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MiR200-upregulated Vasohibin 2 promotes the malignant transformation of tumors by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.

Xue X, Zhang Y, Zhi Q, Tu M, Xu Y, Sun J, Wei J, Lu Z, Miao Y, Gao W - Cell Commun. Signal (2014)

Bottom Line: VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT.Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway.VASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, the First Affiliated Hospital with Nanjing Medical University, 300# Guangzhou Road, Nanjing, 210029, China. xfxue@suda.edu.cn.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) typically relies on tumor transformation and angiogenesis for its malignant behavior, including growth and metastasis. Previously, we reported that Vasohibin2 (VASH2) is preferentially expressed in hepatocellular carcinoma (HCC) tumor tissues and promotes angiogenesis. Here, we further investigated the role of VASH2 in HCC tumor progression.

Results: Bioinformatics analyses and luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200a/b/c. We then used HepG2 and Hep3B cells, two representative hepatic cancer cell lines, to examine the role of VASH2 in tumors. VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT. Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway.

Conclusion: VASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo. These results indicated that VASH2 expression in HCC cells promotes the malignant transformation of tumors by inducing EMT.

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The effect of VASH2 expression on cell invasion. (A) Transwell invasion assay in HepG2 cells with different levels of VASH2 expression. (B) Cell counts in the Transwell invasion assay. qPCR result for CXCR4 (C) and MMP2 (D) in HepG2 cells with different levels of VASH2 expression. (E) Western blot of CXCR4 and MMP2 in HepG2 cells with different levels of VASH2 expression. (F) Promoter luciferase reporter assay for regulation of VASH2 on CXCR4 and MMP2. (*represents p < 0.05 when compared to the control group).
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Fig4: The effect of VASH2 expression on cell invasion. (A) Transwell invasion assay in HepG2 cells with different levels of VASH2 expression. (B) Cell counts in the Transwell invasion assay. qPCR result for CXCR4 (C) and MMP2 (D) in HepG2 cells with different levels of VASH2 expression. (E) Western blot of CXCR4 and MMP2 in HepG2 cells with different levels of VASH2 expression. (F) Promoter luciferase reporter assay for regulation of VASH2 on CXCR4 and MMP2. (*represents p < 0.05 when compared to the control group).

Mentions: According to previous reports, EMT may promote malignant transformation by increasing the invasion ability and proportion of stem cells in tumors. We conducted Transwell invasion assays to investigate whether VASH2 affected the invasion ability of HepG2 and Hep3B cells. Compared to the control cells, the results (Figure 4A-B) showed that VASH2 overexpression increased the invasive ability of HepG2 cells and that VASH2 interference inhibited the invasive ability of HepG2 cells. VASH2 overexpression also promoted the invasive ability of Hep3B cells (Additional file 2: Figure S2 A-B). Taken together, these data indicated that VASH2 contributes to the invasive ability of hepatic cancer cells. Furthermore, we selectively detected invasion-related genes, such as MMP2 and CXCR4, by qPCR and western blot analyses (Figure 4C-E and Additional file 2: Figure S2C-E). VASH2 overexpression increased the expression levels of MMP2 and CXCR4 in HepG2 and Hep3B cells, but VASH2 knockdown downregulated MMP2 and CXCR4 expression levels. Further promoter luciferase reporter gene assay was used to explain the mechanisms of VASH2 in regulating MMP2 and CXCR4. The results (Figure 4F) suggested VASH2 could definitely upregulate MMP2 and CXCR4 expression. These data suggested that VASH2 might promote the invasive ability by positively regulating pro-invasive genes, such as MMP2 and CXCR4, in hepatic cancer cells.Figure 4


MiR200-upregulated Vasohibin 2 promotes the malignant transformation of tumors by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.

Xue X, Zhang Y, Zhi Q, Tu M, Xu Y, Sun J, Wei J, Lu Z, Miao Y, Gao W - Cell Commun. Signal (2014)

The effect of VASH2 expression on cell invasion. (A) Transwell invasion assay in HepG2 cells with different levels of VASH2 expression. (B) Cell counts in the Transwell invasion assay. qPCR result for CXCR4 (C) and MMP2 (D) in HepG2 cells with different levels of VASH2 expression. (E) Western blot of CXCR4 and MMP2 in HepG2 cells with different levels of VASH2 expression. (F) Promoter luciferase reporter assay for regulation of VASH2 on CXCR4 and MMP2. (*represents p < 0.05 when compared to the control group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: The effect of VASH2 expression on cell invasion. (A) Transwell invasion assay in HepG2 cells with different levels of VASH2 expression. (B) Cell counts in the Transwell invasion assay. qPCR result for CXCR4 (C) and MMP2 (D) in HepG2 cells with different levels of VASH2 expression. (E) Western blot of CXCR4 and MMP2 in HepG2 cells with different levels of VASH2 expression. (F) Promoter luciferase reporter assay for regulation of VASH2 on CXCR4 and MMP2. (*represents p < 0.05 when compared to the control group).
Mentions: According to previous reports, EMT may promote malignant transformation by increasing the invasion ability and proportion of stem cells in tumors. We conducted Transwell invasion assays to investigate whether VASH2 affected the invasion ability of HepG2 and Hep3B cells. Compared to the control cells, the results (Figure 4A-B) showed that VASH2 overexpression increased the invasive ability of HepG2 cells and that VASH2 interference inhibited the invasive ability of HepG2 cells. VASH2 overexpression also promoted the invasive ability of Hep3B cells (Additional file 2: Figure S2 A-B). Taken together, these data indicated that VASH2 contributes to the invasive ability of hepatic cancer cells. Furthermore, we selectively detected invasion-related genes, such as MMP2 and CXCR4, by qPCR and western blot analyses (Figure 4C-E and Additional file 2: Figure S2C-E). VASH2 overexpression increased the expression levels of MMP2 and CXCR4 in HepG2 and Hep3B cells, but VASH2 knockdown downregulated MMP2 and CXCR4 expression levels. Further promoter luciferase reporter gene assay was used to explain the mechanisms of VASH2 in regulating MMP2 and CXCR4. The results (Figure 4F) suggested VASH2 could definitely upregulate MMP2 and CXCR4 expression. These data suggested that VASH2 might promote the invasive ability by positively regulating pro-invasive genes, such as MMP2 and CXCR4, in hepatic cancer cells.Figure 4

Bottom Line: VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT.Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway.VASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, the First Affiliated Hospital with Nanjing Medical University, 300# Guangzhou Road, Nanjing, 210029, China. xfxue@suda.edu.cn.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) typically relies on tumor transformation and angiogenesis for its malignant behavior, including growth and metastasis. Previously, we reported that Vasohibin2 (VASH2) is preferentially expressed in hepatocellular carcinoma (HCC) tumor tissues and promotes angiogenesis. Here, we further investigated the role of VASH2 in HCC tumor progression.

Results: Bioinformatics analyses and luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200a/b/c. We then used HepG2 and Hep3B cells, two representative hepatic cancer cell lines, to examine the role of VASH2 in tumors. VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT. Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway.

Conclusion: VASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo. These results indicated that VASH2 expression in HCC cells promotes the malignant transformation of tumors by inducing EMT.

Show MeSH
Related in: MedlinePlus