Immunohistochemical staining of B7-H1 and its correlation with survival in colorectal cancer patients.

<p>(A-D) Representative immunohistochemiscal staining of positive and negative expression of in colorectal cancer or adjacent tissue (original magnification ×100). (A) B7-H1 positive tumour tissue, (B) B7-H1 positive adjacent tissue, (C) B7-H1 negative tumour tissue, and (D) B7-H1 negative adjacent tissue. Representative pictures were shown. (E) Association between the B7-H1 expression and cancer specific death in 143 colorectal cancer specimens.</p

Effect of B7-H1 knockdown on cell proliferation and apoptosis in HCT 116 cells.

<p>(A) MTT analysis to detect cell proliferation. Parental or HCT116 cells were transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in 96-well plates and cell proliferation was detected by MTT. Data were presented as means ± SD, *P<0.05 versus the si-scramble group. (B) Flow cytometric analysis to detect cell apoptosis. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were collected and stained with Annexin-V-FITC and PI before flow cytometric analysis. Data were presented as means ± SD, *P<0.05 versus the si-scramble group.</p

Effect of B7-H1 knockdown on cell migration and invasion in HCT116 cells.

<p>(A) Boyden chamber assay to detect cell migration. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in Boyden chambers without Matrigel-coated membrane, and after another 48 h, migrated cells were stained and counted under a microscope (magnification×10). Representative images were shown. (B) Number of migrated cells shown in A. Data was shown as means ± SD from five fields. *P<0.05 versus the si-scramble group. (C) Boyden chamber assay to detect cell invasion. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were seeded in modified Boyden chambers with Matrigel-coated membrane, and after another 24 h, invasive cells that moved through the Matrigel membrane were stained and counted under a microscope (magnification×10). Representative images were shown. (D) Number of invasive cells shown in C. Data was shown as means ± SD from five fields. *P<0.05 versus the si-scramble group.</p

Effective knockdown of B7-H1 by siRNA in HCT116 cells.

<p>(A) RT-qPCR analysis to show the B7-H1 mRNA level. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were harvested and RT-qPCR was performed; (B) Western blot analysis to detect the B7-H1 protein level. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were harvested and cell lysates were prepared and used for Western blot; (C) Flow cytometric analysis and mean channel fluorescence to show the B7-H1 expression on cell membrane. Parental or HCT116 cells transfected with scrambled siRNA or siRNA targeting B7-H1 for 48 h were harvested and cell surface staining was performed before flow cytometric analysis. Data were presented as means ± SD, *P<0.05 versus the si-scramble group.</p

B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells

<div><p>Background And Objective</p><p>The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells.</p> <p>Methods</p><p>Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells.</p> <p>Results</p><p>Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition.</p> <p>Conclusions</p><p>Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.</p> </div

Regulation of P21 and BIM by the miR-106b-93-25 cluster.

<p>(<b>a</b>) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3′UTR target sequence of miR-106b or miR-25 in the P21 or BIM gene were co-transfected into ECC-1 cells with either miRNA-negative control, miRNA mimics or empty pGL3 control vector (each n = 3). Luciferase activity was determined in the cell extracts after 24 h. In the presence of the wild-type P21 3′UTR, the miR-106b mimics significantly inhibited the luciferase activity compared with vector control. This inhibition was not observed with the mutant 3′UTR reporter construct (left). In the presence of the wild-type BCL2L11 3′UTR, the miR-25 significantly inhibited the luciferase activity compared with vector control. This inhibition was attenuated with the mutant 3′UTR reporter construct (right). (<b>b</b>) qRT-PCR analysis of P21 and BIM mRNA levels after the transfection of ECC-1 cells with miR-106b or miR-25 mimics. P21 mRNA was significantly decreased by miR-106b mimics while BIM mRNA was not significantly changed by miR-25 mimics. (<b>c</b>) Western blot analysis showing downregulated P21 and BIM proteins in ECC-1 and HEC-1A cells transfected with miR-106b and miR-25 mimics. (<b>d</b>) qRT-PCR and Western blot analysis showing upregulated P21 and BIM mRNA and protein level in ECC-1 and HEC-1A cells treated with TSA for 24 h. **, P<0.01; *, P<0.05, paired t-test.</p

TSA downregulated the miR-106b-93-25 cluster by downregulating MYC.

<p>(<b>a</b>) The mRNA and protein levels of MYC was downregulated in ECC-1 and HEC-1A cells treated with TSA for 24 h. (<b>b</b>) Depletion of MYC inhibited the expression of miR-106-93-25, as well as its host gene MCM7, and promoted the expression of P21 and BIM mRNA and protein expressions in ECC-1 and HEC-1A cells. (<b>c</b>) Overexpression of MYC upregulated expressions of miR-106b-93-25 and MCM7, and downregulated P21 and BIM mRNA expressions. (<b>d</b>) P21 mRNA and protein levels were upregulated in ECC-1 cells treated with TSA for 24 h, while it was partially neutralized by miR-106b (left). BIM mRNA was upregulated in ECC-1 cells treated with TSA for 24 h, while it was not neutralized by miR-25, but its protein level was neutralized in miR-25 overexpressing ECC-1 cells. **, P<0.01; *, P<0.05; ns, not significant; paired t-test.</p

TSA Suppresses miR-106b-93-25 Cluster Expression through Downregulation of MYC and Inhibits Proliferation and Induces Apoptosis in Human EMC

<div><p>Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the <em>miR-106b-93-25</em> cluster, together with its host gene <em>MCM7</em>, were obviously down-regulated in EMC cells. <em>p21</em> and <em>BIM</em>, which were identified as target genes of <em>miR-106b-93-25</em> cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified <em>MYC</em> as a regulator of <em>miR-106b-93-25</em> cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of <em>miR-106b-93-25</em> cluster and up-regulation of <em>p21</em> and <em>BIM</em>. More important, we found <em>miR-106b-93-25</em> cluster was up-regulated in clinical EMC samples in association with the overexpression of <em>MCM7</em> and <em>MYC</em> and the down-regulation of <em>p21</em> and <em>BIM</em>. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the <em>miR-106b-93-25</em> cluster and up-regulation of it's target genes <em>p21</em> and <em>BIM</em> via <em>MYC</em>.</p> </div

MYC activation of human <i>MCM7</i> promoter activity was dependent on the E-box sequence.

<p>ECC-1 cells were transfected to detect the human <i>MCM7</i> promoter activity. (<b>a</b>) Schematic of the core promoter region of the <i>MCM7</i> promoter. Location of the mutated sequence for the E box (AGTTTA) is indicated by the black box. (<b>b</b>) Luciferase activity of constructs of the <i>MCM7</i> promoter and its truncations. ECC-1 cells transfected with luciferase reporter constructs containing different 5′flanking segments of hMCM7 alone or co-transfected with pBABE-MYC. The numbers on the left side of the panel represent the relative position of each deletion. The luciferase activities were normalized to that of ECC-1 cells transfected with the pGL3(-) vector (control) was set to 1.0. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045133#s3" target="_blank">Results</a> are means ± S.D. All assays were repeated for at least 3 independent experiments. In each experiment, the individual data points were calculated as the means of triplicates. (<b>c</b>) Effect of mutated E-box site on activity of the <i>MCM7</i> promoter. The MCM7 (−185/+44)/luciferase reporter construct was co-transfection with its E-box site mutant and with or without the MYC expression plasmid. (<b>d</b>) ChIP assays were performed in ECC-1 cells using antibodies directed against MYC protein or an IgG control. The sequences of all primers for ChIP-PCR were based on the MYC-binding sites (−756 to −50 relative to the TSS) identified from TSS analysis. After immunoprecipitation, DNA was eluted and amplified by PCR using primers designed to amplify the minimal promoter region of MCM7. *, <i>P</i><0.05, paired t-test.</p

Screening for cell lines coexpressing both EPR-1 and Survivin.

<p>(A) The expressions of EPR-1 and Survivin in cell lines were analyzed by Western blotting using β-actin for standardization. (B) The expressions of EPR-1 and Survivin in HEK293 cell line were confirmed by RT-PCR using EPR-1 and Survivin specific cDNA primers.</p