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Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma.

Ye ZL, Huang Y, Li LF, Zhu HL, Gao HX, Liu H, Lv SQ, Xu ZH, Zheng LN, Liu T, Zhang JL, Jin HJ, Qian QJ - Acta Pharmacol. Sin. (2015)

Bottom Line: Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion.In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.

ABSTRACT

Aim: Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis.

Methods: Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis.

Results: In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.

Conclusion: A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

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Expression of AGO2 and VEGF was significantly correlated in HCC cell lines. (A and B) RT-PCR was performed to detect the expressions of angiogenesis related factors in Huh7-ctrl and Huh7-siAgo2 cells; (C) Western blot was performed to detect VEGF and AGO2 expressions in HCC cell lines. (D) ELISA was performed to detect VEGF secretion in HCC cell lines suspensions.
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fig1: Expression of AGO2 and VEGF was significantly correlated in HCC cell lines. (A and B) RT-PCR was performed to detect the expressions of angiogenesis related factors in Huh7-ctrl and Huh7-siAgo2 cells; (C) Western blot was performed to detect VEGF and AGO2 expressions in HCC cell lines. (D) ELISA was performed to detect VEGF secretion in HCC cell lines suspensions.

Mentions: In our previous study, we had surprisingly noticed that Ago2-knockdown impaired the tumorigenesis of Huh7 cells in vivo, but barely affected the growth of Huh7 cells in vitro. These results indicate that AGO2 may be involved in HCC angiogenesis. To determine the validity of this hypothesis, we first examined the expression of the angiogenesis-related factors VEGF, TGF-β, PDGFA, PDGFB, FGF1, FGF2 in the Ago2-knockdown HCC cells. The results showed that VEGF expression was remarkably decreased (P=0.0068), while the expression of the other factors was almost unchanged under the condition when AGO2 was specifically reduced (Figure 1A, 1B). Thus, the correlation between AGO2 and VEGF was investigated by examining the expression of AGO2 and VEGF in a list of HCC cell lines (Figure 1C). From the results of quantitative Western blot analysis, we found that the expression of AGO2 and VEGF was significantly correlated at the protein level (r=+0.79, P=0.031). Furthermore, a more significant correlation was revealed when we analyzed AGO2 intracellular expression and VEGF secretion in HCC cell culture suspensions (r=+0.852, P=0.001).


Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma.

Ye ZL, Huang Y, Li LF, Zhu HL, Gao HX, Liu H, Lv SQ, Xu ZH, Zheng LN, Liu T, Zhang JL, Jin HJ, Qian QJ - Acta Pharmacol. Sin. (2015)

Expression of AGO2 and VEGF was significantly correlated in HCC cell lines. (A and B) RT-PCR was performed to detect the expressions of angiogenesis related factors in Huh7-ctrl and Huh7-siAgo2 cells; (C) Western blot was performed to detect VEGF and AGO2 expressions in HCC cell lines. (D) ELISA was performed to detect VEGF secretion in HCC cell lines suspensions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814202&req=5

fig1: Expression of AGO2 and VEGF was significantly correlated in HCC cell lines. (A and B) RT-PCR was performed to detect the expressions of angiogenesis related factors in Huh7-ctrl and Huh7-siAgo2 cells; (C) Western blot was performed to detect VEGF and AGO2 expressions in HCC cell lines. (D) ELISA was performed to detect VEGF secretion in HCC cell lines suspensions.
Mentions: In our previous study, we had surprisingly noticed that Ago2-knockdown impaired the tumorigenesis of Huh7 cells in vivo, but barely affected the growth of Huh7 cells in vitro. These results indicate that AGO2 may be involved in HCC angiogenesis. To determine the validity of this hypothesis, we first examined the expression of the angiogenesis-related factors VEGF, TGF-β, PDGFA, PDGFB, FGF1, FGF2 in the Ago2-knockdown HCC cells. The results showed that VEGF expression was remarkably decreased (P=0.0068), while the expression of the other factors was almost unchanged under the condition when AGO2 was specifically reduced (Figure 1A, 1B). Thus, the correlation between AGO2 and VEGF was investigated by examining the expression of AGO2 and VEGF in a list of HCC cell lines (Figure 1C). From the results of quantitative Western blot analysis, we found that the expression of AGO2 and VEGF was significantly correlated at the protein level (r=+0.79, P=0.031). Furthermore, a more significant correlation was revealed when we analyzed AGO2 intracellular expression and VEGF secretion in HCC cell culture suspensions (r=+0.852, P=0.001).

Bottom Line: Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion.In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.

ABSTRACT

Aim: Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis.

Methods: Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis.

Results: In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.

Conclusion: A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

Show MeSH
Related in: MedlinePlus