Additional file 1: Figure S1. of Overexpression of CTNND1 in hepatocellular carcinoma promotes carcinous characters through activation of Wnt/β-catenin signaling

The expression of CTNND1 was analysised with TCGA data. (JPG 115 kb

Silencing of DLGAP5 by siRNA Significantly Inhibits the Proliferation and Invasion of Hepatocellular Carcinoma Cells

<div><p>Background</p><p>The dysregulation of oncogenes and tumor suppressor genes plays an important role in many cancers, including hepatocellular carcinoma (HCC), which is one of the most common cancers in the world. In a previous microarray experiment, we found that DLGAP5 is overexpressed in HCCs. However, whether the up-regulation of DLGAP5 contributes to hepatocarcinogenesis remains unclear. </p> <p>Methodology/Principal Findings</p><p>In this study, we showed that DLGAP5 was significantly up-regulated in 76.4% (168 of 220) of the analyzed HCC specimens when compared with adjacent liver tissue. DLGAP5 overexpression was evident in 25% (22 of 88) of the HCC specimens without AFP expression, suggesting that DLGAP5 may be a novel biomarker for HCC pathogenesis. The silencing of DLGAP5 gene expression by RNA interference significantly suppressed cell growth, migration and colony formation in vitro. The expression level of DLGAP5 was also found to be related to the methylation level of its promoter in the HCC specimens. </p> <p>Conclusions/Significance</p><p>Taken together, these data suggest that the expression of DLGAP5 is regulated by methylation and that the up-regulation of DLGAP5 contributes to HCC tumorigenesis by promoting cell proliferation.</p> </div

mRNA expression pattern of DLGAP5 in HCC specimens.

<p>(A) Representative results of the semi-quantitative RT-PCR analysis of DLGAP5 in 20 pairs of HCC (C) and adjacent non-cancerous liver tissues (N). β-Actin was used as an internal control. Each PCR was performed for 35 cycles, and the PCR products were visualized by electrophoresis on 2% agarose gels. (B) Real-time RT-PCR analysis of DLGAP5 in 220 paired HCC and adjacent non-cancerous liver tissues. For each sample, the relative mRNA level of DLGAP5 was normalized to the level of β-actin. The line within each box represents the median –ΔCt value; the upper and lower edges of each box represent the 75th and 25th percentile, respectively; the upper and lower bars indicate the highest and lowest values determined, respectively. (C) The distribution of the DLGAP5 and AFP expression levels in the 220 HCC specimens. The numbers indicate the percentages of DLGAP5- and/or AFP-positive HCC specimens, as detected by real-time RT-PCR. (D)/(E) Disease-free survival rate (D) and Overall survival rate (E) of patients with HCC. High expression of DLGAP5 mRNA was significantly associated with worse prognosis (<i>P</i>=0.010). DLGAP5 high: 2^−ΔΔCT>1 (or –ΔΔCT>0); DLGAP5 low: 2^−ΔΔCT≤1 (or −ΔΔCT≤0). </p

Effect of DLGAP5 silencing on HCC cell growth.

<p>(A and B) western blot confirmation of DLGAP5 knockdown in SMMC-7721 (A) and HepG2 (B) cells via transient transfection with siRNA2. siRNA-NC was used as the control. (C and D) The growth curves of the SMMC-7721 (C) and HepG2 (D) cells after DLGAP5 knockdown by siRNA2 were plotted based on the CCK-8 assay. siRNA-NC served as a control. The experiments were repeated at least three times, and the data points represent the average values of triplicate wells, with standard deviations (SDs) included for each mean value.</p

Effect of DLGAP5 on HCC cell migration, invasion and adhesive ratio.

<p>(A and B) Wound closure of SMMC-7721 (A) and HepG2 (B) cells that were transfected with siRNA2 in a wound-healing experiment, with siRNA-NC serving as a control. (C and D) Invasion of SMMC-7721 (C) and HepG2 (D) cells that were transfected with siRNA2 in a Matrigel assay, with siRNA-NC serving as a control. The cell numbers represent the mean values per field (from at least five fields) from three independent experiments (right panel) (mean ± SD). (E and F) Effect of DLGAP5 gene on SMMC-7721 and HepG2 cell adhesive ratio using RNAi.</p

Protein expression pattern of DLGAP5 in HCC specimens.

<p>(A) Representative immunohistochemical DLGAP5 staining of a HCC specimen and its corresponding non-cancerous tissue from a tissue array containing 96 pairs of HCC specimens. The nuclei were counterstained with hematoxylin. Original magnification: The left (×200) and right side (×400) (normal, non-HCC and HCC). (B) Statistical analysis was performed using the chi-square test to compare the relative levels of DLGAP5 between HCC with PVTT and HCC without PVTT (<i>P</i>=0.037).</p

Additional file 5: Figure S5. of HOXA7 plays a critical role in metastasis of liver cancer associated with activation of Snail

HOXA7 promotes expression levels of EMT markers. (A) Western blot analysis of EMT markers in liver cancer cells with silent expression of HOXA7. (B) Western blot analysis of EMT markers in liver cancer cells with overexpression of HOXA7. (C) qRT-PCR analysis of EMT markers in liver cancer cells with silent expression of HOXA7. (D) qRT-PCR analysis of EMT markers in liver cancer cells with overexpression of HOXA7. P < 0.01 in panel C-F based on the Student t test. Error bars, SD. The each number of replicates is 3. (JPG 4138 kb

Additional file 1: Figure S1. of HOXA7 plays a critical role in metastasis of liver cancer associated with activation of Snail

Ectopic HOXA7 expression was related with metastasis of liver cancer. (A) Analysis of HOXA7 levels in noncancerous and tumorous tissues. (B) Analysis of HOXA7 levels in non-metastatic and metastatic liver cancer tissues. P < 0.01 in panel A and B based on the Student t test. Error bars, SD. The each number of replicates is 3. (JPG 976 kb

Additional file 4: Figure S4. of HOXA7 plays a critical role in metastasis of liver cancer associated with activation of Snail

HOXA7 promotes cell proliferation and tumor growth of liver cancer cell. (A) MTT assay of liver cancer cells with silent expression of HOXA7. (B) MTT assay of liver cancer cells with overexpression of HOXA7. (C) Tumor formation assay of liver cancer cells with silent expression of HOXA7. (D) Tumor formation assay of liver cancer cells with overexpression of HOXA7. **P < 0.01 based on the Student t test. Error bars, SD. The each number of replicates is 3. (JPG 3241 kb