AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21

<p>AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.</p

Identification of Serum microRNA Biomarkers for Tuberculosis Using RNA-seq

<div><p>Tuberculosis (TB) remains a significant human health issue. More effective biomarkers for use in tuberculosis prevention, diagnosis, and treatment, including markers that can discriminate between healthy individuals and those with latent infection, are urgently needed. To identify a set of such markers, we used Solexa sequencing to examine microRNA expression in the serum of patients with active disease, healthy individuals with latent TB, and those with or without prior BCG inoculation. We identified 24 microRNAs that are up-regulated (2.85–1285.93 fold) and 6 microRNAs that are down-regulated (0.003–0.11 fold) (P<0.05) in patients with active TB relative to the three groups of healthy controls. In addition, 75 microRNAs were up-regulated (2.05–2454.58 fold) and 11 were down-regulated (0.001–0.42 fold) (P<0.05) in latent-TB infected individuals relative to BCG- inoculated individuals. Of interest, 134 microRNAs were differentially-expressed in BCG-inoculated relative to un-inoculated individuals (18 up-regulated 2.9–499.29 fold, 116 down-regulated 0.0002–0.5 fold), providing insights into the effects of BCG inoculation at the microRNA level. Target prediction of differentially-expressed microRNAs by microRNA-Gene Network analysis and analysis of pathways affected suggest that regulation of the host immune system by microRNAs is likely to be one of the main factors in the pathogenesis of tuberculosis. qRT-PCR validation indicated that hsa-miR-196b and hsa-miR-376c have potential as markers for active TB disease. The microRNA differential-expression profiles generated in this study provide a good foundation for the development of markers for TB diagnosis, and for investigations on the role of microRNAs in BCG-inoculated and latent-infected individuals.</p></div

Demographic characteristics of the study population.

<p>LTBI, individuals with latent TB infection.</p

Venn diagram illustrating the distribution of microRNAs which showed significantly altered expression in TB patients compared with three control groups (LTBI, BCG-inoculated, and un-inoculated individuals).

<p>A, microRNAs which were up-regulated in serum from TB patients compared with the control groups; B, microRNAs which were down-regulated in serum from TB patients compared with the control groups. BCG, BCG-inoculated; LTBI, individuals with latent TB infection; Healthy, un-inoculated controls.</p

microRNA-gene network for hsa-miR-196b, hsa-miR-516b, hsa-miR-376c and hsa-miR-486-5p.

<p>The microRNA-gene network was built using gene expression data and predicted interactions from the TargetScan and PicTar microRNA databases. Red circles represent microRNAs and blue squares represent genes; their relationship is represented by the edges.</p

Effects of miRNA overexpression on NFAT5 mRNA levels (2^−ΔΔCT) in HEK293 cells.

<p>HEK293 cells were transfected respectively with four miRNA mimics (<i>hsa-miR-376c</i>, <i>hsa-miR-516b</i>, <i>hsa-miR-486-5p</i> and <i>cel-miR-67-3p</i>) and incubated at 37°C (5% CO₂) for 24 h. NFAT5 mRNA levels were measured in extracts of total RNA using RT-qPCR. NC: negative control (<i>cel-miR-67-3p</i>). Data presented are mean values from three independent experiments, n = 3. Error bars show the s.e.m. The significance of comparisons was tested using the Student’s <i>t</i>-test; *: P<0.05.</p