Additional file 2: Figure S2. of M2-polarized tumor-associated macrophages facilitated migration and epithelial-mesenchymal transition of HCC cells via the TLR4/STAT3 signaling pathway

Levels of the p-ERK, p-Ikkα/β, total STAT3, ERK, Ikkα, and Ikkβ proteins were detected using western blotting. (A) Levels of the p-STAT3 and STAT3 proteins in the control, LPS, and M2-CM + LPS groups. (B) Levels of the p-ERK and ERK proteins in the control, M2-CM + LPS, and M2-CM groups. (C) Levels of the p-Ikkα/β, Ikkα, and Ikkβ proteins in the control, M2-CM + LPS, and M2-CM groups. (TIFF 601 kb

Goosecoid Promotes the Metastasis of Hepatocellular Carcinoma by Modulating the Epithelial-Mesenchymal Transition

<div><p>The homeobox gene, <i>goosecoid</i> (<i>GSC</i>), is a transcription factor that participates in cell migration during embryonic development. Because cell migration during development has characteristics similar to cell invasion during metastasis, we evaluated the potential role of GSC in the metastasis of hepatocellular carcinoma (HCC). GSC expression in HCC cell lines and tissues was evaluated, and its effects on the migration potential of HCC cells were determined by GSC knock-down and overexpression methods. In addition, the prognostic role of GSC expression in the metastasis of cancer cells in HCC patients was determined. Our data showed that GSC was highly expressed in several HCC cell lines, particularly in a highly metastatic HCC cell line. Overexpression of GSC promoted cell migration and invasion of HCC cells <i>in vitro</i>. Gain-of-function induced the epithelial-mesenchymal transition but not collective cell migration, whereas loss-of-function induced the reverse change. High-level expression of GSC correlated closely with poor survival and lung metastasis in HCC patients; lung metastases showed more upregulated GSC expression than the primary tumor. We conclude that GSC promotes metastasis of HCC potentially through initiating the epithelial-mesenchymal transition. GSC is also a prognostic factor for poor survival and metastasis of HCC, which suggests its potential as a therapeutic target for metastatic HCC.</p></div

GSC expression in HCC tumor tissues.

<p>Immunohistochemistry analysis was performed based on tissue microarray. (A) Top-row shows the staining of GSC from strong to low. Bottom-row shows the controls to confirm the specificity of the GSC antibody. Colon tissue staining was used as the positive control. (B) Six tumor tissues in lung metastases from HCC showed the highly expressed GSC. Comparison of two paired tissues (case 5 and case 6) from lung metastatic cancer and primary foci showed the upregulated GSC expression in lung tissues. (200× original magnification, size bar: 100 µm).</p

Effects of GSC overexpression on migration of HCC.

<p>Hep3B-GSC cells were infected by <i>GSC</i> lentivirus and were selected with puromycin. (A) Wound-healing assay shows that Hep3B-GSC cells migrated faster than normal Hep3B or Hep3B cells without treatment after 24 h and 48 h. The cleared area of Hep3B-GSC was less than the controls. (50× original magnification, size bar: 100 µm) Results were analyzed with the Student <i>t</i> test. (B) Giemsa staining indicated the number of Hep3B-GSC cells that migrated through Matrigel was higher than Hep3B cells without treatment or treated with negative control (100× original magnification, size bar: 100 µm). The data are presented as the mean ± SD of at least three independent experiments. Results were analyzed using Student's <i>t</i> test.</p

GSC expression in HCC cell lines.

<p>(A) Western blot indicates that GSC is abnormally expressed in HCC cell lines, whereas it is not detectable in a normal hepatic cell line. (B) Western blot shows that GSC is strongly expressed in the highly metastatic HCC cell line versus the cell line with low metastatic potential. (C) <i>GSC</i> was overexpressed in HCC cells through lentivirus infection. A stable cell line was selected with 3 mg/mL puromycin. Quantitative real-time RT-PCR shows the prominent overexpression of <i>GSC</i> in Hep3B cells, which was confirmed by western blot. The data are presented as the mean ± SD of at least three independent experiments.</p

GSC expression correlates with poor survival and lung metastasis in HCC.

<p>Immunohistochemistry assay was based on tissue microarray from 112 tumor tissues. (A) Patients with highly expressed GSC had a significantly worse 5-year survival (Kaplan-Meier, log-rank test). (B) Patients with highly expressed GSC had showed early relapse and poor relapse-free survival when compared to other subgroups (Kaplan-Meier, log-rank test). (C) Groups with highly expressed GSC had lower extra-hepatic metastasis-free survival than the group with lower expressed GSC (Kaplan-Meier, log-rank test). (D) Receiver Operating Characteristic (ROC) curve analysis of GSC expression for occurrence of lung metastasis showed areas under the curve (AUC) of >0.5 (<i>P</i><0.05).</p

Univariate and multivariate analysis of factors associated with survival and lung metastasis in HCC patients.

<p>Univariate and multivariate analysis of factors associated with survival and lung metastasis in HCC patients.</p

Additional file 1 of circRanGAP1/miR-27b-3p/NRAS Axis may promote the progression of hepatocellular Carcinoma

Additional file 1: Figure S1. Characterization of circRanGAP1 in HCC. Figure S2. circRanGAP1 expression in HCC cells. Figure S3. CircRanGAP1 acted as a sponge of miR-27b-3p in HCC cells. Figure S4. miR-27b-3p plays tumor-suppressive roles in HCC. Figure S5. Figure S6. The effect of NRAS in cirRanGAP1 knockdown cells. Figure S7. Immune infiltration analysis of miR-27b-3p and NRAS. Figure S8. The effects of circRanGAP1 on immunocytes infiltration. Table S1. Sequences of Primers used for qRT-PCR. Table S2. List of Primary Antibodies Used in the Study. Table S3. Target sequences of hsa_circ_0063513 shRNAs. Table S4. circ_0063513 and miR-27b-3p FISH probe sequences. Table S5. The potential targets of RanGAP1

Expression patterns of eight invasion/metastasis-associated genes (MMP2, MMP7, MMP9, CD44, SPP1, CXCR4, CXCL12, and CDH1).

<p>Time course analysis showed remarkable, dynamic alterations in invasion/metastasis gene expression during the development of the HCC invasion model.</p

Eight typical expression patterns including 201 differential proteins during <i>in vitro</i> HCC invasion.

<p>Proteins obtained from the co-culture spheroids at different time points are labeled as Day 0, Day 5, Day 10, and Day 15.</p