Melanoma Cell Migration Research Articles

Integration Analysis of m6A Related Genes in Skin Cutaneous Melanoma and the Biological Function Research of the SPRR1B.

Melanoma has gradually entered the public view because of its high morbidity and rising prevalence rate, which is a serious threat to human life and health. Recently, N6-methyladenine (m6A) modification has been increasingly confirmed as a potential role in the development of tumogenesis. The purpose of this study is to explore the role and function of m6a-related regulators in the development of melanoma disease at the molecular, cellular and clinical levels through bioinformatics and traditional experiments. We screened and validated differential expression genes (DEGs) in m6A regulators via the GEO, GTEx, TCGA database. The biological processes and signaling pathway involved by DEGs were improved by constructing bioinformational methods such as PPI, GO enrichment, KEGG enrichment, GSEA enrichment, and immune infiltration analysis. And then, we explored the biological function of the key gene, SPRR1B, through cell invasion, migration, infiltration, and tissue chips. The gene IGF2BP3 which was differentially expressed in m6A regulatory factor gene was screened. The results of the enrichment analysis are significantly enriched in the biological processes and pathways of the skin barrier, epidermal differentiation, cytoskeleton, lymphocyte migration and other pathways, pointing to the direction of tumor immunity and tumor metastasis. Tumor immune-related genes YTHDC1, YTHDC2 and ALKBH5 were found. Knock SPRR1B reduction group had a significantly lower invasive ability, the ability to migrate. Nomogram prediction model shows that SPRR1B increased, expressing a worse prognosis. For this purpose, the relationship between m6A regulatory factor and melanoma progression was explored. At the same time, it was found that the abnormal up-regulated expression of SPRR1B before metastasis would lead to poor prognosis of melanoma. SPRR1B promotes the proliferation, invasion and migration of human melanoma cells.

IER2-induced senescence drives melanoma invasion through osteopontin

Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.

Identification of HMGCR as the anticancer target of physapubenolide against melanoma cells by in silico target prediction

Physapubenolide (PB), a withanolide-type compound extracted from the traditional herb Physalis minima L., has been demonstrated to exert remarkable cytotoxicity against cancer cells; however, its molecular mechanisms are still unclear. In this study, we demonstrated that PB inhibited cell proliferation and migration in melanoma cells by inducing cell apoptosis. The anticancer activity of PB was further verified in a melanoma xenograft model. To explore the mechanism underlying the anticancer effects of PB, we carried out an in silico target prediction study, which combined three approaches (chemical similarity searching, quantitative structure-activity relationship (QSAR), and molecular docking) to identify the targets of PB, and found that PB likely targets 3-hydroxy-methylglutaryl CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, which promotes cancer cell proliferation, migration, and metastasis. We further demonstrated that PB interacted with HMGCR, decreased its protein expression and inhibited the HMGCR/YAP pathway in melanoma cells. In addition, we found that PB could restore vemurafenib sensitivity in vemurafenib-resistant A-375 cells, which was correlated with the downregulation of HMGCR. In conclusion, we demonstrate that PB elicits anticancer action and enhances sensitivity to vemurafenib by targeting HMGCR.

The expression, purification, and functional evaluation of the novel tumor suppressor fusion protein IL-24-CN.

Interleukin-24 (IL-24) can specifically induce apoptosis in a broad range of cancer cells without harming normal cells. The interaction of contortrostatin (CN) with integrins on angiogenic vascular endothelial and tumor cells is modulated by the RGD motifs that can significantly inhibit metastasis and angiogenesis. To achieve superior therapeutic efficacy by combining anti-metastasis with tumor-selective apoptosis activity, CN was fused at the C-terminus of IL-24 with a flexible linker (G4S)2, and the recombinant IL-24-CN was expressed in Escherichia coli as a Thioredoxin (Trx)/IL-24-CN fusion protein. The target protein was purified using nickel affinity chromatography. Furthermore, we simplified the purification process by purifying Trx-IL-24-CN and cleaving the Trx tag in one step. The final yield of IL-24-CN was 27.6mg/L based on flask fermentation. In vitro activity assay demonstrated that the recombinant IL-24-CN could more effectively suppress tumor growth and induce apoptosis of melanoma cells. Scratch and transwell assays suggested that IL-24-CN strongly reduced the migration and invasion behavior of melanoma cells. Immunofluorescence analysis and cell adhesion assay showed that CN could evidently improve the tumor inhibition capability of IL-24 by enhancing the affinity of recombinant protein toward cancer cells. In summary, a highly efficient strategy was developed for producing the bioactive IL-24-CN from prokaryotic cells, supporting IL-24-CN in melanoma therapy.Key points• Efficient heterologous production of recombinant IL-24-CN in E. coli using Trx fusion strategy.• Improved tumor growth suppression and apoptosis induction potency of IL-24-CN.• Enhanced cell adhesion ability of IL-24-CN in cancer cells.

Mambalgin-2 Inhibits Growth, Migration, and Invasion of Metastatic Melanoma Cells by Targeting the Channels Containing an ASIC1a Subunit Whose Up-Regulation Correlates with Poor Survival Prognosis.

Melanoma is an aggressive cancer characterized by the acidification of the extracellular environment. Here, we showed for the first time that extracellular media acidification increases proliferation, migration, and invasion of patient-derived metastatic melanoma cells and up-regulates cell-surface expression of acid-sensitive channels containing the ASIC1a, α-ENaC, and γ-ENaC subunits. No influence of media acidification on these processes was found in normal keratinocytes. To control metastatic melanoma progression associated with the ASIC1a up-regulation, we proposed the ASIC1a inhibitor, -mambalgin-2 from Dendpoaspis polylepis venom. Recombinant analog of mambalgin-2 cancelled acidification-induced proliferation, migration, and invasion of metastatic melanoma cells, promoted apoptosis, and down-regulated cell-surface expression of prooncogenic factors CD44 and Frizzled 4 and phosphorylation of transcription factor SNAI. Confocal microscopy and affinity purification revealed that mambalgin-2 interacts with heterotrimeric ASIC1a/α-ENaC/γ-ENaC channels on the surface of metastatic melanoma cells. Using the mutant variant of mambalgin-2 with reduced activity toward ASIC1a, we confirmed that the principal molecular target of mambalgin-2 in melanoma cells is the ASIC1a subunit. Bioinformatic analysis confirmed up-regulation of the ASIC1 expression as a marker of poor survival prognosis for patients with metastatic melanoma. Thus, targeting ASIC1a by drugs such as mambalgin-2 could be a promising strategy for metastatic melanoma treatment.

Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia.

Simple SummaryObstructive sleep apnea has been implicated in deleterious effects on cancer incidence and outcomes. Among the cancers identified as affected by sleep apnea, melanoma has consistently emerged as particularly susceptible. Furthermore, evidence suggests that exosomes, microparticles that originate from all cells and contain a variety of biologically active molecules such as microRNAs, can modify cancer cell properties. Here, we examined whether exosomes from sleep apnea patients can alter melanoma cell properties, and whether such effects were attributable to selected microRNA candidates, and affected melanoma cells harboring different mutations differently. In these experiments, the divergent responses of three melanoma cell lines to exosomes from sleep apnea patients before and after treatment indicated that STK11 gene mutations in melanoma cells make them particularly responsive to sleep apnea by altering cellular metabolism, and that specific microRNAs appear to underlie such effects.Obstructive sleep apnea (OSA) is associated with increased cutaneous melanoma incidence and adverse outcomes. Exosomes are secreted by most cells, and play a role in OSA-associated tumor progression and metastasis. We aimed to study the effects of plasma exosomes from OSA patients before and after adherent treatment with continuous positive airway pressure (CPAP) on melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to-severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity in the presence or absence of a chemical activator. Subsequently, CRL-1424 and CRL-1675 cells were exposed to intermittent hypoxia (IH) and normoxia, and exosomal miRNAs were identified followed by GO and KEG pathways and gene networks. The exosomes from these IH-exposed melanoma cells were also administered to THP1 macrophages to examine changes in M1 and M2 polarity markers. Plasma exosomes from V1 increased CRL-1424 melanoma cell proliferation and migration compared to V2, but not the other two cell lines. Exposure to CRL-1424 exosomes reduced pAMPK/tAMPK in V1 compared to V2, and treatment with AMPK activator reversed the effects. Unique exosomal miRNAs profiles were identified for CRL-1424 and CRL-1675 in IH compared to normoxia, with six miRNAs being regulated and several KEGG pathways were identified. Two M1 markers (CXCL10 and IL6) were significantly increased in monocytes when treated with exosomes from IH-exposed CRL-1424 and CRL-1625 cells. Our findings suggest that exosomes from untreated OSA patients increase CRL-1424 melanoma malignant properties, an effect that is not observed in two other melanoma cell lines. Exosomal cargo from CRL-1424 cells showed a unique miRNA signature compared to CRL-1675 cells after IH exposures, suggesting that melanoma cells are differentially susceptible to IH, even if they retain similar effects on immune cell polarity. It is postulated that mutations in STK-11 gene encoding for the serine/threonine kinase family that acts as a tumor suppressor may underlie susceptibility to IH-induced metabolic dysfunction, as illustrated by CRL-1424 cells.

A Comparative Survey of Anti-Melanoma and Anti-Inflammatory Potential of Usnic Acid Enantiomers-A Comprehensive In Vitro Approach.

Usnic acid (UA) is a chiral lichen metabolite with an interesting pharmacological profile. The aim of this study was to compare the anti-melanoma effect of (+)-UA and (−)-UA in an in vitro model by studying their impact on the cells as well as the processes associated with cancer progression. The effect of UA enantiomers on the viability, proliferation, and invasive potential of three melanoma cell lines (HTB140, A375, WM793) was evaluated. Their interaction with a chemotherapeutic drug—doxorubicin was assessed by isobolographic analysis. Anti-inflammatory and anti-tyrosinase properties of (+)-UA and (−)-UA were also examined. Both UA enantiomers dose- and time-dependently decreased the viability of all three melanoma cell lines. Their synergistic effect with doxorubicin was observed on A375 cells. (+)-Usnic acid at a sub-cytotoxic dose strongly inhibited melanoma cells migration. Both UA enantiomers decreased the release of pro-inflammatory mediators. The cytotoxic effect of (+)-UA and (−)-UA depends greatly on the melanoma cell type; however, the overall anti-melanoma potential is perspective. Our results indicate that the strategy of combining usnic acid enantiomers with cytostatic drugs may be an interesting option to consider in combating melanoma; however, further studies are required.

L-Theanine inhibits melanoma cell growth and migration via regulating expression of the clock gene BMAL1

L-Theanine is a unique non-protein amino acid found in green tea, which has been identified as a safe dietary supplement. It has been reported that L-theanine exerts various biological activities. In this study, we explored the anti-cancer effects of L-theanine on melanoma cells. A375, B16-F10, and PIG1 cell lines were used in the present study. EdU labeling, TUNEL and AnnexinV/PI staining, wound-healing, and transwell migration assay were performed to detect the effects of L-theanine on melanoma cell proliferation, apoptosis, and migration. Brain and muscle Arnt-like protein 1 (BMAL1) was knocked down in melanoma cells to evaluate if L-theanine plays the anti-cancer role through regulating circadian rhythm of melanoma cells. The western blot, qRT-PCR, and dual luciferase assay were performed to explore the mechanism involved in the effects of L-theanine on melanoma cells. L-Theanine apparently reduced the viability of melanoma cells. Further experiments showed that L-theanine attenuated the proliferation and migration, and promoted apoptosis of melanoma cells. L-Theanine significantly enhanced the expression of BMAL1, a clock gene in melanoma cells. Down-regulation of BMAL1 suppressed the anti-cancer effects of L-theanine on melanoma cells. Further experiments indicated that the p53 transcriptional activity raised by L-theanine was dependent on BMAL1 expression in melanoma cells. L-Theanine exerts the anti-cancer effect on melanoma cells through attenuating the proliferation and migration, and promoting apoptosis of them, which is dependent on the regulation of the clock gene Bmal1 in melanoma cells.

The plasma exosomal miR-1180-3p serves as a novel potential diagnostic marker for cutaneous melanoma

BackgroundExosomes are a promising tool in disease detection because they are noninvasive, cost-effective, sensitive and stable in body fluids. MicroRNAs (miRNAs) are the main exosomal component and participate in tumor development. However, the exosomal miRNA profile among Asian melanoma patients remains unclear.MethodsExosomal miRNAs from the plasma of melanoma patients (n = 20) and healthy individuals (n = 20) were isolated and subjected to small RNA sequencing. Real-time PCR was performed to identify the differential miRNAs and to determine the diagnostic efficiency. Proliferation, scratch and Transwell assays were performed to detect the biological behavior of melanoma cells.ResultsExosomal miRNA profiling revealed decreased miR-1180-3p expression as a potential diagnostic marker of melanoma. The validation group of melanoma patients (n = 28) and controls (n = 28) confirmed the diagnostic efficiency of miR-1180-3p. The level of miR-1180-3p in melanoma cells was lower than that in melanocytes. Accordingly, the level of miR-1180-3p was negatively associated with the proliferation, migration and invasion of melanoma cells. Functional analysis and target gene prediction found that ST3GAL4 was a potential target and highly expressed in melanoma tissues and was negatively regulated by miR-1180-3p. Knockdown of ST3GAL4 hindered the malignant phenotype of melanoma cells.ConclusionsThis study indicates that reduced exosomal miR-1180-3p in melanoma patient plasma is a promising diagnostic marker and provides novel insight into melanoma development.

Pituitary Adenylate Cyclase Activating Polypeptide Has Inhibitory Effects on Melanoma Cell Proliferation and Migration In Vitro.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide which is distributed throughout the body. PACAP influences development of various tissues and exerts protective function during cellular stress and in some tumour formation. No evidence is available on its role in neural crest derived melanocytes and its malignant transformation into melanoma. Expression of PACAP receptors was examined in human skin samples, melanoma lesions and in a primary melanocyte cell culture. A2058 and WM35 melanoma cell lines, representing two different stages of melanoma progression, were used to investigate the effects of PACAP. PAC1 receptor was identified in melanocytes in vivo and in vitro and in melanoma cell lines as well as in melanoma lesions. PACAP administration did not alter viability but decreased proliferation of melanoma cells. With live imaging random motility, average speed, vectorial distance and maximum distance of migration of cells were reduced upon PACAP treatment. PACAP administration did not alter viability but decreased proliferation capacity of melanoma cells. On the other hand, PACAP administration decreased the migration of melanoma cell lines towards fibronectin chemoattractant in the Boyden chamber. Furthermore, the presence of the neuropeptide inhibited the invasion capability of melanoma cell lines in Matrigel chambers. In summary, we provide evidence that PACAP receptors are expressed in melanocytes and in melanoma cells. Our results also prove that various aspects of the cellular motility were inhibited by this neuropeptide. On the basis of these results, we propose PACAP signalling as a possible target in melanoma progression.

Overexpression of microRNA-130a represses uveal melanoma cell migration and invasion through inactivation of the Wnt/β-catenin signaling pathway by downregulating USP6.

Uveal melanoma (UM) is a neoplasm arising from melanocytes of the ciliary body, choroid, and iris of the eye, which is the most common primary malignant intraocular tumor. microRNA-130a (miR-130a) has been confirmed to be underexpressed in many types of cancers. Here we aimed to investigate the mechanism whereby miR-130a affects the Wnt/β-catenin signaling pathway by targeting ubiquitin-specific protease 6 (USP6) in UM. Ocular specimens of 62 patients with UM and 42 participants subjected to enucleation due to trauma were collected. In the normal uveal tissues and those from metastatic and non-metastatic UM, we evaluated miR-130a expression by RT-qPCR and then measured mRNA and protein expression of recombinant human mothers against decapentaplegic homolog 4 (SMAD4), USP6, related factors of the Wnt/β-catenin signaling pathway, and epidermal growth factor receptor (EGFR) by RT-qPCR and western blot analysis. Subsequently, the interaction between miR-130a and USP6 was identified by bioinformatics analysis and dual-luciferase reporter gene assay. Next, UM cell migration and invasion abilities, as well as tumor growth in nude mice, were measured through gain- and loss-of-function studies of miR-130a and USP6. miR-130a expression was downregulated in uveal tissues from patients with UM, especially in metastatic uveal tissues. The overall survival of UM patients with low miR-130a expression was shorter than those with high miR-130a expression. USP6 was a target of miR-130a and the overexpression of miR-130a or inhibition of USP6 in UM MUM-2B and MUM-2C cell lines inhibited the expression of Wnt, β-catenin, and EGFR, and activated SMAD4 expression, while reducing UM cell migration and invasion abilities in vitro. The above changes could be reversed by overexpressing USP6 in vitro, whereas overexpressed miR-130a could inhibit the tumor growth in nude mice. Taken together, overexpressed miR-130a inhibited USP6 expression to repress UM cell migration and invasion abilities through inactivating the Wnt/β-catenin signaling pathway, which could be a potential candidate for treatment of UM.

Novel Allosteric Inhibitors of Deoxyhypusine Synthase against Malignant Melanoma: Design, Synthesis, and Biological Evaluation.

Based on the novel allosteric site of deoxyhypusine synthase (DHPS), two series of 30 novel 5-(2-methoxyphenoxy)-2-phenylpyrimidin-4-amine derivatives as DHPS inhibitors were designed and synthesized. Among them, compound 8m, with the best DHPS inhibitory potency (IC50 = 0.014 μM), exhibited excellent inhibition against melanoma cells, which was superior to that of GC7. Besides, molecular docking and molecular dynamics (MD) simulations further proved that compound 8m was tightly bound to the allosteric site of DHPS. Flow cytometric analysis and enzyme-linked immunosorbent assay (ELISA) showed that compound 8m could inhibit the intracellular reactive oxygen species (ROS) level. Furthermore, by western blot analysis, compound 8m effectively activated caspase 3 and decreased the expressions of GP-100, tyrosinase, eIF5A2, MMP2, and MMP9. Moreover, both Transwell analysis and wound healing analysis showed that compound 8m could inhibit the invasion and migration of melanoma cells. In the in vivo study, the tumor xenograft model showed that compound 8m effectively inhibited melanoma development with low toxicity.

Inhibitory Effect of Dihydroartemisinin on the Proliferation and Migration of Melanoma Cells and Experimental Lung Metastasis From Melanoma in Mice.

Melanoma is aggressive and can metastasize in the early stage of tumor. It has been proved that dihydroartemisinin (DHA) positively affects the treatment of tumors and has no apparent toxic and side effects. Our previous research has shown that DHA can suppress the formation of melanoma. However, it remains poorly established how DHA impacts the invasion and metastasis of melanoma. In this study, B16F10 and A375 cell lines and metastatic tumor models will be used to investigate the effects of DHA. The present results demonstrated that DHA inhibited the proliferative capacity in A375 and B16F10 cells. As expected, the migration capacity of A375 and B16F10 cells was also reduced after DHA administration. DHA alleviated the severity and histopathological changes of melanoma in mice. DHA induced expansion of CD8+CTL in the tumor microenvironment. By contrast, DHA inhibited Treg cells infiltration into the tumor microenvironment. DHA enhanced apoptosis of melanoma by regulating FasL expression and Granzyme B secretion in CD8+CTLs. Moreover, DHA impacts STAT3-induced EMT and MMPS in tumor tissue. Furthermore, Metabolomics analysis indicated that PGD2 and EPA significantly increased after DHA administration. In conclusion, DHA inhibited the proliferation, migration and metastasis of melanoma in vitro and in vivo. These results have important implications for the potential use of DHA in the treatment of melanoma in humans.

Pharmacological inhibition of USP7 suppresses growth and metastasis of melanoma cells in vitro and in vivo.

Melanoma is a highly aggressive type of skin cancer. The development of diverse resistance mechanisms and severe adverse effects significantly limit the efficiency of current therapeutic approaches. Identification of the new therapeutic targets involved in the pathogenesis will benefit the development of novel therapeutic strategies. The deubiquitinase ubiquitin–specific protease‐7, a potential target for cancer treatment, is deregulated in types of cancer, but its role in melanoma is still unclear. We investigated the role and the inhibitor P22077 of ubiquitin‐specific protease‐7 in melanoma treatment. We found that ubiquitin‐specific protease‐7 was overexpressed and correlated with poor prognosis in melanoma. Further, pharmacological inhibition of ubiquitin‐specific protease‐7 by P22077 can effectively inhibit proliferation, and induce cell cycle arrest and apoptosis via ROS accumulation–induced DNA damage in melanoma cells. Inhibition of ubiquitin‐specific protease‐7 by P22077 also inhibits melanoma tumour growth in vivo. Moreover, inhibition of ubiquitin‐specific protease‐7 prevented migration and invasion of melanoma cells in vitro and in vivo by decreasing the Wnt/β‐catenin signalling pathway. Taken together, our study revealed that ubiquitin‐specific protease‐7 acted as an oncogene involved in melanoma cell proliferation and metastasis. Therefore, ubiquitin‐specific protease‐7 may serve as potential candidates for the treatment of melanoma.

Loss of miR-1469 expression mediates melanoma cell migration and invasion.

Tumor ulceration is considered one of the most prognostically significant findings in primary cutaneous melanoma, associated with decreased disease-free and overall survival. However, the unique features associated with ulcerated melanoma that contribute to a poor prognosis in affected patients remain poorly defined. microRNAs are small, non-coding RNAs that function to inhibit expression of specific gene targets, therefore altering the functions of cells in which they are expressed. miR-1469 is a novel miR with significantly decreased expression in ulcerated melanoma tissue relative to non-ulcerated tumors. We hypothesized that loss of miR-1469 expression in melanoma contributes to altered tumor cell functions mediating disease progression. Transfection of a miR-1469 mimic resulted in a significant reduction in the migratory and invasive capacity of the CHL1 and MEL39 melanoma cell lines (>58.1% reduction, p < 0.0332), as well as the invasive capacity of the A375 melanoma cell line (>50% reduction, p < 0.0021). Expression of myeloid cell leukemia-1 (MCL1), a miR-1469 target gene, was reduced in the A375 and MEL39 cell lines by immunoblot. No significant differences in viability, resistance to apoptotic stimuli, or proliferation were observed following transfection. These findings together demonstrate how migration and invasion are specific functions through which miR-1469 expression in melanoma cells can contribute to the differences in disease progression associated with tumor ulceration.

Effects of RNA methylation N6-methyladenosine regulators on malignant progression and prognosis of melanoma

BackgroundMelanoma is an extremely aggressive type of skin cancer and experiencing a expeditiously rising mortality in a current year. Exploring new potential prognostic biomarkers and therapeutic targets of melanoma are urgently needed. The ambition of this research was to identify genetic markers and assess prognostic performance of N6-methyladenosine (m6A) regulators in melanoma.MethodsGene expression data and corresponding clinical informations of melanoma patients as well as sequence data of normal controls are collected from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. Quantitative real-time PCR (qRT-PCR) analysis was carried out to detect the RNA expression of IGF2BP3 in A375 cell line, melanoma tissues, and normal tissues. Western blot, cell proliferation, and migration assays were performed to assess the ability of IGF2BP3 in A375 cell line.ResultsDifferently expressed m6A regulators between tumor samples and normal samples were analyzed. A three-gene prognostic signature including IGF2BP3, RBM15B, and METTL16 was constructed, and the risk score of this signature was identified to be an independent prognostic indicator for melanoma. In addition, IGF2BP3 was verified to promote melanoma cell proliferation and migration in vitro and associate with lymph node metastasis in clinical samples. Moreover, risk score and the expression of IGF2BP3 were positively associated with the infiltrating immune cells and these hub genes made excellent potential drug targets in melanoma.ConclusionWe identified the genetic changes in m6A regulatory genes and constructed a three-gene risk signature with distinct prognostic value in melanoma. This research provided new insights into the epigenetic understanding of m6A regulators and novel therapeutic strategies in melanoma.

TCF21 regulates miR-10a-5p/LIN28B signaling to block the proliferation and invasion of melanoma cells.

Background and aimSome research has suggested that miRNA-10a (miR-10a-5p) had an inhibitory function in proliferation and invasion of cancers. Whereas the role of miR-10a-5p in melanoma has not been fully explored. This study aims to confirm LIN28B as the targeted gene of miR-10a-5p which was explored in melanoma cells. In addition, upstream regulatory molecule of miR-10a-5p was also investigated in melanoma cells.MethodsReal-time Quantitative polymerase chain reaction (RT-qPCR) was adopted to analyze miR-10a-5p expression level in melanoma and the normal human epidermal melanocyte cells. Several biological assays were performed to evaluate miR-10a-5p influences on cell proliferation, migration and invasion ability in A375 and B16-F10 cells. Gene prediction of miRNA targeting and a dual luciferase assay were applied to assess miR-10a-5p-targeted LIN28B. Western blot assessed the impacts of miR-10a-5p on the protein expression of LIN28B. Western blot analyzed the TCF21 effects on the expression of LIN28B and RT-qPCR assessed the influence of TCF21 on the expression level of miRNA-10a. In addition, Chromatin Immunoprecipitation (ChIP) Assay and JASPAR databases were employed to explore the regulatory relationship between TCF21 and miR-10a-5p.ResultsWe discovered that miR-10a-5p expression was lower in melanoma cells and high expression of miR-10a-5p suppressed the proliferation, migration and invasion abilities of melanoma cells. We also discovered that miR-10a-5p targeted the LIN28B mRNA 3′UTR area and diminished LIN28B protein expression. We found that LIN28B expression was strongly decreased by TCF21 upregulation in the two melanoma cells. The qRT-PCR assay showed that miR-10a-5p expression level was obviously boosted by increased TCF21 expression. The results also demonstrated that TCF21 directly regulated miR-10a-5p at transcript levels.ConclusionTCF21 induced miRNA-10a targeting LIN28B could affect the progression and growth of melanoma.

ATG5 and ATG7 Expression Levels Are Reduced in Cutaneous Melanoma and Regulated by NRF1.

Autophagy is a highly conserved cellular process in which intracellular proteins and organelles are sequestered and degraded after the fusion of double-membrane vesicles known as autophagosomes with lysosomes. The process of autophagy is dependent on autophagy-related (ATG) proteins. The role of autophagy in cancer is very complex and still elusive. We investigated the expression of ATG proteins in benign nevi, primary and metastatic melanoma tissues using customized tissue microarrays (TMA). Results from immunohistochemistry show that the expression of ATG5 and ATG7 is significantly reduced in melanoma tissues compared to benign nevi. This reduction correlated with changes in the expression of autophagic activity markers, suggesting decreased basal levels of autophagy in primary and metastatic melanomas. Furthermore, the analysis of survival data of melanoma patients revealed an association between reduced ATG5 and ATG7 levels with an unfavourable clinical outcome. Currently, the mechanisms regulating ATG expression levels in human melanoma remains unknown. Using bioinformatic predictions of transcription factor (TF) binding motifs in accessible chromatin of primary melanocytes, we identified new TFs involved in the regulation of core ATGs. We then show that nuclear respiratory factor 1 (NRF1) stimulates the production of mRNA and protein as well as the promoter activity of ATG5 and ATG7. Moreover, NRF1 deficiency increased in vitro migration of melanoma cells. Our results support the concept that reduced autophagic activity contributes to melanoma development and progression, and identifies NRF1 as a novel TF involved in the regulation of both ATG5 and ATG7 genes.

A KLK4 proteinase substrate capture approach to antagonize PAR1

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling.

MicroRNA-9 inhibits the proliferation and migration of malignant melanoma cells via targeting sirituin 1.

[This retracts the article DOI: 10.3892/etm.2017.4595.].