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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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Ago2-knockdown impaired HCC angiogenesis and tumor growth in vivo. (A–G) The growth curve, morphology, and weight of xenograft were measured. (H) Immunochemistry was conducted by using antibody for AGO2, PTEN, and CD31 (200×).
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fig3: Ago2-knockdown impaired HCC angiogenesis and tumor growth in vivo. (A–G) The growth curve, morphology, and weight of xenograft were measured. (H) Immunochemistry was conducted by using antibody for AGO2, PTEN, and CD31 (200×).

Mentions: To examine the function of AGO2 on HCC angiogenesis in vivo, Huh7-siAgo2/Huh7-ctrl and 7721-siAgo2/7721-ctrl cells were subcutaneously transplanted into nude mice. Twelve days post transplantation, the solid tumors could be detected in all mice. However, the tumor growth of the test group (ie, Huh7-siAgo2 and 7721-siAgo2) was significantly slower than those of the control group (ie, Huh7-ctrl and 7721-ctrl) during the following half month (P<0.05, Figure 3A and 3B). On the day of euthanasia, the sizes of xenograft from Huh7-siAgo2 and 7721-siAgo2 group were remarkably smaller than those from Huh7-ctrl and 7721-ctrl, respectively (Figure 3C and 3D). The xenograft tumor weights between the test group and control group also exhibited a significant difference (P<0.0001, Figure 3E and 3F).


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)

Ago2-knockdown impaired HCC angiogenesis and tumor growth in vivo. (A–G) The growth curve, morphology, and weight of xenograft were measured. (H) Immunochemistry was conducted by using antibody for AGO2, PTEN, and CD31 (200×).
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Related In: Results  -  Collection

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

fig3: Ago2-knockdown impaired HCC angiogenesis and tumor growth in vivo. (A–G) The growth curve, morphology, and weight of xenograft were measured. (H) Immunochemistry was conducted by using antibody for AGO2, PTEN, and CD31 (200×).
Mentions: To examine the function of AGO2 on HCC angiogenesis in vivo, Huh7-siAgo2/Huh7-ctrl and 7721-siAgo2/7721-ctrl cells were subcutaneously transplanted into nude mice. Twelve days post transplantation, the solid tumors could be detected in all mice. However, the tumor growth of the test group (ie, Huh7-siAgo2 and 7721-siAgo2) was significantly slower than those of the control group (ie, Huh7-ctrl and 7721-ctrl) during the following half month (P<0.05, Figure 3A and 3B). On the day of euthanasia, the sizes of xenograft from Huh7-siAgo2 and 7721-siAgo2 group were remarkably smaller than those from Huh7-ctrl and 7721-ctrl, respectively (Figure 3C and 3D). The xenograft tumor weights between the test group and control group also exhibited a significant difference (P<0.0001, Figure 3E and 3F).

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