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Bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs) reportedly play an important role in melanoma pathogenesis. This study aimed to explore the mechanisms of EVs-carried long non-coding RNA (lncRNA) NEAT1 involvement in melanoma. Gain- and loss-of-function experiments were performed to determine biological characteristics of A-375 melanoma cells. Bioinfomatic prediction, RNA immunoprecipitation (RIP), and dual luciferase reporter gene experiments were applied to investigate the roles of NEAT1 and microRNA-374a-5p (miR-374a-5p), and leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). A subcutaneous tumor model was constructed using nude mice, and in vivo fluorescence imaging was used to observe the effect of NEAT1 on the growth and metastasis of melanoma cells in vivo. The results indicated that BMSC-EVs could be internalized by macrophages to promote the expression of macrophages M2 markers. M2 type macrophages promoted malignancy of melanoma cells. NEAT1 derived from BMSC-EVs promoted the progression of melanoma by promoting M2 polarization of macrophages. NEAT1 inhibits miR-374 expression, while miR-374 could upregulate LGR4-dependent IQGAP1 expression. The tumor-inhibiting effect of NEAT1 silencing was validated in the nude mouse xenograft model. Collectively, the results demonstrated that BMSC-EVs carrying NEAT1 can promote the progression of melanoma by inducing M2 polarization of macrophages, and thus may be considered as a potential target for melanoma therapeutics.

Metastasis is the hallmark of cancer that is responsible for the greatest number of cancer-related deaths. As a critical regulator of the Hippo pathway, the phosphorylation status of Yes-associated protein 1 (YAP1), mainly at S127, is critical for its oncogenic function. Herein, we aim to investigate the precise molecular mechanism between long noncoding RNA HOX transcript antisense RNA (HOTAIR) and YAP1 phosphorylation in regulating tumor migration and invasion. In this study, we showed that inhibition of HOTAIR significantly decreased the migration and invasion of cancer cells both in vitro and in vivo through elevating the phosphorylation level of YAP1 on serine 127, demonstrating a tumor suppressive role of YAP1 S127 phosphorylation. Through bisulfite sequencing PCR (BSP), we found that inhibition of HOTAIR dramatically increased Large Tumor Suppressor Kinase 1 (LATS1) expression by regulating LATS1 methylation via DNA methyltransferase 3β (DNMT3B). In accordance with this observation, DNMT3B just only altered the distribution of YAP1 in the cytoplasm and the nucleus by inhibiting its phosphorylation, but did not change its total expression. Mechanistically, we discovered that HOTAIR suppressed YAP1 S127 phosphorylation by regulating the methylation of LATS1 via DNMT3B, the consequence of which is the translocation of YAP1 into the nucleus, reinforcing its coactivating transcriptional function, which in turn promotes the migration and invasion of cancer cells. Collectively, our data reveal that the phosphorylation of YAP1 S127 plays a vital role in the function of HOTAIR in tumorigenicity, and should be taken into consideration in future therapeutic strategies for cervical cancer.

ABSTRACTAbnormal DNA methylation has been observed in multiple malignancies, including melanoma. In this study, we initially noticed the overexpression of DNA methyltransferase 1 (DNMT1) in melanoma samples in bioinformatics analysis and, subsequently, validated it in the purchased melanoma cell lines. After treatment with short-hairpin RNAs or Decitabine (a DNA methylation inhibitor), silencing of DNMT1 was demonstrated to suppress cell viability and invasive and migratory potentials as well as to augment apoptosis and autophagy in melanoma cells. To further explore the downstream mechanisms, we revealed that DNMT1 inhibited HSPB8 expression through augmenting HSPB8 methylation, thereby suppressing the binding between HSPB8 and BAG3. Then, we elucidated through a series of gain- and loss- of function assays that the interplay of HSPB8 and BAG3 blocked the PI3K/AKT/mTOR pathway, thereby repressing the malignant phenotypes of melanoma cells and contributing to melanoma cell apoptosis and autophagy. We further established a mouse model of melanoma and substantiated that DNMT1 enhanced the in vivotumorigenesis of melanoma cells via activation of the PI3K/AKT/mTOR pathway through repressing the binding between HSPB8 and BAG3. Taken together, our data supported that DNMT1 repressed the binding between HSPB8 and BAG3 and activated the PI3K/AKT/mTOR pathway, thus playing a tumour-promoting role in melanoma.