Dependence of specificity (Sp, red circle), accuracy (Ac, green triangle), sensitivity (Sn, black rectangle) and Matthews' correlation coefficient (CC, blue reversing triangle) on the threshold value (Thd)

<p><b>Copyright information:</b></p><p>Taken from "A novel method for high accuracy sumoylation site prediction from protein sequences"</p><p>http://www.biomedcentral.com/1471-2105/9/8</p><p>BMC Bioinformatics 2008;9():8-8.</p><p>Published online 8 Jan 2008</p><p>PMCID:PMC2245905.</p><p></p

The CREB-miR-9 Negative Feedback Minicircuitry Coordinates the Migration and Proliferation of Glioma Cells

<div><p>Migration-proliferation dichotomy is a common mechanism in gliomagenesis; however, an understanding of the exact molecular mechanism of this “go or grow” phenomenon remains largely incomplete. In the present study, we first found that microRNA-9 (miR-9) is highly expressed in glioma cells. MiR-9 inhibited the proliferation and promoted the migration of glioma cells by directly targeting cyclic AMP response element-binding protein (CREB) and neurofibromin 1 (NF1), respectively. Our data also suggested a migration-inhibitory role for CREB through directly regulating the transcription of NF1. Furthermore, we found that the transcription of miR-9-1 is under CREB's control, forming a negative feedback minicircuitry. Taken together, miR-9 inhibits proliferation but promotes migration, whereas CREB plays a pro-proliferative and anti-migratory role, suggesting that the CREB-miR-9 negative feedback minicircuitry plays a critical role in the determination of “go or grow” in glioma cells.</p> </div

MiR-9 promotes migration through targeting NF1.

<p>(A) The NF1 3′UTR contains a putative miR-182 binding site which also has the potential to interact with miR-9. The mammalian alignment is shown on the top. A mutated NF1 3′UTR was generated by mutating the miR-182 binding site. (B) Luciferase reporter assays were performed to test the interactions between the miRNAs and the NF1 3′UTR. NF1 3′UTR luciferase reporter constructs were co-transfected with synthetic miRNA mimics (miR-9, miR-182 and miR-23a) or control mimics (miR-NC), and 24 h later the normalized luciferase activity was determined. Data are represented as the mean ± SD, n = 4. (C) MiR-9, miR-182 and miR-23a were knocked-down in T98G and U251 cells by transfection with miRNA antagomirs, and NF1 protein level was determined by western blotting and quantified by densitometric measurement (mean ± SD, n = 3). (D) T98G cells were transfected with miR-9 mimics (or control mimics, miR-NC) or siRNA targeting NF1 (or control non-specific siRNA), and whole cell protein was extracted for determination of NF1 protein levels by western blotting. (E) Over-expression of miR-9 or knockdown of NF1 enhances the migration of T98G cells in a transwell migration assay. Representative photographs are shown on the top. Crystal violet staining was removed and quantified by absorbance measurement (OD570-630) (mean ± SD, n = 4). (F) NF1 knockdown abolished the effect of decreased miR-9 on the migration of T98G cells. Crystal violet staining was removed and quantified by absorbance measurement (OD570-630) (mean ± SD, n = 4). **, <i>P</i><0.01, ***, <i>P</i><0.001, two-tailed unpaired Student's t test. On the bottom is a representative western blot result showing that knocking down miR-9 increased the NF1 protein level, whereas simultaneous transfection of siNF1 abolished the increased NF1 protein level.</p

MiR-9 is highly expressed in glioma cell lines.

<p>(A) Schematic representation showing that miR-9 can be generated by the processing of any of the three primary transcripts encoded by three distinct genes (miR-9-1, miR-9-2 and miR-9-3). (B and C) The expression levels of mature miR-9 as well as pri-miR-9-1, pri-miR-9-2 and pri-miR-9-3 were determined in the human cervical carcinoma cell line (HeLa), normal human glial cell line (HEB) and four glioma cell lines (U87MG, T98G, A172 and U251) by quantitative RT-PCR (mean ± SD, n = 3). (D) Genomic DNA was extracted from the six cell lines (HeLa, HEB, U87MG, T98G, A172 and U251), and the gene copy numbers of miR-9-1, miR-9-2 and miR-9-3 were determined by quantitative real-time PCR (mean ± SD, n = 3). *, P<0.05, two-tailed unpaired Student's t test, relative to HEB and HeLa.</p

CREB regulates NF1 and knocking down CREB inhibits migration.

<p>(A) The migratory capacity of AD-shcreb or AD-shNC-infected U87MG, T98G and U251 cells were analyzed in a transwell migration assay (top). The bound crystal violet staining was released and quantified by measuring the OD570-630 (mean ± SD, n = 3) (bottom). (B) The mRNA and protein levels of NF1 were detected in control T98G and U251 cells or in cells with CREB knocked down by quantitative RT-PCR and western blotting, respectively. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001, two-tailed unpaired Student's t test. (C) T98G cells were transfected with miR-9 antagomirs or controls followed by infection of adenovirus-mediated shRNA for CREB (AD-shcreb) or AD-shNC, and the protein level of NF1 was detected by western blotting.</p

The effects of miR-9 knockdown on glioma cell growth, colony formation and migration.

<p>(A) MTT assays were performed to evaluate the effects of miR-9 knockdown on the growth and survival of glioma cells (U87MG, T98G and U251). Data are represented as the mean ± SD, n = 4. (B) Colony formation assays were utilized to test the effects of knocking down and over-expressing miR-9 on the colony formation ability of T98G and U251 cells. Data are represented as the mean ± SD, n = 4. (C) Transwell migration assays were employed to evaluate the effects of knocking down miR-9 and miR-23a (as a negative control) on the migration of glioma cells. On the top are representative photographs of transwell assays. The bound crystal violet staining was released with 33% glacial acetic acid and quantified by absorbance measurement (OD570-630) (mean ± SD, n = 4). *, <i>P</i><0.05, **, <i>P</i><0.01, two-tailed unpaired Student's t test.</p

MiR-9-1 is under CREB's control.

<p>(A) Location of putative CREs within the 5′ flanking regions of miR-9-1 and miR-9-2. MiR-9-1 is located in an intron of the gene c1orf61 (chromosome 1), whereas miR-9-2 is located in an exon of linc00461 (chromosome 5). Three pairs of primers (miR-9-1-a, miR-9-1-b and miR-9-2) were designed to detect the binding capacity of CREB to the predicted CREs of miR-9-1-a, miR-9-1-b and miR-9-2, respectively, by ChIP-qPCR assays. Both the 5′ flanking sequences (2 kb) and the pre-miRNA bodies of miR-9-1 and miR-9-2 were inserted upstream of the luciferase reporter (gray box shown by LUC). The arrows denote the positions of primers used for ChIP-qPCR. (B) ChIP-qPCR assays were performed in T98G and U251 cells to detect the binding capacity of CREB to the putative CREs of miR-9-1 and miR-9-2 (mean ± SD, n = 3). (C) In AD-shNC/AD-shcreb-infected T98G and U251 cells, ChIP-qPCR was performed to detect the binding capacity of CREB on CRE-miR-9-1-a (mean ± SD, n = 3). (D) CREB enhances the transcription of miR-9-1. The 5′ flanking sequences (−2 kb+miR-9-1; −2 kb, −570 bp; −560 bp+miR-9-1) without mutations or with a mutation of CRE-a (−569+miR-9-1, from TGACGGGC to TGGAGGGC) in miR-9-1 were inserted upstream of the luciferase cassette. The luciferase reporter constructs were co-transfected with CREB expression plasmids or control vectors and the normalized luciferase activity was determined (mean ± SD, n = 4). (E) The mRNA expression levels of CREB, pri-miR-9-1 and mature miR-9 were detected in T98G and U251 cells infected with AD-shcreb or AD-shNC by quantitative RT-PCR (mean ± SD, n = 3). *, <i>P</i><0.05, **, <i>P</i><0.01, two-tailed unpaired Student's t test.</p

Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines

<div><p>Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.</p></div

Identification of the 6 differential proteins in Huh7.5.1 wt and HepG2 cells detected by analysis of label-free, iTRAQ, qRT-PCR and western blot assays (Relative differential proteins: HepG2/Huh7.5.1 wt).

<p>Identification of the 6 differential proteins in Huh7.5.1 wt and HepG2 cells detected by analysis of label-free, iTRAQ, qRT-PCR and western blot assays (Relative differential proteins: HepG2/Huh7.5.1 wt).</p

The effects of six key differential proteins on HCV RNA levels.

<p>(A) qRT-PCR analysis of HCV RNA levels by transfection of siRNA or over-expression of plasmids. (B) Luciferase assay of Jc1-Luc HCVcc in Huh7.5.1 cells transfected with siRNA or over-expression plasmids. (C) QRT-PCR analysis of HCV RNA levels in knockdown of CTSB or over-expression of vimentin in HepG2 cells after transfection with HCV replicon RNA. Western blot results showed the protein level of knockdown and over-expression differential proteins, and β-actin as protein loading control. (D) Huh7.5.1 cells were transfected with siRNA or over-expression plasmids of CTSB, then 24 h later, cells were infected with HCVcc (multiplicity of infection 0.1). At 48 h post infection, immunoblotting was performed with an anti-HCV core antibody. Each bar represents the average of triplicate data points with the standard deviation represented as the error bar. *P<0.05, ** P<0.01 and ***P<0.001 versus negative control</p