1. MicroRNAs (miRNAs) play essential roles in many biological processes. It is known that aberrant miRNA expression contributes to some pathological conditions. However, it is not known whether miRNAs play any role in the development of insulin resistance in adipocytes, a key pathophysiological link between obesity and diabetes. 2. To investigate the function of miRNAs in the development of insulin resistance, using miRNA microarray analysis we compared miRNA expression profiles between normal insulinsensitive 3T3-L1 adipocytes and 3T3-L1 adipocytes rendered insulin resistant following treatment with high glucose (25mmol/L) and high insulin (1 mol/L). Furthermore, adipocytes were transfected with specific antisense oligonucleotides against miRNA-320 (anti-miR-320 oligo) and the effects on the development of insulin resistance were evaluated. 3. We identified 50 upregulated and 29 downregulated miRNAs in insulin-resistant (IR) adipocytes, including a 50-fold increase in miRNA-320 (miR-320) expression. Using bioinformatic techniques, the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) was found to be a potential target of miR-320. In experiments with anti-miR-320 oligo, insulin sensitivity was increased in IR adipocytes, as evidenced by increases in p85 expression, phosphorylation of Akt and the protein expression of the glucose transporter GLUT-4, as well as insulin-stimulated glucose uptake. These beneficial effects of anti-miR-320 oligo were observed only in IR adipocytes and not in normal adipocytes. 4. In conclusion, the miRNA profile changes in IR adipocytes compared with normal 3T3-L1 adipocytes. Anti-miR-320 oligo was found to regulate insulin resistance in adipocytes by improving insulin–PI3-K signalling pathways. The findings provide information regarding a potentially new therapeutic strategy to control insulin resistance.

Hepatic gluconeogenesis is essential for maintenance of normal blood glucose concentrations and is regulated by opposing stimulatory (cyclic adenosine monophosphate, cAMP) and inhibitory (insulin) signaling pathways. The cAMP signaling pathway leads to phosphorylation of cAMP response element-binding (CREB) protein, resulting in recruitment of the coactivators CREB-binding protein (CBP) and p300 and subsequent activation of gluconeogenesis. Insulin signaling leads to phosphorylation of CBP at serine 436, a residue near its CREB-interacting domain, but it is unknown whether this event modulates cAMP signaling. Here, we show in vitro and in 'knock-in' mice that a mutant CBP (S436A) is aberrantly recruited to CREB protein, resulting in inappropriate activation of gluconeogenesis in the fed state and glucose intolerance resulting from increased hepatic glucose production. We propose that insulin signaling may directly regulate many cAMP signaling pathways at the transcriptional level by controlling CBP recruitment.

Background-Thoracic aortic dissection (TAD) is characterized by dysregulated extracellular matrix. Little is known about the alterations of collagen and stimulators of collagen synthesis, eg, connective tissue growth factor (CTGF), in patients with TAD. In this study, we examined their roles in TAD. Methods and Results-Surgical specimens of the aortic wall of TAD patients (nϭ10) and controls (nϭ10) were tested for collagen types I and III and CTGF expression. When compared with controls, protein levels of type I and III collagen and CTGF were significantly increased by 3.2-, 3.7-, and 5.3-fold, respectively (PϽ0.05 for all). Similar patterns were shown in mRNA levels of type I␣ and I␣2 collagen and CTGF. Using immunohistochemistry and trichrome staining, we also observed elevated levels of collagen in the aortic media and adventitia. Treatment with recombinant human CTGF increased collagen synthesis in cultured aortic smooth muscle cells in a dose-and time-dependent fashion, in which expression of collagens increased from 506Ϯ108 counts per minute to 2764Ϯ240 cpm by 50 ng/mL CTGF, and from 30Ϯ43 cpm to 429Ϯ102 cpm at 48 hours. Conclusions-TAD patients exhibited significantly increased expression of aortic collagen types I and III as well as CTGF, which is likely to be responsible for the compromised aortic distensibility and systemic compliance.

SUMMARY Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity. To understand obesity and its associated diseases, it is important to elucidate the molecular mechanisms governing adipogenesis. MiR-375 has been demonstrated to inhibit differentiation of neurites and participate in the regulation of insulin secretion and blood homeostasis. However, it is unknown whether miR-375 plays a role in adipocyte differentiation.To investigate the role of miR-375 in adipocyte differentiation, we compared miR-375 expression level between 3T3-L1 pre-adipocytes and adipocytes using miRNA microarray and quantitative real-time RT-PCR (qRT-PCR) analysis. Furthermore, we evaluated the effects of overexpression or inhibition of miR-375 on 3T3-L1 adipocyte differentiation.In this study, we found that miR-375 expression was increased after induction of adipogenic differentiation. Overexpression of miR-375 enhanced 3T3-L1 adipocyte differentiation: as evidenced by its ability to increase mRNA levels of both CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ2) and induction of adipocyte fatty acid-binding protein (aP2) and triglyceride (TG) accumulation. Furthermore, we found overexpression of miR-375 suppressed phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2). In contrast, Anti-miR-375 increased ERK1/2 phosphorylation levels and inhibited mRNA expression of C/EBPα, PPARγ2 and aP2 in 3T3-L1 adipocyte, accompanied by decreased adipocyte differentiation.Taken together, these data suggest that miR-375 promotes 3T3-L1 adipocyte differentiation, possibly via modulating ERK - PPARγ2 - aP2 pathway.

Repeats (27-nt) in intron 4 have been shown to play a cis-acting role in endothelial nitric oxide synthase (eNOS) promoter activity. We hypothesize that the 27-nt repeats could be the source of small nuclear RNA specifically regulating eNOS expression. In this study, we used synthesized 27-nt RNA duplex and found that the eNOS gene transcriptional efficiency was reduced 63% (0.047 ؎ 0.009 vs. 0.126 ؎ 0.015, P < 0.01) by nuclear run-on assay. In endothelial cells transfected with the 27-nt small RNA duplex, we found that the eNOS mRNA and protein levels were decreased by >64% (P < 0.01). Conversely, a randomly selected 27-nt from luciferase gene had no effect on the eNOS expression. Furthermore, this eNOS silencing effect appeared to be reversible under the stimulation of vascular endothelial growth factor (10 ng͞ml), which is known to upregulate eNOS expression. Using in situ hybridization and Northern blotting, we observed the presence of endogenous eNOS intron 4-derived 27-nt small RNA, which was confined to the nucleus. In summary, we demonstrated that intron-based microRNAs in eNOS can induce significant gene specific transcriptional suppression, which could be an effective negative feedback regulator for gene expression.intron ͉ microRNA ͉ negative feedback regulation ͉ repeat polymorphism M ost eukaryotic genes are interrupted by one or more introns, which must be removed in the process of pre-mRNA splicing within the nucleus. The mature mRNA with intact ORF is then exported to the cytoplasm for translation. Since the discovery of introns as the noncoding sequences (1), they have mostly been regarded as redundant and useless genomic sequences. However, with the advancement in molecular technology, these intervening sequences have recently been recognized to play a significant role in gene evolution and expression (2-4). Introns can function as enhancer͞repressor to regulate cell specific gene transcription; they can modulate the spliceosome conformation and affect the efficiency of pre-mRNA splicing (5, 6). A recent study has shown that the expression of human ␤-globin gene is highly intron-dependent, which regulates both pre-mRNA splicing and translational efficiency (7). Moreover, the intronic binding site for nuclear proteins mediates the p53-dependent transcriptional activation (8); intronic regulatory elements confer to control developmental and cellspecific gene expression (9). Thus, it is clear that introns are not only essential for genomic integrity, but are also important for controlling cell-specific gene expression.Of particular interest, Hui et al. (10) have recently shown that the variable-length CA repeats in the endothelial nitric oxide synthase (eNOS) intron 13 can bind with hnRNP and affect eNOS splicing. Studies of ours and others have further suggested that the 27-nt repeats in the eNOS intron 4 could be potentially functional in regulating eNOS expression (11,12). We have shown that the 27-nt repeats in intron 4 could act as a possible enhancer͞repressor for the eNOS transcription (13...

The present study is to investigate the role of microRNA-21 (miR-21) in nasopharyngeal carcinoma (NPC) and the mechanisms of regulation of PTEN by miR-21. Fifty-four tissue samples were collected from 42 patients with NPC and 12 healthy controls. Human NPC cell lines CNE-1, CNE-2, TWO3 and C666-1 were used for cell assays. To investigate the expression of miR-21, RT-PCR was employed. RT-PCR, Western blotting, and immunohistochemistry were used to measure the expression of STAT3 mRNA and STAT3 protein. To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. TUNEL assay was used to detect DNA fragmentation. To validate whether miR-21 directly recognizes the 3′-UTRs of PTEN mRNA, luciferase reporter assay was employed. miR-21 expression was increased in NPC tissues compared with control and the same result was found in NPC cell lines. Notably, increased expression of miR-21 was directly related to advanced clinical stage and lymph node metastasis. STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines. Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity. Both in vitro and in vivo assays revealed that miR-21 enhanced NPC cell proliferation and suppressed apoptosis. miR-21, activated by STAT3, induced proliferation and suppressed apoptosis in NPC by targeting PTEN-AKT pathway.

We report the emerging role of microRNA (miRNA) deregulation associated with activation of an oncogene SOX4 (a member of the SRY-related HMG-box) in esophageal carcinoma. Paired esophageal cancer and adjacent non-tumor tissues were obtained from 42 patients who underwent primary surgical resection for esophageal cancer. Experiments such as real-time PCR, western blot analysis, luciferase-reporter assay, cell proliferation and colony formation assays, in vitro migration and invasion assays, and a wound-healing assay were performed to determine the effects of miR-129-2. We found that SOX4 expression was elevated (P<0.005) in esophageal tumors (n=42) when compared with its expression in the controls (n=42). Compared with the normal esophageal tissues, the expression of miR-129-2 was downregulated in 27 of 31 primary esophageal tumors, while the expression of SOX4 was upregulated (P<0.001). Restoration of miR-129-2 by transfection with an miRNA expression plasmid led to a decrease in SOX4 expression, which was accompanied by reduced migration and proliferation of the cancer cells. These results suggest that aberrant expression of SOX4 is associated with repression of miR-129-2, and restoration of miR-129-2 suppresses the migration and proliferation of esophageal cancer cells. Our results demonstrated that the deregulation of miR-129-2 leads to aberrant SOX4 expression, presenting a new paradigm in which the restoration of miRNA suppresses its oncogenic target in esophageal cancer.

We have compared the insulin-like activity of bis(acetylacetonato)oxovanadium(IV) [VO(acac)2], bis(maltolato)oxovanadium(IV) [VO(malto)2], and bis(1-N-oxide-pyridine-2-thiolato)oxovanadium(IV) [VO(OPT)2] in differentiated 3T3-L1 adipocytes. The insulin-like influence of VO(malto)2 and VO(OPT)2 was decreased compared with that of VO(acac)2. Also, serum albumin enhanced the insulin-like activity of all three chelates more than serum transferrin. Each of the three VO2+ chelates increased the tyrosine phosphorylation of proteins in response to insulin, including the beta-subunit of the insulin receptor (IRbeta) and the insulin receptor substrate-1 (IRS1). However, VO(acac)2 exhibited the greatest synergism with insulin and was therefore further investigated. Treatment of 3T3-L1 adipocytes with 0.25 mM VO(acac)2 in the presence of 0.25 mM serum albumin synergistically increased glycogen accumulation stimulated by 0.1 and 1 nM insulin, and increased the phosphorylation of IRbeta, IRS1, protein kinase B, and glycogen synthase kinase-3beta. Wortmannin suppressed all of these classical insulin-signaling activities exerted by VO(acac)2 or insulin, except for tyrosine phosphorylation of IRbeta and IRS1. Additionally, VO(acac)2 enhanced insulin signaling and metabolic action in insulin-resistant 3T3-L1 adipocytes. Cumulatively, these results provide evidence that VO(acac)2 exerts its insulin-enhancing properties by directly potentiating the tyrosine phosphorylation of the insulin receptor, resulting in the initiation of insulin metabolic signaling cascades in 3T3-L1 adipocytes.