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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 apoptosis and SP cell proportion. (A) Flow cytometry to determine the apoptosis rate in HepG2 cells with different levels of VASH2 expression after CDDP treatment. Line graph of the apoptosis rate (*represents p < 0.05 when compared to HepG2-wt). qPCR of ABCC1 (B) and Bcl-2 (C) in HepG2 cells with different levels of VASH2 expression. (D) Western blot of ABCC1 and Bcl-2 in HepG2 cells with different levels of VASH2 expression. (E) Promoter luciferase reporter assay for regulation of VASH2 on ABCC1 and BCL2. (F) SP cell proportion of HepG2 cells with different levels of VASH2 expression. (G) SP cell proportion of Hep3B cells with different levels of VASH2 expression. (*represents p < 0.05 when compared to the control group).
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Fig5: The effect of VASH2 expression on cell apoptosis and SP cell proportion. (A) Flow cytometry to determine the apoptosis rate in HepG2 cells with different levels of VASH2 expression after CDDP treatment. Line graph of the apoptosis rate (*represents p < 0.05 when compared to HepG2-wt). qPCR of ABCC1 (B) and Bcl-2 (C) in HepG2 cells with different levels of VASH2 expression. (D) Western blot of ABCC1 and Bcl-2 in HepG2 cells with different levels of VASH2 expression. (E) Promoter luciferase reporter assay for regulation of VASH2 on ABCC1 and BCL2. (F) SP cell proportion of HepG2 cells with different levels of VASH2 expression. (G) SP cell proportion of Hep3B cells with different levels of VASH2 expression. (*represents p < 0.05 when compared to the control group).

Mentions: We next assessed the effect of VASH2 on the anti-apoptotic ability of hepatic cancer cells. The results (Figure 5A) showed that VASH2 overexpression increased the resistance of HepG2 cells to CDDP treatment, and VASH2 silencing attenuated the anti-apoptotic ability of HepG2 cells upon CDDP treatment. The differences between the groups were significant at a CDDP concentration of 2.5 μg/mL (p < 0.05). Furthermore, the expression of anti-apoptotic genes, such as ABCC1 and BCL-2, was measured by qPCR and western blot analyses (Figure 5B-D). We further did the promoter luciferase reporter gene assay to explain the mechanisms of VASH2 in regulating ABCC1 and BCL-2. The results (Figure 5E) showed VASH2, as a promoting factor, increased ABCC1 and BCL-2 expression. These results suggested that VASH2 increased the anti-apoptotic ability of hepatic cancer cells by upregulating ABCC1 and BCL-2. Besides, we added Hoechst33342 staining to reinforce the anti-apoptosis of VASH2 gene. The result (Additional file 3: Figure S3A) showed that VASH2 overexpression had a more capable resistance to CDDP-induced apoptosis than VASH2 knockdown, which was in consistent with the result of flow cytometry. Furthermore, we tested the relative gene of pro-apoptosis such as Bax, Caspase-3, 6, 9. The result (Additional file 3: Figure S3B) showed VASH2 overexpression gave rise to decrease of pro-apoptotic genes such as Bax, Caspase-3, 6, 9, which elucidated the possible mechanisms of VASH2-induced resistance to apoptosis. Besides, we also measured the relative gene of stem cell property like ALDH1A1, Sox2, Oct4, Nanog. The results (Additional file 3: Figure S3C-D) showed VASH2 upregulated the stem cell marker such as ALDH1A1, Sox2, Oct4, Nanog, which reinforced VASH2 promotion of stem cell proportion.Figure 5


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 apoptosis and SP cell proportion. (A) Flow cytometry to determine the apoptosis rate in HepG2 cells with different levels of VASH2 expression after CDDP treatment. Line graph of the apoptosis rate (*represents p < 0.05 when compared to HepG2-wt). qPCR of ABCC1 (B) and Bcl-2 (C) in HepG2 cells with different levels of VASH2 expression. (D) Western blot of ABCC1 and Bcl-2 in HepG2 cells with different levels of VASH2 expression. (E) Promoter luciferase reporter assay for regulation of VASH2 on ABCC1 and BCL2. (F) SP cell proportion of HepG2 cells with different levels of VASH2 expression. (G) SP cell proportion of Hep3B cells with different levels of VASH2 expression. (*represents p < 0.05 when compared to the control group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: The effect of VASH2 expression on cell apoptosis and SP cell proportion. (A) Flow cytometry to determine the apoptosis rate in HepG2 cells with different levels of VASH2 expression after CDDP treatment. Line graph of the apoptosis rate (*represents p < 0.05 when compared to HepG2-wt). qPCR of ABCC1 (B) and Bcl-2 (C) in HepG2 cells with different levels of VASH2 expression. (D) Western blot of ABCC1 and Bcl-2 in HepG2 cells with different levels of VASH2 expression. (E) Promoter luciferase reporter assay for regulation of VASH2 on ABCC1 and BCL2. (F) SP cell proportion of HepG2 cells with different levels of VASH2 expression. (G) SP cell proportion of Hep3B cells with different levels of VASH2 expression. (*represents p < 0.05 when compared to the control group).
Mentions: We next assessed the effect of VASH2 on the anti-apoptotic ability of hepatic cancer cells. The results (Figure 5A) showed that VASH2 overexpression increased the resistance of HepG2 cells to CDDP treatment, and VASH2 silencing attenuated the anti-apoptotic ability of HepG2 cells upon CDDP treatment. The differences between the groups were significant at a CDDP concentration of 2.5 μg/mL (p < 0.05). Furthermore, the expression of anti-apoptotic genes, such as ABCC1 and BCL-2, was measured by qPCR and western blot analyses (Figure 5B-D). We further did the promoter luciferase reporter gene assay to explain the mechanisms of VASH2 in regulating ABCC1 and BCL-2. The results (Figure 5E) showed VASH2, as a promoting factor, increased ABCC1 and BCL-2 expression. These results suggested that VASH2 increased the anti-apoptotic ability of hepatic cancer cells by upregulating ABCC1 and BCL-2. Besides, we added Hoechst33342 staining to reinforce the anti-apoptosis of VASH2 gene. The result (Additional file 3: Figure S3A) showed that VASH2 overexpression had a more capable resistance to CDDP-induced apoptosis than VASH2 knockdown, which was in consistent with the result of flow cytometry. Furthermore, we tested the relative gene of pro-apoptosis such as Bax, Caspase-3, 6, 9. The result (Additional file 3: Figure S3B) showed VASH2 overexpression gave rise to decrease of pro-apoptotic genes such as Bax, Caspase-3, 6, 9, which elucidated the possible mechanisms of VASH2-induced resistance to apoptosis. Besides, we also measured the relative gene of stem cell property like ALDH1A1, Sox2, Oct4, Nanog. The results (Additional file 3: Figure S3C-D) showed VASH2 upregulated the stem cell marker such as ALDH1A1, Sox2, Oct4, Nanog, which reinforced VASH2 promotion of stem cell proportion.Figure 5

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