Melanoma Cell Migration Research Articles

Long Noncoding RNA Small Nucleolar RNA Host Gene 6 Promotes Cell Proliferation, Migration and Invasion in Melanoma by Sponging miR-944

Long noncoding RNA small nucleolar RNA host gene 6 (SNHG6) has been reported to be a tumor promoter in various human cancers. Nevertheless, the detailed functions and clinical value of SNHG6 in melanoma remain elusive. The study aimed to investigate the role and potential mechanism of SNHG6 in melanoma metastasis. Quantitative real-time PCR (qRT-PCR) was used to detect the expressions of SNHG6 and miR-944 in melanoma cells. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay, and cell migration and invasion were measured by wound healing assay and cell invasion assay, respectively. In addition, dual luciferase reporter assay was performed to verify the interaction between SNHG6 and miR-944. The protein expressions of PI3K/Akt pathway were evaluated by western blot assay. The results revealed that SNHG6 expression was significantly increased in melanoma cells. Knockdown of SNHG6 suppressed cell proliferation, migration and invasion in A375 cells. Moreover, miR-944 was identified as a direct target of SNHG6 in melanoma. miR-944 was downregulated in melanoma cells, while SNHG6 silencing improved miR-944 level in A375 cells. Rescue experiments demonstrated that miR-944 overexpression reversed the effects of SNHG6 on A375 cell proliferation, migration and invasion. Altogether, SNHG6 exerted oncogenic effects in melanoma cells, providing a novel promising target for the treatment of melanoma.

Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling.

Patients diagnosed with melanoma have a poor prognosis due to regional invasion and metastases. The receptor tyrosine kinase epidermal growth factor receptor (EGFR) is found in a subtype of melanoma with a poor prognosis and contributes to drug resistance. Aloysia citrodora essential oil (ALOC-EO) possesses an antitumor effect. Understanding signaling pathways that contribute to the antitumor of ALOC-EO is important to identify novel tumor types that can be targeted by ALOC-EO. Here, we investigated the effects of ALOC-EO on melanoma growth and tumor cell migration. ALOC-EO blocked melanoma growth in vitro and impaired primary tumor cell growth in vivo. Mechanistically, ALOC-EO blocked heparin-binding-epidermal growth factor (HB-EGF)-induced EGFR signaling and suppressed ERK1/2 phosphorylation. Myelosuppressive drugs upregulated HB-EGF and EGFR expression in melanoma cells. Cotreatment of myelosuppressive drugs with ALOC-EO improved the antitumor activity and inhibited the expression of matrix metalloproteinase-7 and -9 and a disintegrin and metalloproteinase domain-containing protein9. In summary, our study demonstrates that ALOC-EO blocks EGFR and ERK1/2 signaling, with preclinical efficacy as a monotherapy or in combination with myelosuppressive drugs in melanoma.

Gelsolin Contributes to the Motility of A375 Melanoma Cells and This Activity Is Mediated by the Fibrous Extracellular Matrix Protein Profile.

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)—an actin-binding protein—levels in melanoma cell lines and GSN’s importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin’s BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN’s influence on parameters associated with cells’ ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin’s BM.

LINC01116 Facilitates Melanoma 1 Progression Via Sequestering miR-3612 and Up-regulating GDF11 and SDC3

Melanoma is the deadliest cutaneous malignant tumor with high risks. Though increasing evidence has widely referred to the involvement of long non-coding RNAs (lncRNAs) in the mechanism of tumor development, including melanoma, the functional roles of most lncRNAs in melanoma remain to be explored. In this study, we focus on disclosing the role of long intergenic non-protein coding RNA 1116 (LINC01116) in melanoma. Firstly, we detected LINC01116 expression through RT-qPCR. Functional analysis and animal experiments were carried out to assess the role of LINC01116 in vivo and in vitro. Western blot analysis was employed for detection of important markers regarding epithelial mesenchymal transition (EMT). In addition, RNA pulls down, RIP and luciferase reporter assays were performed to probe into the regulatory mechanism of LINC01116. LINC01116 was significantly up regulated in melanoma cells. LINC01116 deficiency abrogated cell proliferation, migration, invasion and EMT in melanoma. Moreover, LINC01116 enhanced growth differentiation factor 11 (GDF11) and syndecan 3 (SDC3) expression through sponging microRNA-3612 (miR-3612). The oncogenic role of the LINC01116/miR-3612/GDF11/SDC3 axis in melanoma was finally demonstrated. Conclusively, LINC01116 sequestered miR-3612 and targeted GDF11 and SDC3 to contribute to the progression of melanoma.

NCK1-AS1 promotes the progression of melanoma by accelerating cell proliferation and migration via targeting miR-526b-5p/ADAM15 axis

BackgroundLong non-coding RNAs (lncRNAs) are vital regulators of gene expression and cellular processes in multiple cancers, including melanoma. Nevertheless, the function of lncRNA NCK1-antisense 1 (NCK1-AS1) in melanoma remains unknown.MethodsRT-qPCR was used to analyze the expression of NCK1-AS1, microRNA-526b-5p (miR-526b-5p) and ADAM metallopeptidase domain 15 (ADAM15). Cell proliferation was determined by CCK-8, colony formation and EdU assays. Cell migration was assessed by transwell migration and wound healing assays. Mechanism experiments including luciferase reporter, RIP and RNA pull down assays were conducted to demonstrate the interactions between RNAs. Xenograft model was established to verify the function of NCK1-AS1 and miR-526b-5p in melanoma in vivo.ResultsNCK1-AS1 was overexpressed in melanoma cell lines and NCK1-AS1 knockdown hampers the proliferation and migration of melanoma cells. Besides, miR-526b-5p binds to NCK1-AS1 in melanoma and ADAM15 was validated as its downstream target. Further, the inhibitory effects of NCK1-AS1 knockdown on cell proliferation and migration in melanoma were reversed by the depletion of miR-526b-5p and further counteracted by ADAM15 knockdown. The growth of melanoma tumors was hindered by the down-regulation of NCK1-AS1 or up-regulation of miR-526b-5p.ConclusionNCK1-AS1 facilitates cell proliferation and migration in melanoma via targeting miR-526b-5p/ADAM15 axis.

MiR-302a-3p suppresses melanoma cell progression via targeting METTL3

The miRNA-302 family plays a critical role in carcinogenesis. As an enzyme that regulates the N6-methyladenosine modification, methyltransferase-like 3 (METTL3) plays important roles in the development and progression of various tumours. However, the upstream regulatory mechanisms of METTL3 in melanoma have not yet been fully investigated. Herein, we investigated the functions of miR-302a-3p and its target RNA METTL3 on proliferation, apoptosis, and invasion of melanoma. Quantitative real-time PCR and immunofluorescence staining were used to measure the expression of METTL3 mRNA and protein level after transfection. miR-302a-3p expression was determined by quantitative reverse transcription-PCR. The cell proliferation, cell cycle progression, apoptosis, colony formation, migration, and cell invasion ability were determined using MTT assay, propidium iodide (PI) staining, Annexin V/PI flow cytometry, plate clone assay, and Transwell migration and invasion assays, respectively. Melanoma cell metastasis was also evaluated using an in vivo model. The effect of METTL3 on the phosphorylation of PI3K and AKT was measured with western blot analysis. Our results showed that miR-302a-3p was significantly downregulated in melanoma and exerted a tumour suppressive role against melanoma progression. We identified METTL3 as a direct target of miR-302a-3p in melanoma cells using bioinformatics analysis and luciferase assay. Furthermore, the enforced overexpression of METTL3 promoted the proliferation, cell cycle progression, cell invasion, migration, expression of epithelial-to-mesenchymal transition markers, and the PI3K-AKT signalling pathway as well as suppressed the apoptosis of melanoma cells. Meanwhile, silencing the expression of METTL3 with specific shRNA demonstrated reverse outcomes of the above phenotypes in melanoma cells. By rescue experiments, we found that the restoration of METTL3 expression in miR-302a-3p-overexpressing melanoma cells successfully recovered the miR-302a-3p-mediated melanoma suppression. The in vivo results also showed that miR-302a-3p substantially inhibited melanoma cell growth and metastasis. In summary, this study demonstrated that miR-302a-3p targets METTL3 and plays tumour suppressive roles in the proliferation, apoptosis, invasion, and migration of melanoma cells.

Targeting grainyhead-like transcription factor 2/Snail2 with chitosan-lipid nanoparticles inhibits melanoma progression

The grainyhead-like transcription factor 2 (GRHL2) has been reported to be involved in the progression of several malignant tumors; however, its expression and function are still unclear. This study confirmed that GRHL2 expression increased in melanoma tissue and cell lines. We used a novel chitosan-lipid nanoparticle (CS-LNP) nanomaterial to form CS-LNP/plasmids and CS-LNP/siRNAs to upregulate/downregulate GRHL2. The results indicated that GRHL2 promoted melanoma cell proliferation and migration by upregulating the EMT-related snail2 gene, and GRHL2 function in melanoma cells was partially reversed by snail2 knockdown. GRHL2/snail2 could be a potential target in melanoma treatment.

LRG1 Promotes Metastatic Dissemination of Melanoma through Regulating EGFR/STAT3 Signalling.

Simple SummaryMelanoma is a highly metastatic and lethal form of skin cancer. Currently, there is no effective treatment available once melanoma cells spread to other parts of the body. Our study demonstrated that LRG1 regulates multiple aspects of melanoma metastasis through modulating EGFR/STAT3 signalling. Targeting LRG1 may offer an alternative way to control the metastatic spread of melanoma cells.Although less common, melanoma is the deadliest form of skin cancer largely due to its highly metastatic nature. Currently, there are limited treatment options for metastatic melanoma and many of them could cause serious side effects. A better understanding of the molecular mechanisms underlying the complex disease pathophysiology of metastatic melanoma may lead to the identification of novel therapeutic targets and facilitate the development of targeted therapeutics. In this study, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in melanoma development and progression. We first established the association between LRG1 and melanoma in both human patient biopsies and mouse melanoma cell lines and revealed a significant induction of LRG1 expression in metastatic melanoma cells. We then showed no change in tumour cell growth, proliferation, and angiogenesis in the absence of the host Lrg1. On the other hand, there was reduced melanoma cell metastasis to the lungs in Lrg1-deficient mice. This observation was supported by the promoting effect of LRG1 in melanoma cell migration, invasion, and adhesion. Mechanistically, LRG1 mediates melanoma cell invasiveness in an EGFR/STAT3-dependent manner. Taken together, our studies provided compelling evidence that LRG1 is required for melanoma metastasis but not growth. Targeting LRG1 may offer an alternative strategy to control malignant melanoma.

NecroX-5 Can Suppress Melanoma Metastasis by Reducing the Expression of Rho-Family GTPases.

NecroX-5 (NX-5) is a cell-permeable necrosis inhibitor with cytoprotective effects. Although it has been reported to inhibit lung and breast cancer metastasis by modulating migration, its therapeutic effect on melanoma metastasis is still unknown. In this study, we examined the anti-metastatic effect of NX-5 on melanoma cell lines and its related therapeutic mechanism. The anti-metastatic effect of NX-5 on melanoma cell lines was determined using a transwell migration assay. We performed a quantitative real-time polymerase chain reaction and western blot analysis to measure changes in the expression of mRNA and protein, respectively, for major mediators of Rho-family GTPases after NX-5 treatment in melanoma cells. In addition, after constructing the 3D melanoma model, the expression of Rho-family GTPases was measured by immunohistochemistry. NX-5 (10 μM and 20 μM) treatment significantly reduced melanoma cell migration (p < 0.01). Additionally, NX-5 (20 μM) treatment significantly decreased the mRNA and protein expression levels of Cdc42, Rac1, and RhoA in melanoma cells compared with the untreated group (p < 0.001 and p < 0.05, respectively). Immunohistochemistry for our 3D melanoma model showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrate that NX-5 can suppress melanoma metastasis by reducing the expression of Rho-family GTPases.

An In Vitro-In Vivo Evaluation of the Antiproliferative and Antiangiogenic Effect of Flavone Apigenin against SK-MEL-24 Human Melanoma Cell Line.

One of the most important class of natural compounds with successful preclinical results in the management of cancer is the flavonoids. Due to the plethora of biological activities, apigenin (4′,5,7 trihydroxyflavone) is a main representant of the flavone subclass. Although the antiproliferative and antiangiogenic effects of apigenin were studied on a significant number of human and murine melanoma cell lines, in order to complete the data existing in the literature, the aim of this study is to evaluate the in vitro effect of apigenin on SK-MEL-24 human melanoma cell line as well as in vivo on tumor angiogenesis using the aforementioned cell line on the chorioallantoic membrane assay. Results have shown that in the range of tested doses, the phytocompound presents significant antiproliferative, cytotoxic, and antimigratory potential at 30 μM, respectively, 60 μM. Moreover, the phytocompound in both tested concentrations limited melanoma cell growth and migration and induced a reduced angiogenic reaction limiting melanoma cell development.

MiR-34a inhibits melanoma growth by targeting ZEB1.

Melanoma is a highly lethal cutaneous cancer with the tendency for early invasion and metastasis. Integrated miRNA transcriptome sequence analysis of human melanoma tumors and adjacent control tissues identified 17 miRNAs differentially expressed in melanoma tissues: let-7a-5p, let-7b-5p, let-7c, miR-374a-3p, miR-100-5p, miR-7, miR-195, miR-1908, miR-214, miR-221, miR-199a-5p, miR-21, miR-18, miR-34a, miR-199a-3p, miR-92a and miR-106b. Among these, miR-34a was most significantly down-regulated in melanoma tissues, and its expression correlated with TNM melanoma stage. miR-34a overexpression inhibited expression and activity of the transcription factor ZEB1, resulting in decreased proliferation and migration of melanoma cells. Moreover, miR-34a overexpression inhibited ZEB1 expression and melanoma tumor growth in vivo, in a melanoma nude mouse model. Together, these findings demonstrate that miR-34a inhibits melanoma growth by targeting the proto-oncogene ZEB1 and suggest the miR-34a -ZEB1 axis may serve as a novel target for melanoma treatment.

Up-regulation of KISS1 as a novel target of Let-7i in melanoma serves as a potential suppressor of migration and proliferation in vitro.

Melanoma is a kind of skin cancer that is begun by the alteration of melanocytes. miRNAs are small non‐coding RNA molecules that regulate a variety of biological processes. KISS1, the metastasis‐suppressor gene, encodes kisspeptins which inhibits migration and proliferation of cancers. This study was aimed to determine the role of Let‐7i and KISS1 in melanoma cell migration and proliferation. At first, the expression of Let‐7i and KISS1 was determined in patients with melanoma. In the in vitro part of the study, Let‐7i mimics were transfected and the impact of its restoration on target gene expression, proliferation, migration and apoptosis of SK‐MEL‐3 melanoma cell line was assessed by real‐time PCR and Western blotting, MTT assay, wound‐healing assay and flow cytometry, respectively. Besides, KISS1 inhibitor siRNA alone and along with Let‐7i was transfected to determine their probable correlation. The results revealed that either Let‐7i or KISS1 were down‐regulated in patients with melanoma. The results obtained from the in vitro part of the study revealed that restoration of Let‐7i reduced the expression of metastasis‐ and proliferation‐related target genes. Moreover, it was revealed that up‐regulation of Let‐7i attenuated migration and proliferation capability of SK‐MEL‐3 cells. Besides, it was demonstrated that Let‐7i restoration induced apoptosis in melanoma cells. More importantly, the KISS1 inhibitor caused a prominent cell migration and proliferation, attenuated by Let‐7i re‐expression. To sum up, the present study revealed the impressive role of Let‐7i restoration along with its correlation with KISS1 on melanoma carcinogenicity which may be applicable in future in vivo studies.

Zwint facilitates melanoma progression by promoting c-Myc expression.

ZW10 interactor (Zwint) is upregulated in various types of tumors and exerts a carcinogenic effect. However, little is known about the expression profile, function and molecular mechanisms of action of Zwint in melanoma. Therefore, the aim of the present study was to investigate the expression levels of Zwint in melanoma cell lines and tissues. It was revealed that Zwint was highly expressed in melanoma samples. Functional experiments indicated that Zwint knockdown suppressed the proliferation and migration of A375 melanoma cells. Further mechanistic studies demonstrated that Zwint knockdown decreased the protein expression levels of c-Myc, MMP-2, Slug, mTOR, phosphorylated (p)-mTOR, p-p38 and fibronectin, while it increased the protein expression levels of E-cadherin and MMP-9. Among these genes, c-Myc, MMP-2 and Slug were overexpressed to investigate their effects on cell proliferation following Zwint knockdown. The results demonstrated that overexpression of c-Myc, but not MMP-2 or Slug, rescued the effects of Zwint knockdown on melanoma cell proliferation and migration. Taken together, the results of the present study suggested that Zwint may act as an oncogene in melanoma by regulating c-Myc expression.

MiR-509-3p Suppresses Migration, Invasion, and Epithelial- Mesenchymal Transition in Melanoma Cells by Targeting Collagen Triple Helix Repeat Containing 1.

Background: microRNAs (miRNAs) are ubiquitously dysregulated in numerous tumor cell types, including melanoma cells. The anti-tumor effect of miR-509-3p was widely evaluated in various cancers.Aims: To determine the functional role of miR-509-3p in melanoma.Study Design: Cell culture study.Methods: Expression of miR-509-3p in melanoma cell models were assessed by qRT-PCR. Cell migration and invasion were analyzed by wound healing and transwell assays, respectively. Expression levels of biomarkers of epithelial–mesenchymal transition were determined by Western blot. Luciferase vectors containing wildtype or mutant miR-509-3p binding site were constructed, and then dual-luciferase reporter assay.Results: Dysregulated miR-509-3p level was found in melanoma cells. Elevated miR-509-3p expression suppressed melanoma cell migration (P < .001) and invasion (P < .001) capacities. Epithelial–mesenchymal transition of melanoma cells was repressed by miR-509-3p, along with increased α-catenin/E-cadherin (P < .001) and decreased vimentin/fibronectin (P < .001). CTHRC1 (collagen triple helix repeat containing 1) contained a potential binding site for miR-509-3p, and miR-509-3p decreased protein expression of CTHRC1 in melanoma cells (P < .001). CTHRC1 promoted melanoma cell migration and invasion (P < .001), as well as contributed to epithelial–mesenchymal transition. Increased CTHRC1 expression attenuated miR-509-3p-induced inhibition of melanoma cell migration (P < .001), invasion, and epithelial–mesenchymal transition.Conclusion: miR-509-3p suppressed the biological function of melanoma cells through negatively regulating CTHRC1, shedding light on miR-509-3p as a potential candidate for melanoma therapeutics and treatments.

Bergamottin Induces DNA Damage and Inhibits Malignant Progression in Melanoma by Modulating miR-145/Cyclin D1 Axis.

BackgroundMelanoma is a prevalent skin cancer with the high rate of metastasis and mortality, affecting the increasing number of people worldwide. Bergamottin (BGM) is a natural furanocoumarin derived from grapefruits and presents the potential anti-cancer activity in several tumor models. However, the role of BGM in the development of melanoma remains unclear. Here, we aimed to explore the effect of BGM on the DNA damage and progression of melanoma.MethodsThe effect of BGM on the melanoma progression was analyzed by CCK-8 assays, colony formation assays, transwell assays, Annexin V-FITC Apoptosis Detection Kit, cell-cycle analysis, in vivo tumorigenicity analysis. The mechanism investigation was performed using luciferase reporter gene assays, qPCR assays, and Western blot analysis.ResultsWe identified that BGM repressed cell proliferation, migration, and invasion of melanoma cells. BGM induced cell cycle arrest at the G0/G1 phase and enhanced apoptosis of melanoma cells. The DNA damage in the melanoma cells was stimulated by the BGM treatment. Meanwhile, BGM was able to up-regulate the expression of miR-145 and miR-145 targeted Cyclin D1 in the melanoma cells. Furthermore, BGM inhibited the progression of melanoma by targeting miR-145/Cyclin D1 axis in vitro. BGM attenuated the tumor growth of melanoma in vivo.ConclusionThus, we conclude that BGM induces DNA damage and inhibits tumor progression in melanoma by modulating the miR-145/Cyclin D1 axis. Our finding provides new insights into the mechanism by which BGM modulates the development of melanoma. BGM may be applied as a potential anti-tumor candidate for the clinical treatment of melanoma.

Luteolin inhibits the proliferation, adhesion, migration and invasion of choroidal melanoma cells in vitro

Choroidal melanoma is a devastating disease that causes visual loss and a high mortality rate due to metastasis. Luteolin, a potential anticancer compound, is widely found in natural plants. The aim of this study was to evaluate the antiproliferative, antiadhesive, antimigratory and anti-invasive effects of luteolin on choroidal melanoma cells in vitro and to explore its potential mechanism. Cell counting kit-8 (CCK-8) assays, 5-ethynyl-2′-deoxyuridine (EdU) assays, Cell adhesion, migration, and invasion assays were performed to examine the inhibitory effects of luteolin on cell cell viability, proliferation, adhesion, migration and invasion capacities, respectively. Considering the correlation between Matrix metalloenzymes and tumor metastasis, Enzyme-linked immunosorbent assays (ELISAs) were used to assess matrix metalloproteases MMP-2 and MMP-9 secretion. Western blotting was performed to detect p-PI3K P85, Akt, and p-Akt protein expression. The cytoskeletal proteins vimentin were observed with cell immunofluorescence staining. Luteolin can inhibit OCM-1 cell proliferation, migration, invasion and adhesion and C918 cell proliferation, migration, and invasion through the PI3K/Akt signaling pathway. Therefore, Luteolin may have potential as a therapeutic medication for Choroidal melanoma.

Nicotinamide N-methyltransferase gene silencing enhances chemosensitivity of melanoma cell lines.

Melanoma accounts for less than 5% of all cutaneous neoplasms but is responsible for the greater part of skin cancer-related deaths. Therefore, the identification of molecules that could serve as the therapeutic target is urgent. This study focused on the enzyme nicotinamide N-methyltransferase (NNMT). The effect of NNMT knockdown on cell proliferation and migration of A375 melanoma cells was evaluated by MTT and wound healing assays, respectively. Viability of A375 cells downregulating NNMT was also explored under treatment with dacarbazine, a chemotherapeutic drug approved for advanced melanoma treatment. The impact of enzyme knockdown on cell proliferation and chemosensitivity was also investigated in WM-115 melanoma cells. Results obtained demonstrated that NNMT silencing led to a significant reduction of cell proliferation and migration of A375 cells. Moreover, enzyme downregulation was associated with an increase of melanoma cells sensitivity to treatment with dacarbazine. Analogous effects induced by enzyme knockdown on cell proliferation and chemosensitivity were also found in the WM-115 cell line. Our data seem to demonstrate that NNMT could represent a promising molecular target for the effective treatment of this form of skin cancer.

ETS1 promoted cell growth, metastasis and epithelial-mesenchymal transition process in melanoma by regulating miR-16-mediated SOX4 expression.

Melanoma is a malignant tumor with high metastasis and mortality. Epithelial-mesenchymal transition (EMT) was reported to be involved in the growth and metastasis of melanoma. To investigate these sections further, we showed that E26 transformation specific 1 (ETS1) could regulate growth, metastasis and EMT process of melanoma by regulating microRNA(miR)-16/SRY-related HMG box (SOX) 4 expression. MiR-16, ETS1, SOX4 and nuclear factor κB (NF-κB) expression levels in melanoma cells were examined using qPCR. ETS1, SOX4, EMT-related proteins and NF-κB signaling pathway-related proteins were examined using western blot. Cell counting kit-8 assay, transwell assay were applied to evaluate the cell proliferation, migration and invasion of melanoma cells, respectively. Besides, a dual-luciferase reporter assay was employed to verify the binding relationship between ETS1 and miR-16, miR-16 and SOX4, miR-16 and NF-κB1. We showed that ETS1 and SOX4 were upregulated in melanoma cells, while miR-16 was downregulated. MiR-16 overexpression suppressed growth, metastasis and EMT process of melanoma. We found ETS1 could bind to the promoter region of miR-16 and inhibited its transcription. ETS1 silence could inhibit growth, metastasis and EMT process of melanoma, and inhibition of miR-16 could reverse the effects. Besides, miR-16 is directly bound to SOX4 and downregulated its expression. Rescued experiments confirmed that SOX4 overexpression abolished the inhibition effect of miR-16 mimics on growth, metastasis and EMT process of melanoma. Finally, NF-κB1 as the target of miR-16 mediated downstream biological responses. ETS1 activated NF-κB signaling pathway through miR-16 via targeting SOX4, thus promoting growth, metastasis and EMT of melanoma.

Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization

Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxoscelesserine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.

FUT8-AS1 Inhibits the Malignancy of Melanoma Through Promoting miR-145-5p Biogenesis and Suppressing NRAS/MAPK Signaling.

Melanoma is the major lethal skin malignancy. However, the critical molecular drivers governing melanoma progression and prognosis are still not clear. By analyzing The Cancer Genome Atlas (TCGA) data, we identified FUT8-AS1 as a prognosis-related long non-coding RNA (lncRNA) in melanoma. We further confirmed that FUT8-AS1 is downregulated in melanoma. Reduced expression of FUT8-AS1 is correlated with aggressive clinical factors and inferior overall survival. Using in vitro functional assays, our findings demonstrated that ectopic expression of FUT8-AS1 represses melanoma cell proliferation, migration, and invasion. FUT8-AS1 silencing promotes melanoma cell proliferation, migration, and invasion. Furthermore, in vivo functional assays demonstrated that FUT8-AS1 represses melanoma growth and metastasis. Mechanistically, FUT8-AS1 was found to bind NF90, repress the interaction between NF90 and primary miR-145 (pri-miR-145), relieve the repressive roles of NF90 on mature miR-145-5p biogenesis, and thus promote miR-145-5p biogenesis and upregulate mature miR-145-5p level. The expression of FUT8-AS1 is positively correlated with miR-145-5p in melanoma tissues. Via upregulating miR-145-5p, FUT8-AS1 reduces the expression of NRAS, a target of miR-145-5. FUT8-AS1 further represses MAPK signaling via downregulating NRAS. Functional rescue assays demonstrated that inhibition of miR-145-5p reverses the tumor suppressive roles of FUT8-AS1 in melanoma. The oncogenic roles of FUT8-AS1 silencing are also blocked by MAPK signaling inhibitor MEK162. In conclusion, these findings demonstrate that FUT8-AS1 exerts tumor suppressive roles in melanoma via regulating NF90/miR-145-5p/NRAS/MAPK signaling axis. Targeting FUT8-AS1 and its downstream molecular signaling axis represent promising therapeutic strategies for melanoma.