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


CONCLUSIONS
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.
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Zhen-long
ZL
Ye
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Yao
Y
Huang
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Lin-fang
LF
Li
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Hai-li
HL
Zhu
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Hai-xia
HX
Gao
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Hui
H
Liu
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Sai-qun
SQ
Lv
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Zeng-hui
ZH
Xu
Xinyuan Institute of Medicine and Biotechnology College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Luo-ning
LN
Zheng
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Tao
T
Liu
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Jing-lei
JL
Zhang
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Hua-jun
HJ
Jin
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.|Xinyuan Institute of Medicine and Biotechnology College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Qi-jun
QJ
Qian
D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D008819:Mice, Nude; D035683:MicroRNAs / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D000067492:RNAi Therapeutics; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human