<Emphasis Type="Italic">miR-3075</Emphasis> Inhibited the Migration of Schwann Cells by Targeting Cntn2

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.     

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.     

A miRNA signature of prion induced neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion-induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified.     

MicroRNAs Are Part of the Regulatory Network that Controls EGF Induced Apoptosis,Including Elements of the JAK/STAT Pathway,in A431 Cells

MiRNAs are known to regulate gene expression and in the context of cancer have been shown to regulate metastasis, cell proliferation and cell death. In this report we describe potential miRNA regulatory roles with respect to induction of cell death by pharmacologic dose of Epidermal Growth Factor (EGF). Our previous work suggested that multiple pathways are involved in the induction of apoptosis, including interferon induced genes, cytokines, cytoskeleton and cell adhesion and TP53 regulated genes. Using miRNA time course expression profiling of EGF treated A431 cells and coupling this to our previous gene expression and proteomic data, we have been able to implicate a number of additional miRNAs in the regulation of apoptosis. Specifically we have linked miR-134, miR-145, miR-146b-5p, miR-432 and miR-494 to the regulation of both apoptotic and anti-apoptotic genes expressed as a function of EGF treatment. Whilst additional miRNAs were differentially expressed, these had the largest number of apoptotic and anti-apoptotic targets. We found 5 miRNAs previously implicated in the regulation of apoptosis and our results indicate that an additional 20 miRNAs are likely to be involved based on their correlated expression with targets. Certain targets were linked to multiple miRNAs, including PEG10, BTG1, ID1, IL32 and NCF2. Some miRNAs that target the interferon pathway were found to be down regulated, consistent with a novel layer of regulation of interferon pathway components downstream of JAK/STAT. We have significantly expanded the repertoire of miRNAs that may regulate apoptosis in cancer cells as a result of this work.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.     

MicroRNA Expression Differences in Human Hematopoietic Cell Lineages Enable Regulated Transgene Expression

Blood microRNA (miRNA) levels have been associated with and shown to participate in disease pathophysiology. However, the hematopoietic cell of origin of blood miRNAs and the individual blood cell miRNA profiles are poorly understood. We report the miRNA content of highly purified normal hematopoietic cells from the same individuals. Although T-cells, B-cells and granulocytes had the highest miRNA content per cell, erythrocytes contributed more cellular miRNA to the blood, followed by granulocytes and platelets. miRNA profiling revealed different patterns and different expression levels of miRNA specific for each lineage. miR-30c-5p was determined to be an appropriate reference normalizer for cross-cell qRT-PCR comparisons. miRNA profiling of 5 hematopoietic cell lines revealed differential expression of miR-125a-5p. We demonstrated endogenous levels of miR-125a-5p regulate reporter gene expression in Meg-01 and Jurkat cells by (1) constructs containing binding sites for miR-125a-5p or (2) over-expressing or inhibiting miR-125a-5p. This quantitative analysis of the miRNA profiles of peripheral blood cells identifies the circulating hematopoietic cellular miRNAs, supports the use of miRNA profiles for distinguishing different hematopoietic lineages and suggests that endogenously expressed miRNAs can be exploited to regulate transgene expression in a cell-specific manner.     

MicroRNA-143 (miR-143) regulates cancer glycolysis via targeting hexokinase 2 gene

High glycolysis, well known as "Warburg effect," is frequently observed in a variety of cancers. Whether the deregulation of miRNAs contributes to the Warburg effect remains largely unknown. Because miRNA regulates gene expression at both mRNA and protein levels, we constructed a gene functional association network, which allows us to detect the gene activity instead of gene expression, to integratively analyze the microarray data for gene expression and miRNA expression profiling and identify glycolysis-related gene-miRNA pairs deregulated in cancer. Hexokinase 2 (HK2), coding for the first rate-limiting enzyme of glycolysis, is among the top list of genes predicted and potentially regulated by multiple miRNAs including miR-143. Interestingly, miR-143 expression was inversely associated with HK2 protein level but not mRNA level in human lung cancer samples. miR-143, down-regulated by mammalian target of rapamycin activation, reduces glucose metabolism and inhibits cancer cell proliferation and tumor formation through targeting HK2. Collectively, we have not only established a novel methodology for gene-miRNA pair prediction but also identified miR-143 as an essential regulator of cancer glycolysis via targeting HK2.     

Angiotensin II directly impairs adipogenic differentiation of human preadipose cells

microRNAs (miRNAs) are small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. Reduced expression of miR-338 has been reported in several types of cancers; however, the role of miR-338 in oral squamous cell carcinoma (OSCC) has not been elucidated. In this study, we demonstrated that miR-338 was dramatically downregulated in OSCC tissues and cell lines. Overexpression of miR-338 significantly inhibited proliferation, colony formation, migration, and invasion of OSCC cells. In addition, neuropilin1 (NRP1) was identified as a target of miR-338 in OSCC cells and inversely correlated with miR-338 in OSCC tissues. Furthermore, restoration of NRP1 attenuated the tumor-suppressive effects of miR-338. Taken together, miR-338 might inhibit growth and metastasis of OSCC cells by targeting NRP1.     

Identification and expression analysis of miRNAs during batch culture of HEK-293 cells

MicroRNAs (miRNAs) are small non-coding RNAs of about 20–24 nucleotides in length. They regulate gene expression negatively and have been implicated in a wide variety of biological processes. To identify potential miRNAs that may influence the growth and proliferation of mammalian cells cultured in bioreactors, we applied miRNA microarray expression profiling technology to batch cultures of HEK293 cells in protein free media. In our study, we identified miRNAs that were differentially expressed during the exponential and stationary phases, 13 of these showed distinct up regulation trends while 1 exhibited down regulation. These miRNAs have been implicated in cellular differentiation, growth arrest and apoptosis. Specifically, miR-16 and let-7b are potentially useful in the enhancement of bioreactor cell cultures.     

Parallel mRNA and MicroRNA Profiling of HEV71-Infected Human Neuroblastoma Cells Reveal the Up-Regulation of miR-1246 in Association with DLG3 Repression

Human enterovirus 71 (HEV71) has emerged as the leading cause of viral encephalitis in children in most Asian countries. The roles of host miRNAs in the neurological pathogenesis of HEV71 infection remain unknown. In the present study, comprehensive miRNA expression profiling in HEV71-infected human neuroblastoma SH-SY5Y cells was performed using the Affymetrix Gene Chip microarray assay and was validated using real-time RT-PCR. Among the 69 differentially expressed miRNAs, miR-1246 was specifically induced by HEV71 infection in human neuroblastoma cells, but inhibition of miR-1246 failed to affect HEV71 replication. Parallel mRNA and microRNA profiling based on the 35 K Human Genome Array identified 182 differentially regulated genes. Target prediction of miR-1246 and network modeling revealed 14 potential target genes involved in cell death and cell signaling. Finally, a combined analysis of the results from mRNA profiling and miR-1246 target predication led to the identification of disc-large homolog 3 (DLG3), which is associated with neurological disorders, for further validation. Sequence alignment and luciferase reporter assay showed that miR-1246 directly bound with the 3′-UTR of DLG3 gene. Down-regulation of miR-1246 induced significant changes in DLG3 expression levels in HEV71-infected SHSY5Y cells. Together, these results suggested that miR-1246 might play a role in neurological pathogenesis of HEV71 by regulating DLG3 gene in infected cells. These findings provide new information on the miRNA and mRNA profiles of HEV71-infected neuroblastoma cells. The biological significance of miR-1246 and DLG3 during the course of HEV71 infection deserves further investigation.