miRNA-7-5p inhibits melanoma cell migration and invasion

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of melanoma. However, the precise mechanistic role of many of these miRNAs remains unclear. We have investigated the functional role of miR-7-5p in melanoma, and demonstrate that miR-7-5p expression is reduced in metastatic melanoma-derived cell lines compared with primary melanoma cells, and that when ectopically expressed miR-7-5p significantly inhibits melanoma cell migration and invasion. Additionally, we report that insulin receptor substrate-2 (IRS-2) is a target of miR-7-5p in melanoma cells, and using RNA interference (RNAi) we provide evidence that IRS-2 activates protein kinase B (Akt), and promotes melanoma cell migration. Thus, miR-7-5p may represent a novel tumor suppressor miRNA in melanoma, acting at least in part via its inhibition of IRS-2 expression and oncogenic Akt signaling.     

miR-140-5p is negatively correlated with proliferation,invasion, and tumorigenesis in malignant melanoma by targeting SOX4 via the Wnt/β-catenin and NF-κB cascades

MicroRNAs (miRNAs) have been validated as critical regulators in the development of melanoma. miR-140 was abnormally downregulated in uveal melanoma samples. However, the expression level and roles of miR-140-5p remain unclear in melanoma for now. We speculate that miR-140-5p is abnormally expressed and may play an important role in melanoma. The expressions of miR-140-5p and SOX4 messenger RNA were determined by quantitative real-time polymerase chain reaction assays. Western blot assays were employed to detect the expression levels of SOX4, Ki67, MMP-2, MMP-7, p-β-catenin, c-Myc, cyclin D1, p65, and IκBα. Luciferase reporter assays were employed to elucidate the interaction between SOX4 and miR-140-5p. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) and transwell invasion assays were applied to evaluate capabilities of cell proliferation and invasion, respectively. Xenograft models of melanoma were established to verify the role and molecular basis of miR-140-5p. Immunohistochemical (IHC) assays were employed to measure the Ki67 and SOX4 at the protein level in xenografted melanoma tissues. Herein, these observations showed that, miR-140-5p was abnormally downregulated in melanoma tissues and cells, while SOX4 was upregulated. miR-140-5p directly targeted SOX4 and inhibited its expression in melanoma cells. miR-140-5p overexpression repressed melanoma cell proliferation and invasion and its effects were partially restored SOX4 overexpression. Moreover, miR-140-5p hindered melanoma growth in vivo by downregulating SOX4. Mechanistically, miR-140-5p suppressed activation of the Wnt/β-catenin and NF-κB pathways by targeting SOX4. Our study concluded that miR-140-5p hindered cell proliferation, invasion, and tumorigenesis by targeting SOX4 via inactivation of the Wnt/β-catenin and NF-κB signaling pathways in malignant melanoma, which provides an underlying molecular mechanism for the treatment for melanoma with miRNAs.     

miRNA调控恶性黑色素瘤细胞上皮-间充质转化的分子网络及机制

黑色素瘤是一种极易发生转移的恶性皮肤肿瘤,具有高度的致死性。上皮-间充质细胞转化(Epithelial-mesenchymal transition, EMT)在胚胎发育过程中起到非常重要的作用,同时在肿瘤的发生和恶化过程中也扮演着重要的角色。miRNA具有广谱的调节能力,对于肿瘤发生和EMT形成都能产生不同程度的影响。本文整合黑色素瘤细胞系转录组和miRNA组测序数据,在转录组数据中筛选得到参与肿瘤EMT过程的基因,通过Mirsystem软件预测并从miRNA组数据中筛选出与之负相关的11个miRNA,包括miR-130a-3p、miR-130b-3p、miR-125a-5p、miR-30a-3p、miR-195-5p、miR-345-5p、miR-509-3-5p、miR-374a-5p、miR-509-5p、miR-148a-3p和miR-330-3p。经过生物信息学分析miRNA靶基因富集的分子网络和信号途径,发现了两个与细胞发育和细胞间相互作用密切相关的网络,以及多个参与调控EMT过程的信号通路。对11个miRNA进行分子生物学验证,发现miR-195-5p、miR-130a-3p、miR-509-5p和miR-509-3-5p共4个可以调节重要肿瘤基因的miRNA。本研究运用mRNA和miRNA两种转录组的测序数据筛选EMT相关miRNA的方法,为肿瘤多组学数据整合分析提供了新的研究思路,并以期能为肿瘤精准基因组学的发展发挥重要的推进作用。     

Tumor suppressive microRNA-485-5p targets PRRX1 in human skin melanoma cells,regulating epithelial–mesenchymal transition and apoptosis

Melanoma is one of the most aggressive skin cancers. Existing evidence has reported the aberrant expression of microRNAs (miRNAs) in melanoma, but their putative targets and underlying downstream effects remain to be further understood. Herein, we explored the suppressive role of miR-485-5p in melanoma progression. Initial bioinformatics analyses showed that the PRRX1 gene was differentially expressed in melanoma, while miR-485-5p was predicted to be a potential regulatory miRNA binding to PRRX1 mRNA. We confirmed that PRRX1 was upregulated, while miR-485-5p was downregulated in human melanoma samples compared with adjacent normal skin tissues. We then showed that PRRX1 was a target gene of miR-485-5p by dual-luciferase reporter gene assay. Moreover, a reduction in the expression of PRRX1 and downregulation of important proteins of the transforming growth factor-beta (TGFβ) signaling pathway was observed after miR-485-5p overexpression. Furthermore, miR-485-5p overexpression or PRRX1 knockdown suppressed epithelial–mesenchymal transition, cell viability, migration, and invasion, and promoted cell apoptosis in melanoma cells. Our study demonstrates the tumor-suppressive functions of miR-485-5p in the development of human melanoma, providing a potential target for therapy.     

Phosphoinositide-dependent kinase 1–associated glycolysis is regulated by miR-409-3p in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most popular kidney cancer in adults. Metabolic shift toward aerobic glycolysis is a fundamental factor for ccRCC therapy. MicroRNAs (miRNAs) are thought to be important regulators in ccRCC development and progression. Phosphoinositide-dependent kinase 1 (PDK1) is required for metabolic activation; however, the role of PDK1-induced glycolytic metabolism regulated by miRNAs is unclear in ccRCC. So, the purpose of the current study is to elucidate the underlying mechanism in ccRCC cell metabolism mediated by PDK1. Our results revealed that miR-409-3p inhibited glycolysis by regulating PDK1 expression in ccRCC cells. We also found that miR-409-3p was regulated by hypoxia. Our results indicated that PDK1 facilitated ccRCC cell glycolysis, regulated by miR-409-3p in hypoxia.     

MicroRNA-384-5p regulates ischemia-induced cardioprotection by targeting phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (PI3K p110δ)

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. This study identified 16 differentially expressed miRNAs in ischemic myocardium of rats using TaqMan Low Density Array. In addition, bioinformatics analyses, such as Gene ontology and Pathway assays, were applied to determine the apoptosis pathway, only regulated by miR-384-5p, and all the associated target genes (PIK3CD, PPP3CA, PPP3CB, PPP3R1, CASP3 and IL1A). These target genes, besides PIK3CB, were shown to be significantly up-regulated by qRT-PCR assay, which further suggested that PIK3CD, PPP3CA, PPP3R1, CASP3, IL1A could be regulated by miR-384-5p. MTT, Western blot, qRT-PCR and luciferase assays were used to investigate the role of miR-384-5p in myocardial ischemia. We found that cleaved caspase3 expression was up-regulated by miR-384-5p and down-regulated by miR-384-5p inhibitor suggesting that apoptosis pathway was regulated by miR-384-5p. We also found that miR-384-5p suppressed cell viability while miR-384-5p inhibitor improved it, confirming H9c2 cell survival was affected by miR-384-5p. In addition, the PIK3CD protein level in H9c2 cells was up-regulated by miR-384-5p inhibitor. We found that miR-384-5p expression level decreased and PIK3CD protein level increased in both ischemic myocardium of rats and hypoxic H9c2 cells, and that miR-384-5p suppress PIK3CD expression through a miR-384-5p binding site within the 3′ untranslational region of PIK3CD. These results show that miR-384-5p, an important protecting factor, plays a significant role in cardioprotection by regulating PIK3CD in myocardial ischemia.     

PKM2 depletion induces the compensation of glutaminolysis through β-catenin/c-Myc pathway in tumor cells

The metabolic activity in cancer cells primarily rely on aerobic glycolysis. Besides glycolysis, some tumor cells also exhibit excessive addition to glutamine, which constitutes an advantage for tumor growth. M2-type pyruvate kinase (PKM2) plays a pivotal role in sustaining aerobic glycolysis, pentose phosphate pathway and serine synthesis pathway. However, the participation of PKM2 in glutaminolysis is little to be known. Here we demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamine metabolism in colon cancer cells. Treatment with 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor, got consistent results with the above. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities, plays a critical role in regulating β-catenin. Moreover, we found that overexpression of PKM2 negatively regulated β-catenin through miR-200a. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.     

MicroRNA Expression Profiling in Clear Cell Renal Cell Carcinoma: Identification and Functional Validation of Key miRNAs

ObjectiveThis study aims to profile dysregulated microRNA (miRNA) expression in clear cell renal cell carcinoma (ccRCC) and to identify key regulatory miRNAs in ccRCC.ConclusionsThis study identified 11 commonly dysregulated miRNAs in ccRCC, three of which (miR-199a-5p, miR-22 and miR-429) may represent key miRNAs involved in the pathogenesis of ccRCC. Further studies suggested that miR-199a-5p plays an important role in inhibition of cell invasion of ccRCC cells by suppressing expression of TGFBR1 and JunB.     

LINC00958 facilitates cervical cancer cell proliferation and metastasis by sponging miR-625-5p to upregulate LRRC8E expression

Accepted as a malignant tumor worldwide, cervical cancer (CC) has attracted much attention for its high incidence and mortality rates. Previous studies have elucidated the critical regulatory function that long noncoding RNAs (lncRNAs) exert on the tumorigenesis and progression of diverse tumors. Although multiple investigations have depicted that LINC00958 has a great impact on the complex biological process of many cancers, knowledge concerning the regulatory role of LINC00958 in CC remains limited and needs to be further explored. In our study, LINC00958 expression was evidently overexpressed in CC tissues and cells. Besides this, LINC00958 negatively regulated miR-625-5p expression and was verified to bind with miR-625-5p in CC. Subsequently, it was testified by a series of experiments that LINC00958 promotes CC cell proliferation and metastasis by sponging miR-625-5p. Furthermore, the leucine-rich repeat containing the eight family member E (LRRC8E) could bind with miR-625-5p, and its expression was negatively modulated by miR-625-5p, whereas positively regulated by LINC00958 in CC. Final rescue assays verified the effects of LINC0095/LRRC8E interaction and miR-625-5p/LRRC8E interaction on CC cell proliferation and metastasis. Collectively, LINC00958 facilitates CC cell proliferation and metastasis via the miR-625-5p/LRRC8E axis.     

MiR-652-5p elevated glycolysis level by targeting TIGAR in T-cell acute lymphoblastic leukemia

The effect of glycolysis remains largely elusive in acute T lymphoblastic leukemia (T-ALL). Increasing evidence has indicated that the dysregulation of miRNAs is involved in glycolysis, by targeting the genes coding glycolysis rate-limiting enzymes. In our previous studies, we found that overexpression of the ARRB1-derived miR-223 sponge repressed T-ALL progress and reduced the expression of miR-652-5p. However, little is known about miR-652-5p on T-ALL. Here, we showed that impaired miR-652-5p expression inhibited growth, promoted apoptosis of T-ALL cells in vitro and prolonged overall survival (OS) in vivo. Based on the GO enrichment of miR-652-5p target genes, we uncovered that impaired miR-652-5p decreased glycolysis, including reduced the lactate, pyruvate, ATP level and the total extracellular acidification rate (ECAR), elevated oxygen consumption rate (OCR) in T-ALL cell lines. Mechanically, miR-652-5p targeted the 3ʹUTR of Tigar mRNA and inhibited its expression. Furthermore, the alteration of glycosis level was attributed to Tigar overexpression, consistent with the effect of impaired miR-652-5p. Additionally, Tigar suppressed the expression of PFKFB3, a glycolysis rate-limiting enzyme, in vivo and in vitro. Taken together, our results demonstrate that impaired miR-652-5p/Tigar axis could repress glycolysis, thus to slow growth of T-ALL cells, which support miR-652-5p as a novel potential drug target for T-ALL therapeutics.Subject terms: Paediatric cancer, Mechanisms of disease     

Pyruvate kinase type M2 is upregulated in colorectal cancer and promotes proliferation and migration of colon cancer cells

Pyruvate kinase type M2 (PKM2) has been reported to be involved in aerobic glycolysis and cell growth in various tumors. However, the expression pattern of PKM2 in colorectal cancer (CRC) and the correlation between PKM2 expression and CRC remains unclear. The aim of this study is to investigate PKM2 expression and its possible role in CRC. We found that expression of PKM2 was increased in CRC and the increased PKM2 expression was associated with later stage and lymph metastasis of the tumors. Knockdown of PKM2 suppressed the aerobic glycolysis and decreased lactate production of colon cancer RKO cells. Knockdown of PKM2 repressed proliferation and migration of the cells. Inhibition of PKM2 suppressed xenograft tumor growth of RKO cells in vivo. These results suggest that the expression of PKM2 plays a critical role in development of CRC, and it may provide a growth advantage for colon cancer cells. Thus, PKM2 might be a potential therapeutic target for CRC. ? 2012 IUBMB Life, 64(9): 775-782, 2012.     

miR-145-5p inhibits tumor occurrence and metastasis through the NF-κB signaling pathway by targeting TLR4 in malignant melanoma

Compelling evidence shows that deregulated microRNAs (miRNAs) are important regulators in the progression of melanoma. miR-145-5p has been suggested to exhibit antitumorigenic activity in melanoma. However, the molecular mechanism underlying the biological activity of miR-145-5p in melanoma remains to be further understood. Herein, quantitative real-time polymerase chain reaction was used to examine the miR-145-5p expression in malignant melanoma tissues and cells. The interaction between miR-145-5p and toll-like receptor 4 (TLR4) was explored by bioinformatics analyses, luciferase reporter assay, and Western blot. The effects of miR-145-5p or combined with TLR4 on cell proliferation, colony formation, migration, and invasion abilities were investigated by (4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation, wound healing, and transwell assays, respectively. The melanoma xenograft tumor models were established to determine the biological activity of miR-145-5p in melanoma in vivo. In addition, the changes of the nuclear factor kappa B (NF-κB) pathway were analyzed by detecting the NF-κB activity and the NF-κB p65 protein level. We observed that the miR-145-5p expression was underexpressed in melanoma tissues and cells. miR-145-5p suppressed the TLR4 expression by binding to its 3′untranslated region in melanoma cells. Moreover, TLR4 overexpression abolished the inhibition of cell proliferation, colony formation, migration, and invasion abilities induced by miR-145-5p in melanoma cells. Meanwhile, miR-145-5p was confirmed to restrain melanoma tumor growth in vivo by targeting TLR4. Furthermore, miR-145-5p overexpression inactivated the NF-κB pathway in melanoma in vitro and in vivo, which was reversed by TLR4 overexpression. We concluded that miR-145-5p hindered the occurrence and metastasis of melanoma cells in vitro and in vivo by targeting TLR4 via inactivation of the NF-κB pathway.     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

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.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

A microRNA panel that regulates proinflammatory cytokines as diagnostic and prognosis biomarkers in colon cancer

Colon cancer (CC) is the third most common neoplasm and the fourth cause of cancer-related death worldwide in both sexes. It has been established that inflammation plays a critical role in tumorigenesis and progression of CC. Immune, stromal and tumor cells supply the tumor microenvironment with pro-inflammatory cytokines such as interleukin 1β, TNFα, IL-6 and IL-11, to hyperactivate signaling pathways linked to cancerous processes. Recent findings suggest a putative role of microRNAs (miRNAs) in the progression and management of the inflammatory response in intestinal diseases. Moreover, miRNAs are able to regulate expression of molecular mediators that are linking inflammation and cancer. In this work a miRNA panel differentially expressed between healthy, inflammatory bowel disease (IBD) and CC tissue was established. Identified miRNAs regulate signaling pathways related to inflammation and cancer progression. An inflammation associated-miRNA panel composed of 11-miRNAs was found to be overexpressed in CC but not in inflamed or normal tissues (miR-21-5p, miR-304-5p, miR-577, miR-335-5p, miR-21-3p, miR-27b-5p, miR-335-3p, miR-215-5p, miR-30b-5p, miR-192-5p, miR-3065-5p). The association of top hit miRNAs, miR-3065-5p and miR-30b-5p expression with overall survival of CC patients was demonstrated using Kaplan-Meier tests. Finally, differential miRNA expression was validated using an inflammation-associated CC model induced by Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) to compare miRNA expression in normal and inflamed tissue versus CC tissues. Based on these findings we propose the identified inflammatory miRNA panel as a potent diagnostic tool for CC determination.