Normal Skin Samples Research Articles

Expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis and effect of ultraviolet B radiation on its mRNA and protein expression in HaCaT cells

Objective To determine the expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis (CAD) , and to explore the effect of ultraviolet B (UVB) radiation on its mRNA and protein expression in HaCaT cells. Methods In 2016, skin samples were collected from lesions of 10 patients with CAD (test group), 10 patients with eczema (positive control group) and from normal skin of 10 healthy controls after cosmetic surgery (negative control group) in the Department of Dermatology, First Affiliated Hospital of Kunming Medical University. Immunohistochemical staining was performed to determine the expression of caspase-14 in the normal skin, CAD and eczema lesions. Cultured HaCaT cells were divided into several groups: UVB groups irradiated with 0, 30, 60, 90 mJ/cm2 UVB separately, and 5-AzaC groups irradiated with 0, 30, 60, 90 mJ/cm2 UVB separately followed by the treatment with the methylase inhibitor 5-AzaC for 24 hours. Then, the cells were collected, and real-time fluorescence-based quantitative PCR (RT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of caspase-14 respectively in HaCaT cells in the UVB groups and 5-AzaC groups. Statistical analysis was carried out with SPSS22.0 software by using chi-square test for the comparison of rates, and t test and two-factor analysis of variance for the comparison of means. Results In the CAD and eczema lesions, caspase-14 was mainly expressed in the spinous and granular layers, but not in the stratum corneum. However, caspase-14 was markedly expressed in the stratum corneum of the normal skin tissues. Of the 10 CAD samples, 5 were positive for caspase-14, and 9 of 10 normal skin samples were positive for caspase-14. The positive rate of caspase-14 significantly differed between the two above groups (χ2=7.30, P < 0.05) . RT-PCR and Western blot analysis showed significant changes in the mRNA and protein expression of caspase-14 in HaCaT cells after irradiation with different doses of UVB (F=87.54, 23.46, both P < 0.05) , which showed a decreasing trend along with the increase in the dose of UVB. After exposure to 0, 30, 60 and 90 mJ/cm2 UVB, the mRNA and protein expression of caspase-14 was significantly higher in the 5-AzaC groups than in the UVB groups (all P < 0.05) . Conclusions In CAD lesions, the expression of caspase-14 markedly decreased, and was absent in the stratum corneum. UVB radiation can down-regulate the mRNA and protein expression of caspase-14 in HaCaT cells. Key words: Photosensitivity disorders; Eczema; Ultraviolet rays; Caspase 14; Keratinocytes; Chronic actinic dermatitis

Data Independent Acquisition Proteomic Analysis Can Discriminate between Actinic Keratosis, Bowen’s Disease, and Cutaneous Squamous Cell Carcinoma

Actinic keratosis, Bowen's disease and cutaneous squamous cell carcinoma (cSCC) are heterogeneous keratinocytic skin lesions. Biomarkers that can accurately stratify these lesion types are needed to support a new paradigm of personalized and precise management of skin neoplasia. In this paper, we used a data independent acquisition proteomics workflow, sequential window acquisition of all theoretical mass spectra, to analyze formalin-fixed paraffin-embedded samples of normal skin and keratinocytic skin lesions, including well-differentiated, moderately differentiated and poorly differentiated cSCC lesions. We quantified 3,574 proteins across the 93 samples studied. Differential abundance analysis identified 19, 5, and 6 protein markers exclusive to actinic keratosis, Bowen's disease and cSCC lesions, respectively. Among cSCC lesions of various levels of tumor differentiation, 118, 230, and 17 proteins showed a potential as biomarkers of well-differentiated, moderately differentiated and poorly differentiated cSCC lesions, respectively. Bioinformatics analysis revealed that actinic keratosis and cSCC lesions were associated with decreased apoptosis, and Bowen's disease lesions with over-representation of the DNA damage repair pathway. Differential expression of alternatively spliced FGFR2, Rho guanosine triphosphatase signaling, and RNA metabolism proteins were associated with the level of cSCC tumor differentiation. Proteome profiles also separated keratinocytic skin lesion subtypes on principal components analysis. Overall, protein markers have excellent potential to discriminate keratinocytic skin lesion subtypes and facilitate new diagnostic and therapeutic strategies.

Key candidate genes of STAT1 and CXCL10 in melanoma identified by integrated bioinformatical analysis.

The underlying mechanisms and gene signatures of melanoma are unknown. In this study, three expression profile data sets (GSE65568, GSE100050, GSE114445) were integrated to identify candidate genes explaining the pathways and functions of melanoma. Expression data sets including 24 melanoma tumours and 13 normal skin samples were merged and analysed in detail. The three GSE profiles shared 431 differentially expressed genes (DEGs), including 227 upregulated genes, 200 downregulated genes and 4 differentially regulated genes. Moreover, the functions and signalling pathways of the shared DEGs with significant p-values were identified. The two most significant modules were filtered from the DEGs protein-protein interaction (PPI) network, which consisted of 284 nodes. We also plotted the prognostic value of hub genes from an online database. In summary, using integrated bioinformatic analysis, we have identified candidate DEGs and pathways in melanoma that could improve our understanding of the causes and underlying molecular events of melanoma, and these candidate genes and pathways could be therapeutic targets for melanoma.

Dissecting the role of RNA modification regulatory proteins in melanoma

Melanoma is the deadliest form of skin cancer. Despite recent advances in medicine and the development of new treatments for melanoma, cures remain elusive as acquired resistance to both targeted and immunotherapies are becoming common. Therefore, more studies are conducted to dissect underlying molecular mechanisms that drive melanoma growth in order to provide better therapeutic option. Here, employing a comprehensive and unbiased analysis of different RNA modification regulatory proteins using various publicly available databases we identify the most relevant RNA modifying proteins that plays crucial role in melanoma development. Our study started with the analysis of various genetic alterations (amplifications, mutations/deletion) as well as RNA overexpression of these RNA modification regulatory proteins in The Cancer Genome Atlas melanoma database. We then analyzed their expression in The Human Protein Atlas data. The result of analysis revealed that only a subset of RNA modification regulatory proteins are overexpressed in >75% of melanoma patient cases as compared to normal skin. However, when examined in Oncomine dataset we found only two genes (METTL4 and DNMT3A) were significantly overexpressed in melanoma samples versus normal skin samples and matched with the results of The Human Protein Atlas data. Therefore, we functionally validated METTL4 and DNMT3A using shRNA-mediated knockdown and found that their knockdown in melanoma cells led to melanoma cells growth inhibition. Collectively, in this study, we investigated the epitranscriptomic landscape of melanoma using various publicly available database and identified DNMT3A and METTL4 as the most relevant potential regulators of melanoma growth.

Dielectric and Double Debye Parameters of Artificial Normal Skin and Melanoma

The aim of this study is to characterise the artificial normal skin and melanoma by testing samples with different fibroblast and metastatic melanoma cell densities using terahertz (THz) time-domain spectroscopy (TDS) attenuated total reflection (ATR) technique. Results show that melanoma samples have higher refractive index and absorption coefficient than artificial normal skin with the same fibroblast density in the frequency range between 0.4 and 1.6 THz, and this contrast increases with frequency. It is primarily because that the melanoma samples have higher water content than artificial normal skin, and the main reason to melanoma containing more water is that tumour cells degrade the contraction of the collagen lattice. In addition, complex refractive index and permittivity of the melanoma samples have larger variations than that of normal skin samples. For example, the refractive index of artificial normal skin at 0.5 THz increases 4.3% while that of melanoma samples increases 8.7% when the cell density rises from 0.1 to 1 M/ml. It indicates that cellular response of fibroblast and melanoma cells to THz radiation is significantly different. Furthermore, the extracted double Debye (DD) model parameters demonstrate that the static permittivity at low frequency and slow relaxation time can be reliable classifiers to differentiate melanoma from healthy skin regardless of the cell density. This study helps understand the complex response of skin tissues to THz radiation and the origin of the contrast between normal skin and cancerous tissues.

154 Dissecting the keratinocyte lineage of basal cell carcinoma using single cell RNA sequencing

It remains unclear whether basal cell carcinomas (BCCs) originate from the stem cells in the hair follicle bulge or inter-follicular epithelial (IFE) regions. To explore the lineage derivation of BCC tumors, we applied single-cell RNA sequencing to 56219 cells isolated from 4 BCCs and 2 normal skin samples, profiling tumor keratinocyte, immune, stromal, melanocyte and endothelial cell subpopulations. Pseudotime trajectories of keratinocyte and fibroblast clusters displayed a common origin of normal cells, then a bifurcation into two branches in BCCs. In particular, tumor keratinocyte clusters characterized by high levels of EPCAM also expressed high levels of human bulge markers (KRT15, LGR5, DKK3) and low levels of human IFE markers (CD34, KIT). In contrast, keratinocyte clusters with low EPCAM expression had low levels of human bulge markers and high levels of mouse IFE markers (GRHL3, OVOL1). Single-cell analyses also defined distinct tumor keratinocyte-stromal interaction patterns. Our findings support the hair follicle bulge stem cells as the lineage of origin for BCC and elucidate the tumor and microenvironment pathways that evolve during BCC development.

509 Decline in Langerhans cell precursor recruitment into the human skin explains the reduction in epidermal Langerhans cells with age

Aging is accompanied by immunosenescence and increased susceptibility to bacterial and viral infections. Skin provides the first line of physical and immunological defense against environmental insults. However, the age-related changes in the immune function of the human skin are unclear. Here,we investigated the age-related changes in epidermal Langerhans cells (LCs), which play a sentinel role in the initiation of the immune responses in the skin. We screened over 400 normal human skin samples to identify cohorts of young and old sun-protected skin from the same anatomical site. There was a significant reduction in the number of epidermal LCs with age. Among the possible explanations for this reduction, we found a significant reduction in the number of CD14+ CD207+ CCR6+dermal LC precursors with age. In addition, the expression of LC precursor-recruiting chemokine, CXCL14, was markedly down-regulated in the basal layer keratinocytes of the old skin. Importantly, we discovered that young skin recruited more LC precursor-like THP1 cells compared to old skin ex vivo, and this recruitment was inhibited by blocking CXCL14 signaling.Overall, our findings demonstrate an age-related decline in CXCL14-LC precursor axis, which results in the reduction of epidermal LCs under steady-state condition. CXCL14 may provide a novel therapeutic target for prevention of age-related LC reduction.

Overexpression and Implications of Melanoma-associated Antigen A12 in Pathogenesis of Human Cutaneous Squamous Cell Carcinoma.

Melanoma-associated antigen A12 (MAGEA12) has recently been reported as a repressor of tumor-suppressor genes. This study aimed to investigate the implications of MAGEA12 expression in the pathogenesis of cutaneous squamous cell carcinoma (cSCC). MAGEA12 and p21 expression were investigated in 15 samples of normal skin and 111 of cSCC tissues by immunohistochemistry. The biological functions of MAGEA12 in cSCC were also investigated both in vitro and in vivo. Expression of both MAGEA12 and p21 was significantly increased in cSCC. MAGEA12 expression showed a positive correlation, while p21 expression showed negative correlation with the recurrence-free survival of patients with cSCC. In addition, MAGEA12 knockdown significantly attenuated proliferative, migratory, invasive, and tumorigenic activities of cSCC cells and was negatively correlated with p21 expression both in vitro and in vivo. MAGEA12-mediated down-regulation of p21 may be involved in cSCC pathogenesis and MAGEA12 may serve as a molecular biomarker in cSCC.

Weighted correlation network and differential expression analyses identify candidate genes associated with BRAF gene in melanoma

BackgroundPrimary cutaneous malignant melanoma is a cancer of the pigment cells of the skin, some of which are accompanied by BRAF mutation. Melanoma incidence and mortality rates have been rising around the world. As the current knowledge about pathogenesis, clinical and genetic features of cutaneous melanoma is not very clear, we aim to use bioinformatics to identify the potential key genes involved in the expression and mutation status of BRAF.MethodsFirstly, we used UCSC public hub datasets of melanoma (Lin et al., Cancer Res 68(3):664, 2008) to perform weighted genes co-expression network analysis (WGCNA) and differentially expressed genes analysis (DEGs), respectively. Secondly, overlapping genes between significant gene modules and DEGs were screened and validated at transcriptional levels and overall survival in TCGA and GTEx datasets. Lastly, the functional enrichment analysis was accomplished to find biological functions on the web-server database.ResultsWe performed weighted correlation network and differential expression analyses, using gene expression data in melanoma samples. We identified 20 genes whose expression was correlated with the mutation status of BRAF. For further validation, three of these genes (CYR61, DUSP1, and RNASE4) were found to have similar expression patterns in skin tumors from TCGA compared with normal skin samples from GTEx. We also found that weak expression of these three genes was associated with worse overall survival in the TCGA data. These three genes were involved in the nucleic acid metabolic process.ConclusionIn this study, CYR61, DUSP1, and RNASE4 were identified as potential genes of interest for future molecular studies in melanoma, which would improve our understanding of its causes and underlying molecular events. These candidate genes may provide a promising avenue of future research for therapeutic targets in melanoma.

10‑Hydroxycamptothecin induces apoptosis in human fibroblasts by regulating miRNA‑23b‑3p expression.

10-Hydroxycamptothecin (HCPT) effectively controls epidural fibrosis, but the exact underlying mechanisms remain ambiguous. Abnormal microRNA (miR)-23b-3p expression has been detected in various types of fibrotic tissues that are present in different diseases. The aim of the present study was to elucidate the mechanisms through which HCPT induces fibroblast apoptosis. Reverse transcription-quantitative polymerase chain reactions were performed on six traumatic scar samples and matched normal skin samples; traumatic scar formation was revealed to be significantly inversely associated with miR-23b-3p expression. In addition, the miR-23b-3p expression level in human fibroblasts was examined following HCPT treatment. The effects of HCPT and miR-23b-3p on fibroblast apoptosis were assessed using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling, flow cytometry and western blot analysis. The results demonstrated that HCPT treatment notably increased miR-23b-3p expression levels and accelerated fibroblast apoptosis. Therefore, upregulation of miR-23b-3p expression was demonstrated to promote fibroblast apoptosis, consistently with the effects of HCPT. The results of the present study indicated that HCPT may induce fibroblast apoptosis by regulating miR-23b-3p expression.

MiR-181b-5p promotes proliferation and inhibits apoptosis of hypertrophic scar fibroblasts through regulating the MEK/ERK/p21 pathway.

Hypertrophic scar (HS) is a common skin disorder occurring during the wound healing process, and the pathogenesis of HS remains unclear. Previous studies indicated that miRNAs may be involved in the onset and progression of HS. In the present study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to investigate the expression of miR-181b-5p and decorin in HS tissues. Direct interaction between miR-181b-5p and decorin was confirmed using a dual-luciferase assay. Human HS fibroblasts (HSFbs) were cultured and transfected with miR-181b-5p mimics, and MTT assay and Annexin V fluorescein isothiocyanate/propidium iodide staining were performed to investigate the role of miR-181b-5p in the proliferation and apoptosis of HSFbs. Subsequently, the expression levels of mitogen-activated protein kinase kinase (MEK), phospho (p)-extracellular signal-regulated kinase (ERK) and p21 were determined in HSFbs transfected with miR-181b-5p mimics and untransfected cells using RT-qPCR and western blotting. The results indicated upregulation of miR-181b-5p and downregulation of decorin expression in HS tissues compared with normal skin samples. miR-181b-5p may regulate the expression of decorin through direct binding to the 3′-untranslated region, as demonstrated by the results of the dual-luciferase assay. Transfection with miR-181b-5p mimics in HSFbs enhanced cell proliferation, reduced apoptosis and increased the expression of MEK, p-ERK and p21. Furthermore, treatment with MEK inhibitor in HSFbs transfected with miR-181b-5p mimics partially inhibited miR-181b-5p-induced antiapoptotic effects. Taken together, increased expression of miR-181b-5p may serve important roles in the pathogenesis of HS through regulating the MEK/ERK/p21 pathway, suggesting that miR-181b-5p may be a therapeutic target for the treatment of HS.

Gene Expression of Neurotrophins and Their Receptors in Keloids

The aim of this study was to assess gene expression of neurotrophins and their receptors in keloids. Skin samples of normal skin and keloids were obtained from patients in the control (n = 12) and keloid (n = 12) groups, respectively. Ribonucleic acid was extracted from the skin specimens, purified, evaluated by spectrophotometry, and used to synthesize complementary DNA. Real-time quantitative polymerase chain reaction analysis of 84 human neurotrophin genes and their receptors was performed. Twelve genes, including heat shock 27-kDa protein 1, gastrin-releasing peptide receptor, corticotropin-releasing hormone receptor 2, neuropeptide Y Y2 receptor, interleukin 6 signal transducer, nerve growth factor, metallothionein 3, B-cell chronic lymphocytic leukemia/lymphoma 2, cholecystokinin A receptor, persephin, galanin receptor 2, and fibroblast growth factor receptor 3, were down-regulated in keloid tissue compared with normal skin. The genes 27-kDa heat shock protein 1, gastrin-releasing peptide receptor, corticotropin-releasing hormone receptor 2, nerve growth factor, metallothionein 3, B-cell chronic lymphocytic leukemia/lymphoma 2, and persephin protein were considered priority genes associated with keloid formation.

The miRNA 361-3p, a Regulator of GZMB and TNF Is Associated With Therapeutic Failure and Longer Time Healing of Cutaneous Leishmaniasis Caused by L. (viannia) braziliensis

L. (viannia) braziliensis infection causes American Tegumentary Leishmaniasis (ATL), with prolonged time to healing lesions. The potent inflammatory response developed by the host is important to control the parasite burden and infection however an unbalanced immunity may cooperate to the tissue damage observed. The range of mechanisms underlying the pathological responses associated with ATL still needs to be better understood. That includes epigenetic regulation by non-coding MicroRNAs (miRNAs), non-coding sequences around 22 nucleotides that act as post-transcriptional regulators of RNAs encoding proteins. The miRNAs have been associated with diverse parasitic diseases, including leishmaniasis. Here we evaluated miRNAs that targeted genes expressed in cutaneous leishmaniasis lesions (CL) by comparing its expression in both CL and normal skin obtained from the same individual. In addition, we evaluated if the miRNAs expression would be correlated with clinical parameters such as therapeutic failure, healing time as well as lesion size. The miR-361-3p and miR-140-3p were significantly more expressed in CL lesions compared to normal skin samples (p = 0.0001 and p < 0.0001, respectively). In addition, the miR-361-3p was correlated with both, therapeutic failure and healing time of disease (r = 0.6, p = 0.003 and r = 0.5, p = 0.007, respectively). In addition, complementary analysis shown that miR-361-3p is able to identify with good sensitivity (81.2%) and specificity (100%) patients who tend to fail initial treatment with pentavalent antimonial (Sbv). Finally, the survival analysis considering “cure” as the endpoint showed that the higher the expression of miR-361-3p, the longer the healing time of CL. Overall, our data suggest the potential of miR-361-3p as a prognostic biomarker in CL caused by L. braziliensis.

Lesional upregulation of SLURP1 immunostaining parallels disease severity in psoriasis vulgaris patients.

The exact pathogenesis of psoriasis is still unclear. SLURP1 is vital for the normal differentiation of keratinocytes, and could therefore be involved in psoriasis. In this study we investigated the immunohistochemical staining reaction of SLURP1 in psoriasis vulgaris patients and its possible relation to disease pathogenesis. 20 patients with psoriasis vulgaris, 20 patients with psoriasiform dermatitis and 20 normal skin samples were studied. Psoriasis severity was measured with a combination of PASI and DLQI scores. Lesional and non-lesional sites were biopsied for each psoriasis patient. A single biopsy sample was taken for cases with psoriasiform dermatitis and for controls. All sections were immunostained for SLURP1 according to the manufacturer's protocol. Significant differences were noted in SLURP1 immunostaining between lesional and non-lesional biopsies of psoriasis patients and between lesional biopsies of psoriasis patients and lesional sites of psoriasiform dermatitis. However, the differences between non-lesional biopsies of psoriasis patients and normal controls were not significant. Furthermore, the grading of SLURP1 immunostaining paralleled the degree of psoriasis severity. SLURP1 immunostaining is significantly increased in lesional skin of psoriasis vulgaris and not in psoriasiform dermatitis, which demonstrates the role of SLURP1 in the pathogenesis of psoriasis. SLURP1 could be used as a biological marker for psoriasis severity, and this hypothesis warrants further investigation.

NEDD4 Is Involved in Inflammation Development during Keloid Formation

Keloids mark a chronic inflammatory disease characterized by a fibroproliferative disorder of the skin. A genome-wide association study showed that single-nucleotide polymorphism rs8032158 in the neural precursor cell-expressed NEDD4 gene, which has six protein-coding transcript variants (TVs), is genetically linked to keloids. Here, we show that the high frequency of risk allele C in rs8032158 in keloid patients is associated with a selectively higher expression of TV3 of NEDD4 to activate the NF-κB pathway. Comparisons of keloid scars with normal skin samples that do not have the single-nucleotide polymorphism allele and were derived from different anatomical sites showed stronger expressions of NEDD4 TV3 and activated forms of NF-κB and STAT3 in keloid scars. Forced expression or selective knockdown of NEDD4 TV3 increased or decreased NF-κB activation invitro. Furthermore, NEDD4 knockdown suppressed NF-κB-dependent inflammation development invivo. Mechanistic analysis showed that NEDD4 TV3 is involved in NF-κB activation through its association with the adaptor protein RIP. These results suggest that NEDD4 TV3 is a potential diagnostic marker and therapeutic target for chronic skin diseases, including keloid.

Activation and overexpression of the aryl hydrocarbon receptor contribute to cutaneous squamous cell carcinomas: an immunohistochemical study

BackgroundIn vitro studies showed that the aryl hydrocarbon receptor (AHR) contributed to the development of cutaneous squamous cell carcinomas, but supporting clinical data are lacking.MethodsImmunohistochemical analysis was used to detect the expression of AHR, CYP1A1, EGFR, and Ki-67 in 10 actinic keratosis (AK) cases, 10 Bowen disease (BD) cases, 20 cutaneous squamous cell carcinoma (cSCC) cases and 20 normal skin samples. H-scores were used to assess the immunoreactivity.ResultsWeak positive AHR immunoreactivity was found in all normal skin samples, while strong positive AHR immunoreactivity was found in atypical squamous proliferation (AK, BD and cSCC) cases. H-scores and the rate of strong immunostaining of the atypical squamous proliferation cases were higher than those of normal controls (p < 0.01). Nuclear expression of AHR was higher in atypical squamous proliferation cases than in normal controls (p < 0.01). H-scores and the nuclear expression rate of AHR were significantly higher in AK and BD cases than cSCC cases (p < 0.01). CYP1A1 expression was low and showed no differences among the four studied groups (p > 0.05). The H-score of AHR was positively correlated with EGFR expression (r = 0.54, p < 0.01) in atypical squamous proliferation cases but was not correlated with CYP1A1 (r = − 0.17, p = 0.295) and Ki-67 (r = − 0.48, p = 0.222) expression.ConclusionAHR plays a vital role in cSCC pathogenesis. The overexpression and activation of AHR are involved in the early development of skin cancers. AHR expression correlates with EGFR expression and may influence cell proliferation. AHR is a valuable therapeutic target for skin cancers.

Targeting of TRX2 by miR-330-3p in melanoma inhibits proliferation

ObjectiveThis study is intended to identify the key gene from gene expression profile and validate its role and regulatory mechanism in melanoma. MethodsGene expression profile of GSE3189 from GEO database was selected among which 7 are normal skin samples, 18 are benign skin lesion samples, and 45 are melanoma samples. The present study examined the 7 normal skin samples and the 45 melanoma samples. Differentially expressed genes (DEGs) between melanoma patients and health people were performed using Morpheus online tool. The 100 most differentially expressed genes (50 upregulated genes and 50 downregulated genes) were selected as hub genes. Then, expression levels and survival analysis of hub genes were conducted via GEPIA tool to choose target gene. The expression of target gene in melanoma cell lines was examined by RT-qPCR and western blotting. The biological function of target gene on cell proliferation in melanoma was measured in vitro. The predicted target of target gene was validated by dual-luciferase reporter assay and rescue experiment. The gene expression in clinical samples were determined by RT-qPCR, immunohistochemistry (IHC) and in situ hybridization (ISH). The tumor formation study was conducted in vivo. ResultsTargeting protein for Xklp2 (TPX2) was identified as key gene in melanoma. TPX2 could promote the proliferation of melanoma cells. The dual luciferase reporter assay confirmed that miR-330-3p targets TPX2. In rescue experiment, it was proved that miR-330-3p inhibits the proliferation of melanoma cells by negatively regulating the expression of TPX2. The results in vitro were also confirmed in vivo. miR-330-3p/TPX2 pathway expressed differently between melanoma patients and health people. These differences were statistically significant (P < 0.05). ConclusionInhibiting TPX2 by miR-330-3p suppresses the proliferation of melanoma cell lines. miR-330-3p/TPX2 pathway could be a potential therapeutic target in melanoma.

The Role of Human Papillomaviruses and Polyomaviruses in BRAF-Inhibitor Induced Cutaneous Squamous Cell Carcinoma and Benign Squamoproliferative Lesions.

Background: Human papillomavirus (HPV) has long been proposed as a cofactor in the pathogenesis of cutaneous squamous cell carcinoma (cSCC). More recently, the striking clinico-pathological features of cSCCs that complicate treatment of metastatic melanoma with inhibitors targeting BRAF mutations (BRAFi) has prompted speculation concerning a pathogenic role for oncogenic viruses. Here, we investigate HPV and human polyomaviruses (HPyV) and correlate with clinical, histologic, and genetic features in BRAFi-associated cSCC.Materials and Methods: Patients receiving BRAFi treatment were recruited at Barts Health NHS Trust. HPV DNA was detected in microdissected frozen samples using reverse line probe technology and degenerate and nested PCR. HPV immunohistochemistry was performed in a subset of samples. Quantitative PCR was performed to determine the presence and viral load of HPyVs with affinity for the skin (HPyV6, HPyV7, HPyV9, MCPyV, and TSPyV). These data were correlated with previous genetic mutational analysis of H, K and NRAS, NOTCH1/2, TP53, CDKN2A, CARD11, CREBBP, TGFBR1/2. Chromosomal aberrations were profiled using single nucleotide polymorphism (SNP) arrays.Results: Forty-five skin lesions from seven patients treated with single agent vemurafenib in 2012–2013 were analyzed: 12 cSCC, 19 viral warts (VW), 2 actinic keratosis (AK), 5 verrucous keratosis/other squamoproliferative (VK/SP) lesions, one melanocytic lesion and 6 normal skin samples. Significant histologic features of viral infection were seen in 10/12 (83%) cSCC. HPV DNA was detected in 18/19 (95%) VW/SP, 9/12 (75%) cSCC, 4/5 (80%) SP, and 3/6 (50%) normal skin samples and in 1/12 cases assessed by immunohistochemistry. HPyV was co-detected in 22/30 (73%) of samples, usually at low viral load, with MCPyV and HPyV7 the most common. SNP arrays confirmed low levels of chromosomal abnormality and there was no significant correlation between HPV or HPyV detection and individual gene mutations or overall mutational burden.Conclusion: Despite supportive clinicopathologic evidence, the role for HPV and HPyV infection in the pathogenesis of BRAFi-induced squamoproliferative lesions remains uncertain. Synergistic oncogenic mechanisms are plausible although speculative. Nonetheless, with the prospect of a significant increase in the adjuvant use of these drugs, further research is justified and may provide insight into the pathogenesis of other BRAFi-associated malignancies.

MicroRNA expression in the affected skin of adult patients with IgA vasculitis.

IgA vasculitis (IgAV) represents a common systemic vasculitis in pediatric and adult population. Our current knowledge of disease pathogenesis is still very limited, without information on miRNAs in IgAV. The aim of our study was to determine the expression of five pre-selected miRNAs (miRNA-146a-5p, miRNA-148-3p, miRNA-155-5p, miRNA-223-3p, and let-7b) in the affected skin of adult IgAV patients. The study included 65 skin samples from consecutive, untreated IgAV patients (61.5% male, median age 67.6years, range 29-91), diagnosed between October 2014 and September 2016, and 20 samples of normal skin from healthy volunteers. Total RNA was isolated from tissue sections of formalin-fixed, paraffin-embedded samples. Expression of miRNAs was measured using qRT-PCR. To present relative miRNA expression, the ΔΔCT method was used. Skin miRNA expression was correlated to clinical characteristics of adult IgAV patients. We found significantly higher levels of miRNA-155-5p, miRNA-223-3p, and let-7b in the affected skin compared to controls (18.6-fold, 6.4-fold, and 7.9-fold higher respectively). Contrary, the miRNA 148-3p expression was significantly lower (2.2-fold). The expression of the miRNA-146-5p showed near normal levels. Patients with necrotic skin lesions had significantly higher miRNA-223 tissue expression than those with non-necrotic purpura (p = 0.029). Gastrointestinal tract involvement inversely correlated with the expression of miRNA-155-5p and/or miRNA-146a-5p in affected skin. Altered expression of miRNA-148b-3p, miRNA-155-5p, miRNA-223-3p, and let-7b was found in vasculitic skin lesions in IgAV. Additionally, we found a positive association between the severity of purpura and skin miRNA-223-3p expression. Aberrantly expressed miRNAs could represent a biomarker in adult IgAV.

Analysis of KRT1, KRT10, KRT19, TP53 and MMP9 expression in pediatric and adult cholesteatoma.

Cholesteatoma is an epidermal cyst with still unknown pathomechanism. The aim of the current study was to investigate molecular differences in the background of the hyperproliferative property and aggressive behavior typical of the cholesteatoma epithelium. The expression of three cytokeratin genes (KRT1, KRT10 and KRT19), the matrix metalloproteinase 9 gene (MMP9) and the tumor suppressor TP53 gene was measured by qRT-PCR in surgical samples of pediatric and adult cholesteatoma cases and their expression level was compared to that of normal skin samples from the retroauricular region of control individuals. Cholesteatoma samples were stratified according to the age of onset and recurrence for more detailed analysis. Our results showed identical expression pattern for KRT1 and KRT10, their expression was higher in pediatric cases than in adults, especially in pediatric recurrent samples. The expression level of KRT19 was inversely proportional to that of KRT1/KRT10, it was lower in the more invasive recurrent cases both in our pediatric and adult groups. As it was expected from the bone destructive behavior of cholesteatoma, a significantly elevated expression of MMP9 was measured in cholesteatoma samples, the highest level was found in adult recurrent cases. Low expression levels characterize the TP53 gene without significant differences in our samples. These findings demonstrate that cytokeratin expression distinguishes between pediatric/adult, nonrecurrent/recurrent cases, suggesting that distinct differentiation state and cell division potential characterize these cholesteatoma cases. KRT19 with a tumor suppressor potential might restrict the recurrence of cholesteatoma. The differences observed in gene expression profiles between cholesteatoma and control samples support the notion that cholesteatoma is a cystic lesion with tumor-like behavior because it is characterized by invasive, destructive growth and high tendency for recurrence.