibitor ruxolitinib, along with cells from patients with STAT3 loss of function (STA3 LOF; autosomal dominant hyper IgE syndrome) we examined the role of JAK/STAT signaling in the hyperproliferation and metabolic dysregulation seen in keloid fibroblasts. Although ruxolitinib inhibited hyperactivity in the scratch assay in keloid fibroblasts, it paradoxically exacerbated the hyper-glycolytic state, possibly by further limiting OxPhos via alterations in mitochondrial phosphorylated STAT3 (pSTAT3Ser727). In healthy volunteer fibroblasts, folic acid exposure recapitulated the exaggerated closure and hyper-glycolytic state of keloid fibroblasts through JAK1/2- and STAT3-dependent pathways. Although additional studies are needed before extrapolating from a representative cell line to keloids writ large, our results provide novel insights into the metabolic consequences of STAT3 dysfunction, suggest a possible role for folate metabolism in the pathogenesis of keloid scars, and offer in vitro pre-clinical data supporting considerations of clinical trials for ruxolitinib in keloid disorder.