Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma

HepatologyVolume 57, Issue 5 p. 1906-1918 Hepatobiliary MalignanciesFree Access Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma†‡ Na Cheng, Na Cheng Shanghai Institute of Digestive Surgery & Human Genome Center, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China These authors contributed equally to this study.Search for more papers by this authorYandong Li, Yandong Li Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China These authors contributed equally to this study.Search for more papers by this authorZe-Guang Han, Corresponding Author Ze-Guang Han [email protected] Shanghai Institute of Digestive Surgery & Human Genome Center, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China fax: +86-21-50800402Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, 351 Guo Shou-Jing Road, Shanghai 201203, China===Search for more papers by this author Na Cheng, Na Cheng Shanghai Institute of Digestive Surgery & Human Genome Center, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China These authors contributed equally to this study.Search for more papers by this authorYandong Li, Yandong Li Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China These authors contributed equally to this study.Search for more papers by this authorZe-Guang Han, Corresponding Author Ze-Guang Han [email protected] Shanghai Institute of Digestive Surgery & Human Genome Center, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China fax: +86-21-50800402Shanghai-MOST Key Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, 351 Guo Shou-Jing Road, Shanghai 201203, China===Search for more papers by this author First published: 19 December 2012 https://doi.org/10.1002/hep.26202Citations: 53 † Potential conflict of interest: Nothing to report. ‡ Supported by grants from Chinese National Key Program on Basic Research (2010CB529204 and 2010CB529206), China National Key Projects for Infectious Disease (2012ZX10002012-008 and 2013ZX10002010-006), and National Natural Science Foundation of China (81071722) and the Shanghai Postdoctoral sustentation Fund (11R21420500). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers and shows a propensity to metastasize and infiltrate adjacent and more distant tissues. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effect of Argonaute2 (Ago2), a member of the Ago gene family that plays a role in short interfering RNA-mediated gene silencing, on HCC tumorigenesis, and metastasis. We found that Ago2 was frequently up-regulated in HCC specimens compared to that in corresponding adjacent nontumor liver. Interestingly, Ago2 overexpression can promote proliferation, colony formation in an anchor-independent manner, migration, tumorigenicity, and metastasis of HCC cells in vivo; in contrast, Ago2 knockdown can restrict anchor-independent colony formation, migration, and tumor metastasis of HCC cells in vivo. However, known microRNAs related to tumor metastasis appeared not be deregulated with Ago2 overexpression in HCC cells; even the knockdown of Dicer, which is responsible for microRNA biosynthesis, did not abolish the actions of Ago2 in HCC cells. Significantly, focal adhesion kinase (FAK), a well-known molecule associated with tumor metastasis, was up-regulated as a result of Ago2 overexpression. Chromatin immunoprecipitation assay showed that Ago2 can bind to the FAK promoter and then trigger its transcription. Moreover, an increased DNA copy number of Ago2 on chromosome 8q24, one of the most frequent DNA amplified regions, was validated and shown by way of fluorescence in situ hybridization. Conclusion: Our data demonstrate that Ago2 overexpression, as a result of genomic DNA amplification, promotes HCC tumorigenesis and metastasis by way of up-regulation of FAK transcription, thereby providing new insight into HCC progression and Ago2 function. (HEPATOLOGY 2013) Argonaute (Ago) proteins are ∼100-kD highly basic molecules found in many species and contain two common functional domains, namely, PAZ and PIWI.1-3 In humans, there are eight members in the Argonaute family, four of which belong to the eIF2C/AGO subfamily (EIF2C1/AGO1, EIF2C2/AGO2, EIF2C3/AGO3, and EIF2C4/AGO4).4 All the members are known to be involved in the effector phase of RNA interference (RNAi) either at the stage of translational initiation or elongation.5-8 Among the eIF2C/AGO subfamily, human Ago2, also named EIF2C2, is the only member with an intrinsic endonuclease activity, endowed by its PIWI domain.9 Ago2 may function as an RNA-induced silencing complex (RISC) slicer by way of its RNase activity, cleaving targeted messenger RNA (mRNA)s that are complementary to the guiding small interference RNA (siRNA) or microRNA (miRNA).10-13 Ago2 can specifically generate an additional miRNA precursor, ac-pre-miRNA, which harbors a single strand cleavage in the 3′-arm of the pre-miRNA, and thus form a shortened hairpin bound to a fragment of about 11-12 nucleotides.14 Most interestingly, the Ago family has recently been found to be in human tumors. Although human Ago1 is highly expressed in the embryonic kidney,15 Ago1 expression is often reduced in Wilms' tumors.16 In neuroblastoma cell lines, Ago1 overexpression resulted in a significant reduction of the proliferation rate, along with an increase of differentiation and a less invasive phenotype.17 Based on a tissue microarray analysis on colon cancer, Ago1 up-regulation was associated with the occurrence of colon cancer, whereas Ago2-4 were associated with distant metastasis.18 Ago2 also was up-regulated in head and neck squamous cell carcinoma.19 Ago2 overexpression was involved in high-risk myeloma by way of elevating global miRNAs.20 Moreover, the elevated Ago2 expression could raise the possibility that it augments tumorigenic potential in breast cancer by enhancing miRNA activity.21 It has also been reported that Ago2 knockdown induced apoptosis in myeloid leukemia cells and inhibited siRNA-mediated silencing of transfected oncogenes in HEK-293 cells.22 Nevertheless, little is known about the role of Ago members, especially Ago2, in hepatocellular carcinoma (HCC), one of the most common cancers, which shows a propensity to metastasize and infiltrate adjacent and more distant tissues. In this study we evaluated the expression of all four members of the Ago gene family in HCC and found that Ago2 was frequently overexpressed in HCC. Significantly, Ago2 overexpression can promote HCC cell metastasis by way of the up-regulation of focal adhesion kinase (FAK); this finding provides new insight into HCC metastasis and Ago2 function. Abbreviations Ad-Ago2, recombinant adenovirus expressing both Ago2 and GFP; Ad-GFP, recombinant adenovirus expressing green fluorescent protein; ChIP, chromatin immunoprecipitation; FAK, focal adhesion kinase; FISH, fluorescence in situ hybridization; HCC, hepatocellular carcinoma; MOI, multiplicity of infection; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA. Materials and Methods Tissue Specimens and Cell Lines. All HCC specimens were obtained from HCC patients by way of surgery and with informed consent. The primary tumors were strictly paired and frozen at −80°C until RNA extraction. All the liver tumor-derived cell lines employed in this study are listed in the Supporting Materials and Methods. Studies involving humans and animals were approved by the Ethics Committee of the Chinese National Human Genome Center at Shanghai. Immunohistochemistry Staining. Tissue array containing HCC samples (Shanghai OUTDO Biotech, China) and mouse anti-Ago2 monoclonal antibody (ab57113, Abcam) were used in this study, as described.23 More detail is provided in the Supporting Materials and Methods. Construction of Recombinant Plasmids and Adenoviral Vectors. More detail is provided in the Supporting Materials and Methods. RNA Interference. Two siRNAs or short hairpin RNA (shRNA) against Ago2 were designed. More detail is provided in the Supporting Materials and Methods. Colony Formation. G418 (Life Technologies, Grand Island, NY) was employed to select the transfected HCC cells. More detail is provided in the Supporting Materials and Methods. Chromatin Immunoprecipitation (ChIP). ChIP assay was performed using the EZ ChIP Kit (Millipore) according to the manufacturer's protocol. More detail is provided in the Supporting Materials and Methods. Dual Luciferase Reporter Assay. Dual-luciferase assay was performed with the Dual-Luciferase Reporter Assay System kit (Promega) according to the manufacturer's instructions. More detail is provided in the Supporting Materials and Methods. Fluorescence In Situ Hybridization (FISH). To detect Ago2 amplification, FISH was carried out using a Tissue Array (Shanghai OUTDO Biotech) containing HCC samples with the BAC clone RP11-642A1 (red), which was verified for the Ago2 gene by polymerase chain reaction (PCR), as previously described.23 The probe specific to the centromere for chromosome 8 (CEN8) (green) was used as a control. More detail is provided in the Supporting Materials and Methods. Cell Transwell Assay. More detail is provided in the Supporting Materials and Methods. In Vivo Tumorigenicity. HCC cells expressing ectopic Ago2 were subcutaneously injected into the flank of nude mice, while HCC cells containing empty vector were injected into the opposite flank of the same mice as a control. Growth curves were plotted based on mean tumor volume within each experimental group at the indicated timepoints. Tumor dimensions were measured every 3-4 days using a digital caliper, and the tumor volume was calculated using the following formula: V = (larger diameter) × (smaller diameter) × (smaller diameter). The tumorigenic experiments in vivo were performed with eight mice in each group. In Vivo Metastasis. We employed the subcutaneous split-spleen reservoir model and the tail vein injection assay to assess the effect of Ago2 on tumor metastasis. The operation to establish split-spleen reservoirs was conducted as follows: 8- to 10-week-old athymic nude mice were anesthetized with 2.5% sodium pentobarbital (40 mg/kg; Sigma-Aldrich), and the left side of the abdomen was sterilely prepped. The abdomen was penetrated through a small opening in the muscle and the peritoneum. The upper part of each spleen was then injected with 100 μL of HCC cells for Ago2 overexpression or knockdown, while those with empty vector were used for the control. Then the skin was sealed. These mice were observed for about 8 weeks to assess liver metastasis. In addition, to assess long-distance lung metastasis the treated HCC cells were injected into athymic nude mice by way of their tail veins, while those with empty vector were used as a control. The mice were then observed for about 16 weeks. Statistical Analysis. An analysis of variance (ANOVA) and Student's t test were used for comparison among groups. The Mann-Whitney U test was used for comparison of tumor volume. Categorical data were evaluated with the chi-square test or Fisher's exact test. P < 0.05 was considered significant. Results Ago2 Is Frequently Up-regulated in HCC. To assess the potential effect of Ago members on HCC, we first determined the relative transcriptional levels of these known Ago genes, Ago1-Ago4, in 31 pairs of human HCC specimens by real-time RT-PCR. The data showed that only the mRNA level of Ago2 was significantly up-regulated in HCC specimens, as compared to that of corresponding noncancerous livers (Fig. 1A). To confirm this result, Ago2 was further evaluated in 47 paired additional human HCC specimens and noncancerous livers by real-time RT-PCR, where Ago2 was shown to be up-regulated in 25/47 (53.2%) of the HCC specimens at more than 1.5-fold higher levels (Fig. 1B). Furthermore, we evaluated the expression levels of Ago2 in HCC cell lines by western blotting. The result showed that Ago2 was highly expressed in a majority of the HCC cell lines examined (Fig. 1C). mRNA levels of Ago2 in HCC cell lines were also detected by RT-PCR and real-time PCR (Supporting Fig. 1A,B). In addition, a tissue array containing an additional 72 pairs of HCC samples was examined by immunohistochemical staining with a specific antibody against Ago2. The resulting data showed that Ago2 was significantly overexpressed in 41 (56.9%) of 72 HCC specimens compared with that of the corresponding noncancerous livers (Fig. 1D; Supporting Fig. 1C). Collectively, these data indicated that Ago2 was frequently up-regulated in HCC samples and cells. Figure 1Open in figure viewerPowerPoint Expression pattern of Ago2 in HCC specimens and cell lines. (A) Quantitative real-time RT-PCR was performed to detect the expression level of Ago1-Ago4 in 31 pairs of HCC and the corresponding adjacent noncancerous liver (non-HCC), where β-actin was used as an internal control. Each plot displays the expression level of individual HCC and adjacent liver sample, where the longer lines represent the median with interquartile range of −ΔCt value; P values as indicated in these panels were calculated by paired t test. (B) Real-time RT-PCR was carried out on additional 47 paired HCC samples and adjacent noncancerous tissues. The columns show the up-regulated fold change of Ago2 mRNA level in HCCs, as compared to that in the corresponding adjacent noncancerous liver, where β-actin was used as an internal control. (C) Western blot assay was performed to evaluate the Ago2 expression level in HCC cell lines, and actin was used as loading control. (D) A representative immunohistochemical staining on a tissue array containing HCC samples with anti-Ago2 antibody. Magnification: ×40 (upper) and ×200 (bottom). Ago2 Can Promote Cellular Proliferation, Colony Formation, and In Vivo Tumorigenicity. To investigate the role of Ago2 in HCC, we first observed the effect of Ago2 overexpression in HCC cells. Here, we transiently transfected the recombinant plasmid pcDNA3.1-Ago2 containing the Ago2 gene into HCC cell lines, including Huh-7, Hep3B, SK-hep-1, and HepG2 cells, expressing Ago2 at a relatively low level. The results indicated that Ago2 overexpression can significantly promote cell viability of all the four examined HCC cell lines compared to that of the empty vector pcDNA3.1 control (P < 0.05) (Fig. 2A). Moreover, Ago2 overexpression can significantly promote colony formation of Huh-7 and SK-hep-1 (P < 0.05) (Supporting Fig. 2A,B). Furthermore, we performed an anchorage-independent colony formation assay in soft agar to evaluate the effect of Ago2 on malignant behavior. Here, Ago2 overexpression significantly enhanced the anchorage-independent growth of Huh-7, SK-hep-1, and HepG2 cells in soft agar (Fig. 2B). Figure 2Open in figure viewerPowerPoint The effect of Ago2 overexpression on cell proliferation, colony formation, and tumorigenicity in vitro and in vivo. (A) Ectopic Ago2 can promote proliferation of Huh-7, Hep3B, SK-hep-1, and HepG2 cells; cells carrying empty vector were used as a control. The experiments were repeated at least three times, and the points represent the mean values of triplicate tests (mean ± SD). Western blot assay was used to detect ectopic Ago2 in these cell lines, as indicated in the upper panel, where actin was used as a loading control. (B) Ago2 overexpression can enhance colony formation of Huh-7, SK-hep-1, and HepG2 cells in soft agar. Representative results show the increased anchorage-independent colony formation. The experiments were repeated at least three times, and the histograms represent the mean numbers of colonies from triplicate tests (mean ± SD). (C,D) HepG2 and Hep3B cells infected with Ad-Ago2 were injected subcutaneously into nude mice (n = 8 and n = 4, respectively), while those with empty vector were used as a control. Tumor growth was monitored at every 4-day interval by measuring the tumor diameters (mean ± SD, left); tumors were removed from the nude mice after about 1 month (right). (E) Tumor growth was also observed in nude mice injected with stably expressing Ago2 Sk-hep-1 cells (mean ± SD, left); tumors were removed from the nude mice after about 1 month (right). A t test was used to evaluate the statistical significance of these experiments, as compared to the control. *P < 0.05; **P < 0.01. To further evaluate the effect of Ago2 overexpression on tumorigenicity, recombinant adenovirus harboring Ago2 (Ad-Ago2) was transfected into HepG2 (4.5 × 106 cells) and Hep3B (4 × 106 cells) cells; then the cells expressing ectopic Ago2 were subcutaneously injected into the flank of athymic nude mice, and an equal volume of cells transfected with empty vector (Ad-GFP), used as controls, was injected into the opposite flank of the same mice. Interestingly, HepG2 and Hep3B cells infected with Ad-Ago2 resulted in significant tumorigenicity in vivo in 6/8 and 4/4 of the nude mice, respectively. However, the corresponding controls infected with empty Ad-GFP had no visible tumors in the same mice, which were under observation for about 1 month (P < 0.05) (Fig. 2C,D). To certify the effect of ectopic Ago2 on tumorigenicity, green fluorescence was observed after Ad-GFP and Ad-Ago2 infection, and the recombinant Falg-tagged Ago2 protein expressed by adenovirus was detected using anti-Flag antibody in the removed xenograft tumors at the end of whole observation period (Supporting Fig. 3A,B). These data indicated that Ago2 overexpression can significantly promote tumorigenicity in vivo. To test this hypothesis, SK-hep-1 cells stably expressing Ago2 and empty vector for control were subcutaneously injected into two flanks of the same nude mice. The result showed that these stably expressing Ago2 SK-hep-1 cells formed larger tumors than the controls with empty vector when observed for about 1 month (P < 0.05) (Fig. 2E). These data revealed that Ago2 overexpression could play an important role in promoting growth, colony formation, and tumorigenicity of HCC cells. Ago2 Can Enhance Tumor Metastasis. To investigate the role of Ago2 in HCC progression, we first employed wound-healing assays to assess the effect of Ago2 overexpression on cell migration. The results showed that Hep3B and SK-hep-1 cells, transiently transfected with pcDNA3.1-Ago2, had enhanced mobility, as compared to the control empty vector (Supporting Fig. 4A,B). Subsequently, the transwell assay was carried out to further evaluate the effect of overexpression. The data showed that Ago2 overexpression did indeed significantly promote migration of Hep3B and SK-hep-1 cells across the membrane when FBS was used as an attractant (Fig. 3A,B; P < 0.05). Figure 3Open in figure viewerPowerPoint The effect of Ago2 overexpression on migration and in vivo metastasis of HCC cells. (A,B) The migration of Hep3B (A) and SK-hep-1 (B) cells transfected with Ago2 were assessed by transwell assay, where those with empty vector were used as a control. The expression level of ectopic Ago2 was confirmed by immunoblotting (upper right). The experiments were repeated at least three times, and the histograms (right) represent mean numbers of the transferred cells from triplicate tests (mean ± SD). The representative views (left) show the transferred cells. (C) Effect of ectopic Ago2 on liver metastasis of stably expressing Ago2 SK-hep-1 cells by way of the portal vein, where those with empty vector were used as a control. Representative massive and microscopic images of hepatic metastases are shown at the top. The scattergram (bottom) shows the numbers of tumor nodules in each of the four nude mice during 8 weeks of observation. (D) Effect of ectopic Ago2 on lung metastasis of stably expressing Ago2 SK-hep-1 cells by way of tail vein injection. Representative images of lung metastases are shown at the top. The scattergram (bottom) shows the numbers of tumor nodules in each of the four nude mice during 16 weeks of observation. Arrows indicate metastatic tumors. Original magnification, ×100 (A,B); ×400 (C,D, right panels). A t test was used to evaluate the statistical significance of these experiments, as compared to control. P values between groups are indicated. Next, to test whether Ago2 overexpression can enhance tumor metastasis in vivo, we injected SK-hep-1 cells (2 × 106 cells) stably expressing ectopic Ago2 into the spleen of nude mice and then observed the metastatic tumors in the liver through their portal vein; mice injected with those carrying empty vector were used as a control. Significantly, after 8 weeks of observation all four mice injected with SK-hep-1 cells with ectopic Ago2 developed metastatic liver tumors with larger and greater numbers of nodules (Fig. 3C; Supporting Fig. 5A; P < 0.05), whereas 1/4 of the control mice displayed metastatic tumors that were both fewer and smaller in number. Histological analysis confirmed the presence of liver tumors in these mice. The results suggested that Ago2 overexpression could significantly promote tumor metastasis of HCC cells by way of their portal vein. To further strengthen the evidence supporting the contribution of Ago2 overexpression to HCC metastasis, we employed the tail-vein injection assay to observe long-distance tumor metastasis in vivo. We injected SK-hep-1 cells stably expressing Ago2 (2 × 106 cells) into nude mice by way of tail veins and then checked lung metastasis after 16 weeks. Intriguingly, metastatic tumors were observed in significant numbers in the lungs of 3/4 mice injected with SK-hep-1 cells overexpressing Ago2, whereas no visible metastatic tumors were found in the control mice (Fig. 3D; Supporting Fig. 5B; P < 0.05)). Histological analysis also confirmed the lung metastasis in these mice. The results supported that Ago2 overexpression can significantly enhance the long-distance tumor metastasis of HCC cells. Ago2 Knockdown Suppresses Tumor Metastasis. To further address the effect of Ago2 on tumor metastasis, chemically synthesized siRNAs were initially employed to effectively knock down endogenous Ago2 in HCC cell lines with relatively high expression. After careful evaluation, siR-1083 and siR-3422 were considered to be the efficacious siRNAs for Ago2 knockdown and were then used in subsequent experiments. The transwell assay showed that Ago2 knockdown induced by both siRNAs can significantly inhibit the migration of YY-8103 and PLC/PRF/5 cells as compared to that of control siRNA-NC (Fig. 4A,B), and the same effect was observed in LM6 cells (Supporting Fig. 6). Figure 4Open in figure viewerPowerPoint The effect of Ago2 knockdown on HCC cell migration and metastasis in vitro and in vivo. (A,B) Migration of YY-8103 (A) and PLC/PRF/5 (B) cells was evaluated by transwell assay using Ago2 knockdown with siRNA-1083 and siRNA-3422, where siRNA-NC was used as a control. RNAi effect was confirmed by immunoblotting (upper right). The experiments were repeated at least three times, and the histograms (right) represent mean numbers of the transferred cells from triplicate tests (mean ± SD). Representative views (left) show the transferred cells. (C,D) Effect of Ago2 knockdown on liver metastasis of YY-8103 and PLC/PRF/5 cells by way of portal vein, which were performed in four and five nude mice, respectively, and were observed for 8 weeks. Representative images of hepatic metastases are shown on the left. The scattergram (bottom) shows the numbers of tumor nodules in each of the four nude mice. (E,F) Effect of Ago2 knockdown on lung metastasis of YY-8103 and PLC/PRF/5 cells by way of tail vein injection. Representative massive and microscopic images of lung metastases are shown at the top. The scattergram (bottom) shows the numbers of tumor nodules in each of the four nude mice during 20 weeks of observation. Arrows indicate metastatic tumors. Original magnification, ×100 (A,B); ×400 (C-F, microscopic images). A t test was used to evaluate the statistical significance of these experiments, as compared to the control. P values between groups are indicated. Next, to evaluate the effect of Ago2 knockdown on tumor metastasis in vivo, we established two stable subclones of YY-8103 and PLC/PRF/5 cell lines, which were stably transfected by a plasmid encoding shRNA-1083 for Ago2 knockdown. The YY-8103 and PLC/PRF/5 offspring subclones, YY-1083-9 (2 × 106 cells) and PLC-1083-11 (3 × 106 cells), were injected into nude mice by way of the spleen and tail vein, respectively. As expected, after 8 weeks of observation metastatic liver tumors derived from these two offspring subclones with reduced Ago2 (knockdown) were significantly fewer and smaller than that of the control shRNA-NC (Fig. 4C,D; Supporting Fig. 7A,B). Significantly, after 20 weeks of observation no visible metastatic tumors formed in the lungs of these mice injected with YY-8103 and PLC/PRF/5 offspring subclones with Ago2 knockdown (Fig. 4E,F; Supporting Fig. 7C-D), whereas control subclones produced notable metastatic lung tumors. Histological analysis confirmed the presence of metastatic tumors in the livers and lungs of these mice. These collective data suggested that Ago2 overexpression could promote tumor metastasis, and therefore, the enzyme can serve as a potential therapeutic target for preventing HCC invasion and metastasis. However, it should be noted that Ago2 knockdown did not appear to inhibit proliferation and tumorigenicity in vivo of these YY-8103 and PLC/PRF/5 offspring subclones (data not shown), implying the possibility that the effect of Ago2 knockdown on proliferation and tumorigenicity could be compensation and counteract other Ago family members. This result also suggested that the effect of Ago2 on cell migration and tumor metastasis is independent of cell proliferation. Ago2 Can Regulate the Expression of FAK. To explore the molecular mechanisms by which Ago2 contributes to HCC cell migration and metastasis, we first examined cell morphology with Ago2 overexpression or knockdown. However, no visible epithelial to mesenchymal transition (EMT) was found. We further examined some known EMT-related molecules, including E-cadherin, vimentin, ZEB-1, ZEB-2, and Twist in HCC cell lines through immunoblotting and real-time RT-PCR with Ago2 overexpression or knockdown (Fig. 5A,B; Supporting Fig. 8A,B). The data indicated that the transcription and protein levels of these molecules were not obviously perturbed with Ago2 overexpression or knockdown, suggesting that EMT could not be involved in the Ago2-induced HCC tumor metastasis. Figure 5Open in figure viewerPowerPoint Ago2 regulates the expression of FAK. (A,B) The expression levels of metastasis-related molecules were detected by western blotting of Ago2 overexpression in Huh-7, Hep3B, and SK-hep-1 (A) or Ago2 knockdown in YY-8103 and PLC/PRF/5 cells (B), where actin was used as a control. (C,D) mRNA levels of FAK were measured by real-time RT-PCR of Ago2 overexpression in Huh-7, Hep3B, and SK-hep-1(C) or Ago2 knockdown in YY-8103 and PLC/PRF/5 cells (D), where β-actin was used as an internal control. A t test was used to evaluate the statistical significance of these experiments, as compared to the control. *P < 0.05; **P < 0.01. (E) The relationship between Ago2 and the FAK expression level in the same 24 HCC samples examined was statistically analyzed using the chi-square test. A previous study showed that Ago2 plays a role in the maturation and effect of miRNA. To determine whether the Ago2-induced HCC tumor metastasis depends on miRNAs, we sought to determine the expression levels of some miRNAs, including miR-103, miR-107, miR-221, miR-200a, and miR-451, which have been reported to be relevant in tumor metastasis. Real-time RT-PCR result revealed that none of these miRNAs were significantly regulated by Ago2 overexpression (Supporting Fig. 9A,B). To further exclude the effect of miRNAs on Ago2-induced metastasis, we chemically synthesized siRNA to knock down endogenous Dicer, which is responsible for miRNA biosynthesis, and then observed the effects of Dicer knockdown on Ago2-induced migration of SK-hep-1 cells. The transwell assay indicated that Dicer knockdown cannot significantly block the migration of stably expressing Ago2 SK-hep-1 cells (Supporting Fig. 9C,D), suggesting that miRNAs also could not be involved in the Ago2-induced HCC metastasis. Subsequently, we focused on FAK, a well-known molecule associated with tumor progression and metastasis. The transcription and pro