Long noncoding RNA OIP5-AS1 acts as a competing endogenous RNA to promote glutamine catabolism and malignant melanoma growth by sponging miR-217

The long noncoding RNA (lncRNA) OIP5-AS1 has been considered to promote the growth and metastasis of many human tumors. However, the role of OIP5-AS1 in melanoma has not been reported. In this study, we found that OIP5-AS1 levels were significantly elevated in melanoma tissue and that high OIP5-AS1 expression was an independent risk factor for the poor survival of patients with melanoma. miR-217 suppressed glutamine catabolism in melanoma cells by targeting glutaminase (GLS), the rate-limiting enzyme of glutamine catabolism. We also demonstrated that OIP5-AS1 acted as a sponge of miR-217 to upregulate GLS expression, thus promoting glutamine catabolism and melanoma growth. Overall, this result elucidates a new mechanism for OIP5-AS1 in metabolism in melanoma and provides a potential therapeutic target for patients with melanoma.     

miR-137 inhibits melanoma cell proliferation through downregulation of GLO1

Late-stage melanoma is refractory to current therapies. MicroRNAs (miRNAs) can modulate many physiological and pathological processes of melanoma. Studies have demonstrated that miR-137 acts as a tumor suppressor by inhibiting the proliferation of melanoma cells through targeting multiple mRNAs. The glyoxalase system member glyoxalase 1 (GLO1) is the principal scavenging enzyme of methylglyoxal (MG), a toxic byproduct of glycolysis. Using ³⁵S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD), we found that miR-137 downregulated the expression of GLO1 in melanoma cells. Bioinformatics analysis predicted that GLO1 is a direct target of miR-137. This was validated by dual luciferase reporter assay. Quantitative RT-PCR (qRT-PCR) and western blot analysis indicated that miR-137 could decrease endogenous GLO1 expression. Furthermore, siRNA targeting of GLO1 mimicked inhibition of melanoma cell proliferation caused by miR-137 overexpression. Re-expression of GLO1 was able to restore miR-137-mediated suppression of melanoma cell proliferation. Therefore, these results suggest that miR-137 inhibits the proliferation of melanoma cells by targeting GLO1.     

The NF-κB member p65 controls glutamine metabolism through miR-23a

Cancer cells have elevated aerobic glycolysis that is termed the Warburg effect. But several tumor cells, including leukemic cells, also increase glutamine metabolism, which is initiated by glutaminase (GLS). The microRNA (miRNA) miR-23 targets GLS mRNA and inhibits expression of GLS protein. Here we show that in human leukemic Jurkat cells the NF-κB p65 subunit binds to miR-23a promoter and inhibits miR-23a expression. Histone deacetylase (HDAC) inhibitors release p65-induced inhibition. Jurkat cells growing in glutamine decrease proliferation due to cell accumulation in G0/G1 phase. Nevertheless, cells get used to this new source of energy by increasing GLS expression, which correlates with an increase in p65 expression and its translocation to the nucleus, leading to a higher basal NF-κB activity. Jurkat cells overexpressing p65 show increase basal GLS expression and proliferate faster than control cells in glutamine medium. Overexpressing miR-23a in leukemic cells impaired glutamine use and induces mitochondrial dysfunction leading to cell death. Therefore, p65 activation decreases miR-23a expression, which facilitates glutamine consumption allowing leukemic cells to use this alternative source of carbon and favoring their adaptation to the metabolic environment.     

Propofol suppresses proliferation,invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9

Propofol is an intravenous anesthetic widely used in clinical surgeries, such as tumor resection. Propofol affects the growth of many cancers, though its effect on melanoma is unknown. Our study aimed to explore how propofol affects melanoma cells. Melanoma cells A2058 and WM793B were cultured with propofol for 24 hr. Propofol significantly suppressed proliferation, migration, and invasion of A2058 and WM793B cells. Lower miR-137 level was observed in A2058 and WM793B cells, compared with normal human epidermal melanocyte HEMa-LP cells. Propofol-induced miR-137 upregulation and decreased proliferation, invasive ability, and migrated ability of A2058 and WM793B cells. Transfection with the miR-137 inhibitor reversed these effects. Additionally, miR-137 was verified to target and negatively regulate fibroblast growth factor 9 (FGF9) expression. Propofol efficiently downregulated FGF9 protein expression by upregulating miR-137. Furthermore, FGF9 overexpression abrogated propofol's repressive effects on the malignant potential of A2058 and WM793B cells. These findings indicate that propofol suppressed melanoma cell proliferation, invasion, and migration by regulating miR-137 and FGF9.     

Intronic miR-211 assumes the tumor suppressive function of its host gene in melanoma

When it escapes early detection, malignant melanoma becomes a highly lethal and treatment-refractory cancer. Melastatin is greatly downregulated in metastatic melanomas and is widely believed to function as a melanoma tumor suppressor. Here we report that tumor suppressive activity is not mediated by melastatin but instead by a microRNA (miR-211) hosted within an intron of melastatin. Increasing expression of miR-211 but not melastatin reduced migration and invasion of malignant and highly invasive human melanomas characterized by low levels of melastatin and miR-211. An unbiased network analysis of melanoma-expressed genes filtered for their roles in metastasis identified three central node genes: IGF2R, TGFBR2, and NFAT5. Expression of these genes was reduced by miR-211, and knockdown of each gene phenocopied the effects of increased miR-211 on melanoma invasiveness. These data implicate miR-211 as a suppressor of melanoma invasion whose expression is silenced or selected against via suppression of the entire melastatin locus during human melanoma progression.     

Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 protein in human melanoma cells

MicroRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of their complementary mRNA. We recently reported that miR-203 is down-regulated, and its exogenous expression inhibits cell growth in canine oral malignant melanoma tissue specimens as well as in canine and human malignant melanoma cells. A microRNA target database predicted E2F3 and ZBP-89 as putative targets of microRNA-203 (miR-203). The expression levels of E2F3a, E2F3b, and ZBP-89 were markedly up-regulated in human malignant melanoma Mewo cells compared with those in human epidermal melanocytes. miR-203 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequence of either E2F3 or ZBP-89 complementary to miR-203. The ectopic expression of miR-203 in melanoma cells reduced the levels of E2F3a, E2F3b, and ZBP-89 protein expression. At the same time, miR-203 induced cell cycle arrest and senescence phenotypes, such as elevated expression of hypophosphorylated retinoblastoma and other markers for senescence. Silencing of E2F3, but not of ZBP-89, inhibited cell growth and induced cell cycle arrest and senescence. These results demonstrate a novel role for miR-203 as a tumor suppressor acting by inducing senescence in melanoma cells.     

MicroRNA 211 Functions as a Metabolic Switch in Human Melanoma Cells

MicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1α (HIF-1α) protein levels and decreased cell growth during hypoxia. HIF-1α protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1α protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations—a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.     

MicroRNA-203 reinforces stemness properties in melanoma and augments tumorigenesis in vivo

One of the challenges encountered in microRNA (miRNA) studies is to observe their dual role in different conditions and cells. This leads to a tougher prediction of their behavior as gene expression regulators. miR-203 has been identified to play a negative role in the progression of malignant melanoma; however, it has been reported, with dual effect, as both an oncomiR and tumor suppressor miRNA in some malignancies, such as breast cancer, meanwhile, the role of miR-203 in melanoma stem cells or even metastatic cells is unclear. In the present study, after observation of upregulation of miR-203 in melanoma patient's serum and also melanospheres as cancer stem cells model, we examined its overexpression on the stemness potential and migration ability of melanoma cells. Our data demonstrated that the increased miR-203 level was significantly associated with significant increase in the ability of proliferation, colony and spheres formation, migration, and tumorigenesis in A375 and NA8 cells. All of these changes were associated with enhancement of BRAF, several epithelial to mesenchymal transition factors, and stemness genes. In conclusion, our results clearly determined that miR-203 could be down-regulateddownregulated in melanoma tissues but be overexpressed in melanoma stem cells. It has an important role as oncomiR and promote repopulation, tumorigenicity, self-renewal, and migration. Therefore, we suggested overexpression of miR-203 as biomarker for early detection of metastasis. However, more studies are needed to validate our data.     

Inhibition of the oxidative stress-induced miR-23a protects the human retinal pigment epithelium (RPE) cells from apoptosis through the upregulation of glutaminase and glutamine uptake

The degeneration of retinal pigment epithelium (RPE) cells in the sub retinal pigment epithelial space and choroid is an initial pathological characteristic for the age-related macular degeneration which is the leading cause of severe vision loss in old people. Moreover, oxidative stress is implicated as a major inducer of RPE cell death. Here, we assessed the correlation between the H₂O₂-induced RPE cell death and glutamine metabolism. We found under low glutamine supply (20 %), the ARPE-19 cells were more susceptive to H₂O₂-induced apoptosis. Moreover, the glutamine uptake and the glutaminase (GLS) were suppressed by H₂O₂ treatments. Moreover, we observed miR-23a was upregulated by H₂O₂ treatments and overexpression of miR-23a significantly sensitized ARPE-19 cells to H₂O₂. Importantly, Western blotting and luciferase assay demonstrated GLS1 is a direct target of miR-23a in RPE cells. Inhibition of the H₂O₂-induced miR-23a by antagomiR protected the RPE cells from the oxidative stress-induced cell death. In addition, recovery of GLS1 expression in miR-23a overexpressed RPE cells rescued the H₂O₂-induced cell death. This study illustrated a mechanism for the protection of the oxidative-induced RPE cell death through the recovery of glutamine metabolism by inhibition of miR-23a, contributing to the discovery of novel targets and the developments of therapeutic strategies for the prevention of RPE cells from oxidative stress.     

MicroRNA-137 Contributes to Dampened Tumorigenesis in Human Gastric Cancer by Targeting AKT2

MiRNAs play important roles in tumorigenesis. This study focused on exploring the effects and regulation mechanism of miRNA-137 on the biological behaviors of gastric cancer. Total RNA was extracted from tissues of 100 patients with gastric cancer and from four gastric cancer cell lines. Expression of miR-137 was detected by real-time PCR from 100 patients. The effects of miR-137 overexpression on gastric cancer cells’ proliferation, apoptosis, migration and invasion ability were investigated in vitro and in vivo. The target gene of miR-137 was predicted by Targetscan on line software, screened by dual luciferase reporter gene assay and demonstrated by western blot. As a result, the expression of miR-137 was significant reduced in gastric cancer cell line HGC-27, HGC-803, SGC-7901 and MKN-45 as well as in gastric cancer tissues compared with GES-1 cell or matched adjacent non-neoplastic tissues (p<0.001). The re-introduction of miR-137 into gastric cancer cells was able to inhibit cell proliferation, migration and invasion. The in vivo experiments demonstrated that the miR-137 overexpression can reduce the gastric cancer cell proliferation and metastasis. Bioinformatic and western blot analysis indicated that the miR-137 acted as tumor suppressor roles on gastric cancer cells through targeting AKT2 and further affecting the Bad and GSK-3β. In conclusion, the miR-137 which is frequently down-regulated in gastric cancer is potentially involved in gastric cancer tumorigenesis and metastasis by regulating AKT2 related signal pathways.     

miR-137 effects on gastric carcinogenesis are mediated by targeting Cox-2-activated PI3K/AKT signaling pathway

The discovery of microRNAs (miRNAs) provided a new avenue for early diagnosis and treatment of GC. MiR-137 has been reported to be under-expressed and involved in various cell processes. However, the role of miR-137 in GC is less known. In this study, we show that miR-137 is under-expressed in GC and functions as a tumor suppressor through targeting Cyclooxygenase-2 (Cox-2), which subsequently suppresses the activation of PI3K/AKT signaling pathway both in vitro and in vivo. Moreover, restored Cox-2 expression partially abolished the tumor suppressive effects of miR-137 in GC cells, suggesting miR-137 may suppress GC carcinogenesis by targeting Cox-2.     

miRNA-205 suppresses melanoma cell proliferation and induces senescence via regulation of E2F1 protein

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.     

miR-137 suppresses cell growth in ovarian cancer by targeting AEG-1

Astrocyte elevated gene-1 (AEG-1) is an oncogene overexpressed in multiple types of human cancers including ovarian cancer (OC). However, the underlying mechanism of AEG-1 up-regulation in OC is not well understood. In this study, we showed that miR-137 downregulated AEG-1 expression through interaction with its 3′ untranslated region (3′UTR) and that miR-137 expression was inversely correlated with AEG-1 levels in OC specimens. Similar to the downregulation of AEG-1, overexpression of miR-137 in OC cell lines decreased in vitro cell growth, clonogenicity, and also induced G1 arrest. Importantly, miR-137 overexpression suppressed in vivo tumor growth in nude mice models. Furthermore, we found that restoring the AEG-1 (without the 3′UTR) significantly rescued miR-137-induced cell growth inhibition and cell-cycle arrest. Taken together, these findings indicate that miR-137 functions as a tumor suppressor by inhibition of AEG-1. These molecules might be targets for prevention or treatment of OC.     

MicroRNA miR-21 regulates the metastatic behavior of B16 melanoma cells

MicroRNA-21 (miR-21) is overexpressed in many human tumors and has been linked to various cellular processes altered in cancer. miR-21 is also up-regulated by a number of inflammatory agents, including IFN, which is of particular interest considering the close relationship between inflammation and cancer. Because miR-21 appears to be overexpressed in human melanoma, we examined the role of miR-21 in cancer development and metastasis in B16 mouse melanoma cells. We found that miR-21 is a member of an IFN-induced miRNA subset that requires STAT3 activation. To characterize the role of miR-21 in melanoma behavior, we transduced B16 cells with lentivirus encoding a miR-21 antagomir and isolated miR-21 knockdown B16 cells. miR-21 knockdown or IFN treatment alone inhibited B16 cell proliferation and migration in vitro, and in combination they had an enhanced effect. Moreover, miR-21 knockdown sensitized B16 cells to IFN-induced apoptosis. In B16 cells miR-21 targeted tumor suppressor (PTEN and PDCD4) and antiproliferative (BTG2) proteins. To characterize the role of miR-21 in vivo, empty vector- and antagomiR-21-transduced B16 melanoma cells were injected via tail vein into syngeneic C57BL/6 mice. Although empty vector-transduced B16 cells produced large lung metastases, miR-21 knockdown cells only formed small lung lesions. Importantly, miR-21 knockdown tumor-bearing mice exhibited prolonged survival compared with empty vector tumor-bearing mice. Thus, miR-21 regulates the metastatic behavior of B16 melanoma cells by promoting cell proliferation, survival, and migration/invasion as well as by suppressing IFN action, providing important new insights into the role of miR-21 in melanoma.     

MiR-144 Inhibits Uveal Melanoma Cell Proliferation and Invasion by Regulating c-Met Expression

MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs. Recent studies have demonstrated that miRNAs are implicated in the development of cancer. However, the role of miR-144 in uveal melanoma metastasis remains largely unknown. MiR-144 was downregulated in both uveal melanoma cells and tissues. Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion. After identification of two putative miR-144 binding sites within the 3'' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites. In addition, the expression of c-Met protein was inhibited by miR-144. Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells. In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.     

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.Subject terms: Ligand-gated ion channels, Metastasis, Melanoma, Ion channel signalling, miRNAs