MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas

MicroRNAs (miRNAs) are single-stranded, 18- to 23-nt RNA molecules that function as regulators of gene expression. Previous studies have shown that microRNAs play important roles in human cancers, including gliomas. Here, we found that expression levels of miR-181b were decreased in gliomas, and we identified IGF-1R as a novel direct target of miR-181b. MiR-181b overexpression inhibited cell proliferation, migration, invasion, and tumorigenesis by targeting IGF-1R and its downstream signaling pathways, PI3K/AKT and MAPK/ERK1/2. Overexpression of IGF-1R rescued the inhibitory effects of miR-181b. In clinical specimens, IGF-1R was overexpressed, and its protein levels were inversely correlated with miR-181b expression. Taken together, our results indicate that miR-181b functions in gliomas to suppress growth by targeting the IGF-1R oncogene and that miR-181b may serve as a novel therapeutic target for gliomas.     

miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2

In chronic alcoholism, brain shrinkage and cognitive defects because of neuronal death are well established, although the sequence of molecular events has not been fully explored yet. We explored the role of microRNAs (miRNAs) in ethanol-induced apoptosis of neuronal cells. Ethanol-sensitive miRNAs in SH-SY5Y, a human neuroblastoma cell line, were identified using real-time PCR-based TaqMan low-density arrays. Long-term exposure to ethanol (0.5% v/v for 72 h) produced a maximum increase in expression of miR-497 (474-fold) and miR-302b (322-fold). Similar to SH-SY5Y, long-term exposure to ethanol induced miR-497 and miR-302b in IMR-32, another human neuroblastoma cell line. Using in silico approaches, BCL2 and cyclin D2 (CCND2) were identified as probable target genes of these miRNAs. Cotransfection studies with 3'-UTR of these genes and miRNA mimics have demonstrated that BCL2 is a direct target of miR-497 and that CCND2 is regulated negatively by either miR-302b or miR-497. Overexpression of either miR-497 or miR-302b reduced expression of their identified target genes and increased caspase 3-mediated apoptosis of SH-SY5Y cells. However, overexpression of only miR-497 increased reactive oxygen species formation, disrupted mitochondrial membrane potential, and induced cytochrome c release (mitochondria-related events of apoptosis). Moreover, ethanol induced changes in miRNAs, and their target genes were substantially prevented by pre-exposure to GSK-3B inhibitors. In conclusion, our studies have shown that ethanol-induced neuronal apoptosis follows both the mitochondria-mediated (miR-497- and BCL2-mediated) and non-mitochondria-mediated (miR-302b- and CCND2-mediated) pathway.     

Multifaceted roles of miR-1s in repressing the fetal gene program in the heart

miRNAs are an important class of regulators that play roles in cellular homeostasis and disease. Muscle-specific miRNAs, miR-1-1 and miR-1-2, have been found to play important roles in regulating cell proliferation and cardiac function. Redundancy between miR-1-1 and miR-1-2 has previously impeded a full understanding of their roles in vivo. To determine how miR-1s regulate cardiac function in vivo, we generated mice lacking miR-1-1 and miR-1-2 without affecting nearby genes. miR-1 double knockout (miR-1 dKO) mice were viable and not significantly different from wild-type controls at postnatal day 2.5. Thereafter, all miR-1 dKO mice developed dilated cardiomyopathy (DCM) and died before P17. Massively parallel sequencing showed that a large portion of upregulated genes after deletion of miR-1s is associated with the cardiac fetal gene program including cell proliferation, glycolysis, glycogenesis, and fetal sarcomere-associated genes. Consistent with gene profiling, glycogen content and glycolytic rates were significantly increased in miR-1 dKO mice. Estrogen-related Receptor β (Errβ) was identified as a direct target of miR-1, which can regulate glycolysis, glycogenesis, and the expression of sarcomeric proteins. Cardiac-specific overexpression of Errβ led to glycogen storage, cardiac dilation, and sudden cardiac death around 3-4 weeks of age. We conclude that miR-1 and its primary target Errβ act together to regulate the transition from prenatal to neonatal stages by repressing the cardiac fetal gene program. Loss of this regulation leads to a neonatal DCM.     

Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.     

MicroRNA-33b regulates hepatocellular carcinoma cell proliferation,apoptosis, and mobility via targeting Fli-1-mediated Notch1 pathway

MicroRNAs (miRNAs) have been confirmed to play pivotal roles in hepatocellular carcinoma (HCC) carcinogenesis. However, the underlying function of microRNA-33b (miR-33b) in HCC remains unclear. Here, we found that miR-33b level was significantly reduced in both HCC tissues and tumor cell lines. Further, luciferase reporter assay and western blot analysis confirmed that Friend leukemia virus integration 1 (Fli-1) was a direct target of miR-33b. Overexpression of miR-33b dramatically suppressed HCC tumor cell proliferation and cell mobility, but facilitated tumor cell apoptosis in vitro. Besides, restoration of Fli-1 partially attenuated miR-33b-mediated inhibition of cell growth and metastasis via activating Notch1 signaling and its downstream effectors. Our findings demonstrate the important role of miR-33b/Fli-1 axis in HCC progression and provide novel therapeutic candidates for HCC clinical treatment.     

Tumor-suppressive microRNA-10a inhibits cell proliferation and metastasis by targeting Tiam1 in esophageal squamous cell carcinoma

Aberrant microRNAs (miRNAs) expressions could contribute to the progression of numerous cancers, including esophageal squamous cell carcinoma, while miR-10a participates in multiple biological processes on cancers. However, the molecular mechanism of miR-10a in esophageal squamous cell carcinoma (ESCC) has not been investigated. Herein, miR-10a was significantly reduced in ESCC clinical tissues and ESCC cell lines (EC109 and TE-3). In addition, immunohistochemistry indicated that the expressions of α-SMA, Ki-67, and PCNA in tumor tissues were higher than that of controls. In vitro, overexpression of miR-10a dramatically suppressed cell proliferation and enhanced cell apoptosis, while the decrease of miR-10a expressed the opposite outcome. Specially, overexpression of miR-10a caused a G0/G1 peak accumulation. Moreover, miR-10a also negatively regulated ESCC cell migration and invasion. Furthermore, targetscan bioinformatics predictions and the dual-luciferase assay confirmed that Tiam1 was a direct target gene of miR-10a. The statistical analysis showed Tiam1 was negatively in correlation with miR-10a in ESCC patient samples. And silencing Tiam1 could lead to a decline on cell growth, invasion, and migration in ESCC cell lines, while it could enhance cell apoptosis and cause a G0/G1 peak accumulation. In vivo, it revealed that miR-10a notably decreased the tumor growth and metastasis in xenograft model and pulmonary metastasis model. And it showed a lower expressions of Tiam1 in the miR-10a mimics group by immunohistochemistry. Taken together the results, they indicated that miR-10a might function as a novel tumor suppressor in vitro and in vivo via targeting Tiam1, suggesting miR-10a to be a candidate biomarker for the ESCC therapy.     

Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation.     

TGF-β1 Reduces miR-29a Expression to Promote Tumorigenicity and Metastasis of Cholangiocarcinoma by Targeting HDAC4

Transforming growth factor β1 (TGF-β1) and miRNAs play important roles in cholangiocarcinoma progression. In this study, miR-29a level was found significantly decreased in both cholangiocarcinoma tissues and tumor cell lines. TGF-β1 reduced miR-29a expression in tumor cell lines. Furthermore, anti-miR-29a reduced the proliferation and metastasis capacity of cholangiocarcinoma cell lines in vitro, overexpression of miR-29a counteracted TGF-β1-mediated cell growth and metastasis. Subsequent investigation identified HDAC4 is a direct target of miR-29a. In addition, restoration of HDAC4 attenuated miR-29a-mediated inhibition of cell proliferation and metastasis. Conclusions: TGF-β1/miR-29a/HDAC4 pathway contributes to the pathogenesis of cholangiocarcinoma and our data provide new therapeutic targets for cholangiocarcinoma.     

Functional Characterization of Drosophila microRNAs by a Novel in Vivo Library

Animal microRNAs (miRNA) are implicated in the control of nearly all cellular functions. Due to high sequence redundancy within the miRNA gene pool, loss of most of these 21- to 24-bp long RNAs individually does not cause a phenotype. Thus, only very few miRNAs have been associated with clear functional roles. We constructed a transgenic UAS-miRNA library in Drosophila melanogaster that contains 180 fly miRNAs. This library circumvents the redundancy issues by facilitating the controlled misexpression of individual miRNAs and is a useful tool to complement loss-of-function approaches. Demonstrating the effectiveness of our library, 78 miRNAs induced clear phenotypes. Most of these miRNAs were previously unstudied. Furthermore, we present a simple system to create GFP sensors to monitor miRNA expression and test direct functional interactions in vivo. Finally, we focus on the miR-92 family and identify a direct target gene that is responsible for the specific wing phenotype induced by the misexpression of miR-92 family members.     

HDAC inhibitors sodium butyrate and sodium valproate do not affect human ncor1 and ncor2 gene expression in HL-60 cells

AIM. This study was designed to examine whether the class I and class IIa histone deacetylase (HDAC) inhibitors, sodium butyrate and sodium valproate alter the expression of human NCOR1 and/or NCOR2 genes coding for N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), respectively. METHODS: Human leukemia HL-60 cells were treated for 24 h with 0.5 and 1 mM sodium butyrate, 1 to 3 mM sodium valproate, 1 mcM all-trans retinoic acid (ATRA) or cotreated with 1 mcM ATRA and 0.5 mM sodium butyrate. The acetylation of histones H3 and H4 was analysed by western blotting. The levels of NCOR1 and NCOR2 mRNA were determined by quantitative real-time PCR. Expression of NCF2 gene coding for the NADPH oxidase subunit p67phox was evaluated as a marker of myeloid differentiation. Results. Both butyrate and valproate increased the acetylation of histone H3 at Lys9 and/or Lys14 as well as histone H4 at Lys12. Both HDAC inhibitors caused a significant increase in NCF2 mRNA levels without affecting NCOR1 or NCOR2 mRNA levels. Similarly, ATRA alone or in combination with butyrate induced NCF2 gene expression without any significant influence on the expression of NCOR1 or NCOR2 genes. CONCLUSION: We conclude that inhibitors of class I and class IIa HDACs do not alter the expression of human NCOR1 or NCOR2 genes and that the onset of myeloid differentiation is not accompanied by induction or repression of these genes in HL-60 cells.     

MicroRNA expression profile in RAW264.7 cells in response to Brucella melitensis infection

MicroRNA (miRNA) is small non-coding RNA with approximate 22 nt in length. Recent studies indicate that miRNAs play significant roles in pathogen-host interactions. Brucella organisms are Gram-negative facultative intracellular bacteria that cause Brucellosis. Brucella strains infect macrophages and establish chronic infection by altering host life activities including apoptosis and autophagy. Here, we report a comprehensive analysis of miRNA expression profiles in mock- and Brucella-infected RAW264.7 cells using high-throughput sequencing approach. In total, 344 unique miRNAs were co-expressed in the two libraries, in which 57 miRNAs were differentially expressed. Eight differentially expressed miRNAs with high abundance were subjected to further analysis. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are involved in apoptosis, autophagy and immune response. In particular, a total of 25 target genes are involved in regulating apoptosis and autophagy, indicating that these miRNAs may play important regulatory roles in the Brucella-host interactions. Furthermore, the interactions of miR-1981 and its target genes, Bcl-2 and Bid, were validated by luciferase assay. The results show that miR-1981 mimic up-regulated the luciferase activity of psiCHECK-2 Bcl-2 3' UTR, but the luciferase activity of psiCHECK-2 Bid 3' UTR was not changed significantly. Taken together, these data provide valuable framework on Brucella induced miRNA expression in RAW264.7 cells, and suggest that Brucella may establish chronic infection by regulating miRNA expression profile.     

microRNA-10b confers cisplatin resistance by activating AKT/mTOR/P70S6K signaling via targeting PPARγ in esophageal cancer

It is well known that the acquisition of chemoresistance is a major obstacle for the effective treatment of human cancers. It is reported that microRNAs (miRNAs) are implicated in chemotherapy resistance of various malignancies. miR-10b was previously proved as an oncogene in multiple malignancies, including esophageal cancer. However, its biological significance in regulating cisplatin (DDP) resistance in esophageal cancer is still elusive. Here, we observed that miR-10b expression was upregulated and peroxisome proliferator-activated receptor-γ (PPARγ) expression was downregulated in esophageal cancer tumor tissues and cells. PPARγ was proved as a functional target of miR-10b. Moreover, suppression of miR-10b enhanced the chemosensitivity of esophageal cancer cells to DDP in vitro and in vivo. In addition, PPARγ-mediated DDP sensitivity was weakened by miR-10b overexpression. Furthermore, miR-10b-activated AKT/mTOR/p70S6K signaling pathway through targeting PPARγ. Inactivation of AKT/mTOR/p70S6K by AKT inhibitor (GSK690693) attenuated miR-10b-induced DDP resistance in esophageal cancer cells. Taken together these observation, miRNA-10b-mediated PPARγ inhibition enhanced DDP resistance by activating the AKT/mTOR/P70S6K signaling in esophageal cancer, suggesting a potential target to improve therapeutic response of patients with esophageal cancer to DDP.     

MicroRNA-10b Promotes Migration and Invasion through KLF4 in Human Esophageal Cancer Cell Lines

Recently, microRNAs have emerged as regulators of cancer metastasis through acting on multiple signaling pathways involved in metastasis. In this study, we have analyzed the level of miR-10b and cell motility and invasiveness in several human esophageal squamous cell carcinoma cell lines. Our results reveal a significant correlation of miR-10b level with cell motility and invasiveness. Overexpression of miR-10b in KYSE140 cells increased cell motility and invasiveness, whereas inhibition of miR-10b in EC9706 cells reduced cell invasiveness, although it did not alter cell motility. Additionally, we identified KLF4, a known tumor suppressor gene that has been reported to suppress esophageal cancer cell migration and invasion, as a direct target of miR-10b. Furthermore, overexpression of miR-10b in KYSE140 and KYSE450 cells led to a reduction of endogenous KLF4 protein, whereas silencing of miR-10b in EC9706 cells caused up-regulation of KLF4 protein. Coexpression of miR-10b and KLF4 in KYSE140 cells and coexpression of small interfering RNA for KLF4 mRNA and miR-10b-AS in EC9706 cells partially abrogated the effect of miR-10b on cell migration and invasion. Finally, analyses of the miR-10b level in 40 human esophageal cancer samples and their paired normal adjacent tissues revealed an elevated expression of miR-10b in 95% (38 of 40) of cancer tissues, although no significant correlation of the miR-10b level with clinical metastasis status was observed in these samples.     

Combinatorial Action of MicroRNAs let-7 and miR-34 Effectively Synergizes with Erlotinib to Suppress Non-small Cell Lung Cancer Cell Proliferation

Lung cancer represents the leading cause of cancer-related deaths in men and women worldwide. Targeted therapeutics, including the epidermal growth factor receptor (EGFR) inhibitor erlotinib, have recently emerged as clinical alternatives for the treatment of non-small cell lung cancer (NSCLC). However, the development of therapeutic resistance is a major challenge, resulting in low 5-year survival rates. Due to their ability to act as tumor suppressors, microRNAs (miRNAs) are attractive candidates as adjuvant therapeutics for the treatment of NSCLC. In this study, we examine the ability of 2 tumor suppressor miRNAs, let-7b and miR-34a to sensitize KRAS;TP53 mutant non-small cell lung cancer cells to the action of erlotinib. Treatment with these miRNAs, individually or in combination, resulted in synergistic potentiation of the anti-proliferative effects of erlotinib. This effect was observed over a wide range of miRNA and erlotinib interactions, suggesting that let-7b and miR-34a target oncogenic pathways beyond those inhibited by EGFR. Combinatorial treatment with let-7b and miR-34a resulted in the strongest synergy with erlotinib, indicating that these miRNAs can effectively target multiple cellular pathways involved in cancer cell proliferation and resistance to erlotinib. Together, our findings indicate that NSCLC cells can be effectively sensitized to erlotinib by supplementation with tumor suppressor miRNAs, and suggest that the use of combinations of miRNAs as adjuvant therapeutics for the treatment of lung cancer is a viable clinical strategy.