MALAT1 Knockdown Inhibits the Proliferation, Migration, and Collagen Deposition of Human Hypertrophic Scar Fibroblasts via Targeting miR-29a-3p/Smurf2 Axis.

Purpose: Hypertrophic scarring (HS) is commonly described as an abnormal post-traumatic tissue repair characterized by excessive hypercellularity and extracellular matrix (ECM) deposition. Mounting evidence suggests that MALAT1 is maladjusted in many fibrotic diseases, but its contribution to HS progression remains poorly understood. Hence, we sought to elucidate the fundamental role of MALAT1 in HS. Methods: The expression of MALAT1, miR-29a-3p, and Smurf2 in skin tissues and fibroblasts was assessed by RT-qPCR and Western blotting. Furthermore, lentiviruses, RNAi, or plasmids were utilized to transfect hypertrophic scar fibroblasts (HSFs) for gene overexpression or downregulation. The biological behaviors of HSFs were quantified by the CCK-8 assay, wound healing assay, transwell assay, and flow cytometry. Mechanistically, bioinformatics analysis, dual-luciferase reporter assays, and rescue experiments were performed to verify the relationship between miR-29a-3p and MALAT1 or Smurf2. Results: Our data indicate that MALAT1, Smurf2 were overexpressed while miR-29a-3p was suppressed in HS tissues and fibroblasts. Downregulation of MALAT1 may lead to decreased proliferation, migration, and invasion of fibroblasts, accompanied by enhanced apoptosis, reduced TGF-ß signal transduction, and ECM accumulation in HSFs, by enhancing miR-29a-3p and suppressing Smurf2 expression. Mechanistically, MALAT1 acted as a sponge for miR-29a-3p, while miR-29a-3p directly targeted Smurf2. More importantly, rescue experiments suggested that MALAT1 downregulation induced impact on the proliferation, migration, and invasion of HSFs could be partially overturned through miR-29a-3p knockdown or Smurf2 overexpression. Conclusion: MALAT1 knockdown inhibits the proliferation, migration, invasion, and collagen deposition of HSFs via targeting the miR-29a-3p/Smurf2 axis, which may reveal a promising therapeutic exploitable vulnerability to HS.

USP15 Enhances the Proliferation, Migration, and Collagen Deposition of Hypertrophic Scar-Derived Fibroblasts by Deubiquitinating TGF-ßR1 In Vitro.

BACKGROUND: Hypertrophic scar is a fibroproliferative disorder caused by skin injury. The incidence of hypertrophic scar following trauma or burns is 40 to 70 percent or 70 percent, respectively. It has been shown that transforming growth factor (TGF) ß1/Smad signaling plays a crucial role in hypertrophic scar, and that USP15 can regulate the activity of TGFß1/Smad signaling to affect the progression of the disease. However, the underlying mechanism of USP15 in hypertrophic scar remains unclear. The authors hypothesized that USP15 was up-regulated and enhanced the proliferation, migration, invasion, and collagen deposition of hypertrophic scar-derived fibroblasts by deubiquitinating TGF-ß receptor I (TßRI) in vitro. METHODS: Fibroblasts were isolated from human hypertrophic scars in vitro. The knockdown and overexpression of USP15 in hypertrophic scar-derived fibroblasts were performed using lentivirus infection. The effect of USP15 on hypertrophic scar-derived fibroblast proliferation, migration, and invasion, and the expression of TßRI, Smad2, Smad3, α-SMA, COL1, and COL3, were detected by Cell Counting Kit-8, scratch, invasion, quantitative real-time polymerase chain reaction, and Western blot assays. The interaction between USP15 and TßRI was detected by co-immunoprecipitation and ubiquitination assays. RESULTS: The authors demonstrated that USP15 knockdown significantly inhibited the proliferation, migration, and invasion of hypertrophic scar-derived fibroblasts in vitro and down-regulated the expression of TßRI, Smad2, Smad3, α-SMA, COL1, and COL3; in addition, USP15 overexpression showed the opposite trends (p < 0.05). Co-immunoprecipitation and ubiquitination assays revealed that USP15 interacted with TßRI and deubiquitinated TßRI. CONCLUSION: USP15 enhances the proliferation, migration, invasion, and collagen deposition of hypertrophic scar-derived fibroblasts by deubiquitinating TßRI in vitro.

Detection and analysis of angiogenesis pathway­associated lncRNA expression profiles in human skin fibroblasts under high­glucose conditions.

Accumulating evidence has indicated that long non­coding RNAs (lncRNAs) have crucial roles in wound healing and that vascular lesions in diabetic wounds are frequently difficult to heal. However, the role of angiogenesis pathway­associated lncRNAs in wound healing in diabetic patients has remained to be fully elucidated. In the present study, human skin fibroblasts were cultured under high­glucose conditions in vitro to mimic a diabetic environment and the angiogenesis pathway­associated lncRNA expression profile in the high­ and normal­glucose groups was examined. The microarray data indicated that 14 lncRNAs and 22 mRNAs were differentially expressed. Several candidate lncRNAs and mRNAs were then analyzed by reverse transcription­quantitative PCR and the results were consistent with the microarray data. Furthermore, the University of California Santa Cruz Genome Browser was used to identify mRNAs linked to angiogenesis pathways near the transcriptional region of lncRNAs. The results suggested that lncRNAs RP4­791C19.1 and CTD­2589O24.1 may act on their target genes epidermal growth factor receptor and p21 (RAC1) activated kinase 1, respectively, as enhancers and cis­regulate their expression. Therefore, the present study confirmed that several angiogenesis pathway­associated lncRNAs were differentially expressed under high­glucose conditions, which may have a key role in wound healing in patients with diabetes.

Long non­coding RNA HOTAIR promotes burn wound healing by regulating epidermal stem cells.

Local transplantation of epidermal stem cells (ESCs) exerts a therapeutic effect on burn wounds. However, cell viability can impede their clinical application. HOX antisense intergenic RNA (HOTAIR) is involved in regulating adult tissue stem cells, as well as in developmental patterning and pluripotency. However, little is known about its role in regulating ESCs. The present study was performed to investigate the effects of HOTAIR in the modulation of ESCs and wound repair. Firstly, reverse transcription­quantitative PCR was used to detect the relative expression of HOTAIR during burn wound healing in mice to determine whether HOTAIR is associated with wound healing. Subsequently, ESCs derived from mouse skin were transfected with a lentiviral vector to overexpress or knockdown HOTAIR. The effects of HOTAIR on cell proliferation and differentiation were measured by 5­bromodeoxyuridine and MTT assays, and by assessing NANOG mRNA expression. Lastly, mice with burns were administered a subcutaneous injection of HOTAIR­overexpressing ESCs. Images were captured and histological analyses were performed to evaluate wound healing. The results revealed that the expression of HOTAIR gradually increased and peaked at day 7 post­burn and maintained at relatively high levels until day 14 post­burn during wound healing. Furthermore, overexpression of HOTAIR promoted ESC proliferation and maintained the stem cell state in vitro. By contrast, suppression of HOTAIR inhibited cell proliferation and cell stemness. It was also identified that HOTIR­overexpressing ESCs accelerated re­epithelialization and facilitated burn wound repair. In conclusion, the present findings confirmed an essential role of HOTAIR in the regulation of ESC proliferation and stemness. Therefore, targeting HOTAIR in ESCs may be a potentially promising therapy for burn wound healing.

tRNA­derived small RNAs: A novel class of small RNAs in human hypertrophic scar fibroblasts.

tRNA­derived small RNAs (tsRNAs) have been shown to play regulatory roles in many physiological and pathological processes. However, their roles in hypertrophic scars remain unclear. The present study investigated differentially expressed tsRNAs in human hypertrophic scar fibroblasts and normal skin fibroblasts via high­throughput sequencing. Several dysregulated tsRNAs were validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, target prediction, coexpression networks and competing endogenous RNA (ceRNA) networks were evaluated to discover the principal functions of significantly differentially expressed tsRNAs. In total, 67 differentially expressed tsRNAs were detected, of which 27 were upregulated and 40 downregulated in hypertrophic scar fibroblasts. The GO analysis indicated that the dysregulated tsRNAs are associated with numerous biological functions, including 'nervous system development', 'cell adhesion', 'focal adhesion', 'protein binding', 'angiogenesis' and 'actin binding'. KEGG pathway analysis revealed that the most altered pathways include 'Ras signaling pathway', 'Rap1 signaling pathway' and 'cGMP­PKG signaling pathway'. The target genes of the differentially expressed tsRNAs participate in several signaling pathways important for scar formation. The results of RT­qPCR were consistent with those of sequencing. MicroRNA (miR)­29b­1­5p was identified as a target of tsRNA­23678 and was downregulated in hypertrophic scar fibroblasts, constituting a negative regulatory factor for scar formation. Furthermore, tsRNA­23761 acted as a ceRNA and bound to miR­3135b to regulate the expression of miR­3135b targets, including angiotensin­converting enzyme. Collectively, these findings reveal that tsRNAs are differentially expressed in human hypertrophic scar fibroblasts, and may contribute to the molecular mechanism and treatment of hypertrophic scars.

Aberrantly expressed long noncoding RNAs in hypertrophic scar fibroblasts in vitro: A microarray study.

A hypertrophic scar is the result of abnormal repair of the body after trauma. Histopathologically, it is mostly the result of the excessive proliferation of fibroblasts and the accumulation of extracellular matrix. Accumulating evidence has demonstrated that long non­coding RNAs (lncRNAs) have a critical role in the regulation of gene expression and in the pathogenesis of diseases. However, the roles of lncRNAs in hypertrophic scars have remained elusive. The present study investigated the profiles of differentially expressed lncRNAs between fibroblasts derived from a hypertrophic scar and normal skin, and explored the possible mechanisms underlying the development of hypertrophic scars. Microarray data indicated that 6,104 lncRNAs and 2,952 mRNAs were differentially expressed. A set of differentially expressed transcripts as confirmed by reverse transcription­quantitative polymerase chain reaction. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to determine the principal functions of the significantly deregulated genes. Furthermore, associated expression networks, including subgroup analysis, competing endogenous RNAs (ceRNAs) and coding­noncoding co­expression networks were constructed using bioinformatics methods. The homology between differentially expressed lncRNAs and mRNAs was assessed and two exon lncRNA were selected to explore their regulatory mechanisms. The ceRNA network inferred that NR_125715 acted as a competing endogenous RNA, bound to microRNA (miR)­141­3p, miR­200a­3p and miR­29 to regulate the expression of the miRs' targets, including transforming growth factor ß2 (TGFB2). Similarly, NR_046402 acted as a competing endogenous RNA, which bound to miR­133a­3p.1 and miR­4469 to then regulate the expression of the miRs' targets, including DNA polymerase Î´1, catalytic subunit (POLD1). In addition, co­expression analysis indicated that the expression of lncRNAs NR_125715 and NR_046402 was correlated with that of TGFB2 and POLD1 mRNA. The identification of these differentially expressed lncRNAs in the hypertrophic scar­derived fibroblasts in the present study, may provide novel insight into the functional interactions of lncRNA, miRNA and mRNA, and lead to novel theories for the pathogenesis and treatment of hypertrophic scars.

Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell­based skin tissue engineering in the future.