<i>EZH2</i> and <i>CTNNB1</i> are downstream targets of miR-214 and both are upregulated in human HCC tissue samples.

<p>(A) Effect of miR-214 on <i>EZH2</i> and <i>CTNNB1</i> expression, as shown by a luciferase reporter assay. The data were normalized by the ratio of Firefly and Renilla luciferase activities measured at 48 h post-transfection. The bar graph showed the mean ± SD in three independent transfection experiments. *<i>P</i><0.05. (B) Western blotting analysis of EZH2, β-catenin, and E-cadherin expression in P-miR-control- and P-miR-214-transfected SK-HEP-1 cells. (C-E) Validation of the expression of <i>EZH2</i> (C), <i>CTNNB1</i> (D) and CDH1 (E) in 20 paired human HCC tissue samples and 10 samples of histologically normal liver tissues were validated by qRT-PCR.</p

Downregulation of miR-214 is associated with the early recurrence of human HCC.

<p>The level of expression of miR-214 was analyzed by qRT-PCR and normalized to U6. (A) Expression of miR-214 in 20 paired of HCC tumor tissues was significantly lower compared to 20 matched histologically normal tissues as well as 10 histologically normal liver tissues from colorectal cancer patients with liver metastases (<i>P</i><0.01). (B) Low miR-214 expression was associated with early recurrent HCC disease when studied in an independent cohort of 50 HCC samples. The average expression level of miR-214, analyzed by qRT-PCR, was lower in HCC patients with early recurrence (≤2 years) (n = 29) than patients with no recurrence in the same time period (n = 21). (C) The expression of miR-214 was associated with survival in patients with HCC. The median expression value obtained for miR-214 of the 50 samples studied was employed as the cut-off point. Fisher’s exact test and Kaplan-Meier analysis were used to demonstrate that high miR-214 expression was significantly associated with early recurrent disease and a relative poorer disease-free survival rate.</p

Paper-Based Microfluidic Electrochemical Immunodevice Integrated with Nanobioprobes onto Graphene Film for Ultrasensitive Multiplexed Detection of Cancer Biomarkers

To the best of our knowledge, this was the first report on the integration of a signal amplification strategy into a microfluidic paper-based electrochemical immunodevice for the multiplexed measurement of cancer biomarkers. Signal amplification was achieved through the use of graphene to modify the immunodevice surface to accelerate the electron transfer and the use of silica nanoparticles as a tracing tag to label the signal antibodies. Accurate, rapid, simple, and inexpensive point-of-care electrochemical immunoassays were demonstrated using a photoresist-patterned microfluidic paper-based analytical device (μPAD). Using the horseradish peroxidase (HRP)–<i>O</i>-phenylenediamine–H₂O₂ electrochemical detection system, the potential clinical applicability of this immunodevice was demonstrated through its ability to identify four candidate cancer biomarkers in serum samples from cancer patients. The novel signal-amplified strategy proposed in this report greatly enhanced the sensitivity of the detection of cancer biomarkers. In addition, the electrochemical immunodevice exhibited good stability, reproducibility, and accuracy and thus had potential applications in clinical diagnostics

Roles of <i>EZH2</i>, <i>CTNNB1</i> and <i>CDH1</i> on the growth and invasion of HCC cells.

<p>(A) Silencing of <i>EZH2</i> significantly inhibited the growth and significant decreased the ability of SK-HEP-1 cells to invade. (i) Western blots showing the silencing of <i>EZH2</i> by pLVTHM-shEZH2. (ii) The effect of silencing <i>EZH2</i> on cell growth at different time points. (iii) The inhibitory effect of silencing <i>EZH2</i> on cell invasion. (B) Silencing of <i>CTNNB1</i> significantly inhibited the growth of SK-HEP-1 cells. (i) Western blots showing the reduction of β-catenin after transfection with shRNA-β-catenin. (ii) Effects of silencing β-catenin on cell growth at different time points. (C) Over-expression <i>CDH1</i> significantly inhibited the ability of SK-HEP-1 cells to invade. (i) Western blots showing the overexpression of CDH1 by pcDNA3.1-CDH1 plasmid transfection. (ii) The inhibitory effects of overexpressing <i>CDH1</i> on SK-HEP-1 cell invasion. (D) Western blots showing the silencing of EZH2 significantly decreased the expression of CTNNB1 and induced CDH1 expression.</p

Expression of <i>EZH2, CTNNB1,</i> and <i>CDH1</i>, downstream targets of miR-214, is associated with early recurrent disease and survival of patients with HCC.

<p>The level of expression of <i>EZH2</i>, <i>CTNNB1,</i> and <i>CDH1</i> in the 50 HCC samples described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044206#pone-0044206-g001" target="_blank">Figure 1</a> was analyzed by qRT-PCR and normalized by <i>HPRT1</i>. (A–C) The average expression level of <i>EZH2</i> and <i>CTNNB1</i> was significantly higher in samples from HCC patients with early recurrence (≤2 years) (n = 29) than patients with no recurrence over the same period (n = 21). In comparison, the average expression level of <i>CDH1</i> was significantly lower in in samples from HCC patients with early recurrence (C). (D–F) The median expression value obtained for <i>EZH2</i>, <i>CTNNB1</i>, and <i>CDH1</i> of the 50 samples studied was employed as the cut-off point and employed independently in Kaplan-Meier analysis to study disease-free survival rate. (G) Combining the expression of <i>EZH2</i>, <i>CTNNB1</i> and <i>CDH1</i> to predict tumor recurrence and disease-free survival rate using Kaplan-Meier analysis.</p

Re-expression of miR-214 significantly inhibits cell growth in vitro and tumorigenic properties of HCC cells.

<p>(A and B) Growth of HLE (A) and SK-HEP-1 (B) cells <i>in vitro</i> at different time points following the re-expression of miR-214 mediated by transfection with P-miR-214, *<i>P</i><0.05. (C) Stable expression of miR-214 inhibited the anchorage-independent growth of SK-HEP-1 cells in soft agar. The upper section shows images of colony formation. The bar graph below the figures showed the mean number of colonies formed (± SD) and counted under the microscope in three randomly selected fields (magnification, ×40). *<i>P</i><0.05. (D) Stable expression of miR-214 inhibited tumorigenicity of SK-HEP-1 cells. The upper section showed images of the tumors obtained in mice at the end of the eighth week. The bar graph indicated the average of overall tumor volume measured each week (n = 6 mice per group).</p

Hematoxylin and Eosin stained dorso lateral prostate of TRAMP mice.

<p><b>A</b>, H&E stained section of DLP of Group 2 control (i) and UA-treated (ii) TRAMP prostate. Arrow indicates LG-PIN, notched right arrow indicates HG-PIN, and block right arrow indicates WDC. (iii) Bar chart shows the incidence of LG-PIN, HG-PIN, WDC, MDC and PDC in DLP of control and Group 2 mice. Reductions in PIN, WDC and MDC were observed after UA treatment. <b>B</b>, H&E stained section of DLP of Group 3 control (i) and UA-treated (ii) TRAMP prostate. As described in A; Arrow head indicates MDC and chevron indicates PDC. <b>C</b>, Western blot of pSTAT3, pAKT, pIKKα/β, and p65 proteins in DLP in Groups 1, 2 and 3 mice. DLP lysates was prepared and immunoblotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032476#s4" target="_blank">Materials and Methods</a>. Equal protein loading for pAKT and pIKKα/β was determined by stripping and probing for total AKT and IKKα, p65 and STAT3 blots were stripped and reprobed for β-actin to determine equal protein loading. The band density was quantitated using Image J software. NT = non-TRAMP-mice; T = TRAMP controls; T+UA = UA-treated TRAMP-mice.</p

Mass spectrometric analysis of UA in serum.

<p><b>A,</b> (i) Ion chromatograms of serum extracts from mice that had not received UA (blank). (ii) Full scan spectrum showing the m/z peak with a molecular mass of 469 corresponds to internal standard (IS) glycyrrhetinic acid. (iii) Full scan spectrum showing the m/z peak with a molecular mass of standard UA at 455 and IS at 469 and (iv) Full scan spectrum of sample, retention time was 1.7 and 1.96 min respectively for both IS and sample. The chromatograms are representative of 3 independent experiments. <b>B,</b> Serum concentrations of UA was calculated using Analyst software 1.4.2 from the linear regression equation of the peak area ratio against the concentration of the calibration curve. Error bars represent SEM.</p

Enzyme linked immunosorbent assay.

<p><b>A.</b> Suppression of serum TNF-α (i) and IL-6 (ii) by UA-treatment. Groups 1–3 mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032476#s4" target="_blank">Materials and Methods</a>. Sandwich ELISA assay was performed as per manufacturers' instruction (R&D systems, USA) to determine the levels of TNF-α and IL-6. *Statistical significance (P<0.05). <b>B,</b> Immunohistochemical analysis of cyclin D1, caspase 3 and COX-2 in tumor tissues in Group 3 TRAMP mice. DLP tumor tissues, embedded in paraffin blocks, were cut into 5 µM tissue sections and probed for cyclin D1, caspase 3 and COX-2 immunoreactivities as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032476#s4" target="_blank">Materials and Methods</a>. UA-treatment (1% w/w) decreased the expression of cyclin D1 and COX-2 but increased caspase-3 expression when compared to controls. Images were taken using a Olympus BX51 microscope (magnification, 20×). Positive cells (brown) were quantitated using The Image-Pro plus v6.3 software package (Media Cybernetics, Inc.).</p

UA inhibits tumor growth in Group 3 TRAMP mice.

<p><b>A,</b> Mice were euthanized at 36 weeks and the tumor volume was measured as described in Material and Methods. Error bars represent SEM. * Statistical significance (P<0.05). <b>B,</b> Body weight of Groups 1–3 TRAMP mice and their controls. <b>C,</b> The effect of UA-treatment on the long term survival of TRAMP mice. Kaplan-Meier survival analysis was performed for the control and UA-treated TRAMP mice. Median survival time was 55 weeks for control (n = 5). Median survival time for group 1, group 2, group 3 and group 4 was 75, 75, 68, and 72 weeks, respectively. Overall significance was determined using log-rank (Mantel-Cox) test. UA enriched diet significantly prolonged survival.</p