Research On The Function Of Argonaute-2 In HCC Vascularization And Vasculogenic Mimicry

Hepatocellular carcinoma(HCC) is still a most common cause for cancer-related mortality. Highly vascularization provides nutrients, major metastasis path and shelter against therapies. And vascularization has been proved complicated but intricately regulated by VEGF, PDGF, FGF, Endostatin and other related factors, and recent studies have revealed that numerous miRNAs involved in this regulation. In addition to vacularization, vasculogenic mimicry formed by tumor cells, is a newly found complementary method for blood supply and metastasis. However, mechanisms about vascularization or vasculogenic mimicry of HCC have not been fully elucidated yet.As an indispensable factor in miRNAs regulation and maturation, AGO2(Argonaute-2, also termed as EIF2C2) has been proved as the only member in AGO protein family with catalytic activity and essential role in RISC(RNA-induced silencing complex) assembly as well as the regulation of small non-coding RNAs guided gene silencing processes through various ways. Recently, AGO2 has been indicated over-expressed in and associated with carcinomas by interaction with tumor factors including EGFR, AKT3, MAPK, FAK, etc. In 2008, AGO2 was found directly related to angiogenesis of endothelial cells by revealing that Ago2 knock-down caused disappearance of the angiogenic potential of endothelial cells. In our previous study, we found AGO2 over-expressed in both HCC cells and samples, Ago2 knock-down attenuated HCC angiogenesis as well as tumor growth in vivo instead of in vitro. But so far, the association of AGO2 and angiogenesis in HCC has not been reported.Therefore, in this study, we provide evidence for the association of AGO2 and HCC angiogenesis through knocking down Ago2 in HCC cell lines Huh7 and SMMC-7721 by small RNA interference(RNAi) technology. The major methods and results are listed below: In vitro study of the impact on angiogenesis related factor expression in HCC cells caused by Ago2 knock-downEstablishments of Ago2 knock-down and relative control cell lines(Huh7/7721-ctrl/siAgo2) were carried out by cooperation with biological company. RT-PCR was used to detect the expressions of angiogenesis related factors on mRNA level and showed that VEGF was severely reduced. Western Blot and ELISA were used to test the expressions of VEGF and AGO2 on protein level in a series of HCC cell lines and revealed the association between AGO2 and VEGF. To confirme this, the secretion of VEGF in cell culture suspensions, the ability to induce capillary tubule structures formation with Human Umbilical Vein Endothelial Cells(HUVEC) in Huh7/7721-ctrl/siAgo2 cells were also tested, results showed that Ago2 knock-down in Huh7 and SMMC-7721 cells by RNAi decreased VEGF expression on both mRNA and protein levels, and impaired their ability to induce capillary tubule structure formation with HUVECs. In vivo study of the impact on HCC angiogenesis caused by Ago2 knock-downAll animal experiments were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scienti?c Investigation Board of the Second Military Medical University, Shanghai. Huh7, SMMC-7721 and Huh7-siAgo2, 7721-siAgo2 were injected subcutaneously into nude mice(1×107/mouse, 6 per group). Twelve days post injection, tumor volumes were measured every 4 days since the xenograft tumors were formed [volume =(W2×L)/2; W, width; L, length, in cubic millimeters]. The mice were sacrificed four weeks post transplantation to weight the xenograft tumors and the tumor tissues cells were separated and stored in 4% paraformaldehyde for AGO2(1:250, CST, USA), CD31(1:200, Abcam, USA) and PTEN(1:250, CST, USA) immunohistological analyses. Results showed significant reduce in tumor growth, weight, volume, AGO2 and CD31 expression related to Ago2 knock-down. Mechanism study for the interaction between AGO2 and VEGFBased on our previous founding that PTEN was up-regulated by Ago2 knockdown and reports made by others that PTEN is an important angiogenesis inhibitor, PTEN was firstly concerned as the mediator of the interaction between AGO2 and VEGF. RT-PCR, Western Blot, IHC and Immunofluorescence were used to test the expression of PTEN on mRNA and protein levels in Huh7/7721-ctrl/siAgo2 and tumor tissues. Results showed that PTEN was un-regulated by Ago2 knock-down. By culturing Huh7/7721-siAgo2 cells with four different concentrations of PTEN specific inhibitor BPV, the expression of VEGF and the ability to induce capillary tubule structure formation from HUVECs of Huh7/7721-siAgo2 were partially restored. Study of the impact on the vasculogenic mimicry of HCC cells caused by Ago2 knock-down6.0 × 104 Huh7/7721-ctrl/siAgo2 cells were collected and seeded in 24-well plates, each of which was coated with 50μl growth factor-reduced matrigel(BD, USA). Photos were taken 24 hours later. By deep sequencing, we found that an important vasculogenic mimicry factor TWIST1 was reduced in Huh7-siAgo2 cells compared to Huh7-ctrl cells, based on this, RT-PCR, Western Blot and Immunofluorescence were used to detect the expressions of AGO2, TWIST1, VEcadherin and E-cadherin. Results showed that Ago2 knock-down attenuated the vasculogenic mimicry process of Huh7 and SMMC-7721 cells by reducing TWIST1 expression.