Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

AGO2 (Argonaute RISC Catalytic Component 2) plays an important role in small RNA-guided gene silencing processes. It has been implied in tumorigenesis of different types of tumors. In this study, we found that AGO2 expression was remarkably increased in human hepatocellular carcinoma (HCC) tissues when compared with adjacent noncancerous tissues. High expression of AGO2 was associated with poor prognosis in HCC patients. The CRISPR/Cas9-mediated knockout of AGO2 in SMMC-7721 cells inhibited cell proliferation and induced significant G1 phase arrest of cell cycle. Inhibition of cell migration was also observed in SMMC-7721 AGO2−/− cells. In vivo experiments showed that tumors grew slower in nude mice transplanted with AGO2−/− cells than in SMMC-7721 cell-derived xenograft mice. Microarray analysis and western blot analysis revealed that AGO2 depletion decreased expression of Survivin, Vimentin, and Snail. Overexpression of AGO2 in SMMC-7721 and Huh-7 cells could reverse the knockout-induced inhibition effects on either cell behaviors or expression of Survivin, Vimentin, and Snail Therefore, our data demonstrated that AGO2 might facilitate HCC tumorigenesis and metastasis through modulating expression of Survivin, Vimentin, and Snail.


Introduction
Hepatocellular carcinoma (HCC), a malignant epithelial liver tumor, is the third leading cause of cancer-associated death globally [1]. The development of HCC is a multistage process involving chronic liver injury, inflammation, hepatocellular degeneration/regeneration, necrosis, and small-cell dysplasia [2]. Genetic and epigenetic aberrations are implied in HCC initiation and progression [2].
Argonaute (AGO) proteins are expressed at high levels in a wide range of organisms. The human Argonaute family has eight members, four of which belong to the eIF2C/AGO subfamily. These eIF2C/AGO subfamily members are essential components of the RNA-induced silencing complex (RISC), which is involved in RNA silencing processes of RNA interference (RNAi) [3]. Among them, only AGO2 has intrinsic endonuclease activity. AGO2 can bind small interfering RNA (siRNA) or microRNA (miRNA), which guides AGO2 to target specific messenger RNA (mRNA) and cause the mRNA cleavage [4,5]. It has been well documented that AGO2 plays important roles in multiple biological or pathological processes [6][7][8]. Recently, AGO2 has been demonstrated as a potential oncogene in human tumorigenesis, including head and neck squamous cell carcinoma [9], nasopharyngeal carcinoma [10], bladder cancer [11], and glioma [12]. It was also reported that AGO2 could promote tumor metastasis [13] and enhance angiogenesis of HCC [14]. However, the underlined mechanisms of AGO2 in HCC progression still need to be clarified.
In the present study, we found that high expression of AGO2 was an independent prognostic indicator for HCC patients with poor outcome. In vitro and in vivo studies would further reveal the function and molecular mechanisms of AGO2 in HCC tumorigenesis and progression.

Patients.
On institutional review board approval, we identified 90 patients with hepatocellular carcinoma (HCC) treated with surgery between 2011 and 2019 at Renmin Hospital of Wuhan University and Tongji Hospital of Huazhong University of Science and Technology. None of the patients received adjuvant therapy. Data collected from each patient included gender, age at diagnosis, grade, stage, and overall survival time. Pairs of cancer tissues and adjacent epithelium tissues from the same HCC patients were obtained by surgical removal. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University (approval No.: WDRY2018-K024). Informed consent (written or verbal) was obtained from the patients in this study. All the samples were anonymous.

Tissue Microarray (TMA).
The TMA slide HLiv-HCC180Sur-04 (Outdo Biotech Co., Ltd., Shanghai, China) contained 90 cases of HCC tissues and paired paracarcinoma tissues. The formalin-fixed and paraffinembedded tissue slides were stained by hematoxylin and eosin according to standard protocols. The target tissue cores were then labeled and punched (Beecher Instruments Inc., Silver Spring, MD, USA) with a diameter of 1.5 mm and a thickness of 4 μm. A total of 180 cores were arrayed on the recipient block.
2.4. Immunohistochemistry. The HCC sections were washed with xylene and then rehydrated in a series of ethanol (100%, 90%, 80%, and 70%) before being fully hydrated in deionized water. After antigen retrieval using citrate buffer, the tissue sections were immersed in 0.3% hydrogen peroxide solution to inactivate endogenous peroxidase and subsequently incubate with 5% BSA for blocking non-specific binding. Next, the sections were incubated with the primary anti-AGO2 antibody (1 : 500) at 4°C overnight followed by incubation with an HRP-conjugated secondary antibody. After washing with PBS, the slides were visualized by staining with 3,3h-diaminobenzidine and hematoxylin. The immunoreactive score was calculated according to the staining intensity in three randomly selected fields (40x). The scores for staining intensity were as follows: 0 was negative, 1 was weak, 2 was moderate, and 3 was strong. All slides were examined and scored by two senior pathologists independently. According to the average scores (AS), high expression level of AGO2 was defined when AS > 0:5. Low expression level of AGO2 was defined when the AS ≤ 0:5. . Several cell lines were used in this study,  including human liver cancer cell lines (SMMC-7721,  HepG2, Huh-7, and Hep3B), HEK293T, and human breast  cancer cell lines (HCC-1937, ZR-75-30, MCF-7, and MDA-MB-231). Cells were incubated with DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; ScienCell, Carlsbad, CA, USA). Cells were maintained in a cell incubator with 5% CO 2 at 37°C. All cell lines were purchased from ATCC. Microsart® Mycoplasma Kit (Sartorius Inc., Gottingen, Germany) was used to monitor cells for Mycoplasma contamination routinely.

Cell
Transfection. The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China). According to the manufacturer's protocol, pCMV3-AGO2 and control pCMV3 plasmids were transfected into Huh-7 cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific), respectively. After 24-48 h transfection, the expression of AGO2 protein was examined.

CCK8 Assay.
For the detection of cell proliferation, 2000 cells were planted at each well of the 96-well plate. Fresh DMEM medium (100 μl) and CCK8 solution (10 μl) were added into each well of the 96-well plate following incubation at 37°C for 2 hours. The absorbance was tested at 450 nm each day.   Mice were randomly assigned as the control group and model group (n = 10 mice per group). In the model group, SMMC-7721 cells (control and AGO2 knockout) were subcutaneously injected in the flank of BALB/c nude mice with 2 × 10 6 cells. Mice in the control group only received sterile PBS injection. About 7 days post injection, the tumor size was measured every other day. The tumor size was calculated as ðlength × width 2 Þ/2. At the indicated time, all mice were sacrificed with CO 2 inhalation and the tumors were photographed.
2.13. Western Blot. Cells were collected and lysed in radioimmunoprecipitation assay buffer (RIPA Buffer) containing protease inhibitors. The protein concentration was determined with the BCA kit (Thermo Fisher Scientific). The same amount (30 μg) of proteins was loaded on 10% SDS/PAGE gels and transferred to a piece of the nitrocellulose membrane (0.45 μM) (Amersham, Piscataway, NY). After blocking by 5% nonfat milk in TBST containing 0.05% (V/V) Tween-20 at room temperature for 1 hour, the membranes were incubated overnight at 4°C with the appropriate primary antibody (1 : 2000). Blots were then incubated at room temperature for 1 hour with a horseradish peroxidase-(HRP-) conjugated secondary antibody (1 : 20000). The peroxidase activity was detected with a chemiluminescent HRP substrate (Millipore, Billerica, MA) and imaged by a chemiluminescence system (Fujifilm LAS-4000, Tokyo, Japan).
2.14. Statistical Analysis. The correlation between AGO2 expression and HCC clinical parameters was calculated by Spearman's correlation analysis. Survival curves were plotted using the method of Kaplan-Meier, and the significance of observed differences was calculated with a logrank test. Student's t-test was used for two paired groups. One-way ANOVA followed by post hoc Turkey's test was performed for multiple comparisons. All assays were repeated for at least three times. Data are shown as average values ðmeanÞ ± SD (standard deviation) from one representative experiment. The P value < 0.05 was considered statistically significant.

AGO2 Associated with Poor Prognosis in HCC Patients.
To explore AGO2 expression in HCC, we first checked the AGO2 expression in the HCC tissue microarray. Immunohistochemical staining was used to detect AGO2 protein in matched pairs of HCC and noncancerous tissues. The representative images (Figure 1(a)) showed high expression of AGO2 in HCC tissue and low expression in normal tissue. The AGO2 expression levels were scored according to the staining intensity ranging from 0 to 3 (0, 1, 2, and 3). Several missing or damaged samples were excluded during immunohistochemistry tissue preparation, and a final total of 85 cases were included in the statistical analysis. Result showed that in 80 of 85 (94%) matched tissue sets, AGO2 expression was significantly higher in tumor tissues than in normal tissues (Figure 1(b), Table 1). Table 1 shows the correlation of AGO2 expression with clinical parameters of HCC patients. Data demonstrated that AGO2 expression in the human HCC tissues was only associated with tumor size (P = 0:011) of the patients. Subsequently, the correlation between AGO2 expression and outcome of patients was also assessed. Kaplan-Meier analysis revealed that high level of AGO2 was related to significantly poor overall survival (OS) (Figure 1(c)). Other clinicopathological characteristics, such as tumor size, could also affect the OS, while age and gender showed no significant correlation with OS (Figures 1(d)-1(f)). Altogether, these data support that AGO2 associated with poor prognosis in HCC patients.

Knockout of AGO2 Inhibited Cell Proliferation and
Migration in HCC In Vitro. To explore the biological function of AGO2 in HCC, we evaluated the AGO2 expression in several cell lines (Figures 2(a) and 2(b)). Interestingly, western blot analysis and qRT-PCR examination revealed different expression patterns of AGO2 protein and mRNA in several cell lines. For example, Huh-7 had the lowest AGO2 protein abundance (Figures 2(a) and 2(b)) and the highest mRNA expression (Supplementary Figure 1A and 1B). In addition, compared with other cell lines, HepG2 and ZR-75-30 cells showed high AGO2 protein expression but conversely low mRNA levels (P < 0:05, Figure 2(a) and Supplementary Figure 1B). This result suggested AGO2 protein abundance was not correlated well with mRNA expression in these cell lines. In comparison, relatively high 3 BioMed Research International correlation between AGO2 protein and mRNA levels was observed in two liver cancer cell lines. SMMC-7221 and Hep3B had moderately high expression of AGO2 at both protein and mRNA levels.

Knockout of AGO2 Suppressed HCC Tumor Growth and
Size In Vivo. To further investigate the AGO2 role in regulating xenograft tumor growth, nude mice were injected subcutaneously with SMMC-7721 and AGO2 -/cells, respectively. The body weights of mice bearing the tumors were not significantly changed (not shown) within 2 weeks. Tested nude mice (n = 10/group) developed subcutaneous tumors with a size of approximately 0.90 to 840 mm 3 after injection with SMMC-7721 or SMMC-7721 AGO2 -/cells (2 × 10 6 /mouse). SMMC-7721 control cells generated visible tumors at day 3 and formed a continuously growing mass. However, SMMC-7721 AGO2 -/cells generated visible tumors at day 6, and the tumor growth rate of SMMC-7721 AGO2 -/cells was lower compared with control cells (Figure 3(a)). These findings suggested that AGO2 promoted tumor growth in vivo, while the knockout of AGO2 appeared to not affect the mice growth (Figure 3(b)). The tumor sizes of mice bearing with SMMC-7721 control cells were obviously smaller than those with SMMC-7721 AGO2 -/cells (Figure 3(c)). Accordingly, the average tumor weight in the SMMC-7721 control group was heavier than that in the SMMC-7721 AGO2 -/group (Figure 3(d)). Therefore, knockout of AGO2 suppressed tumor growth and size in HCC in vivo.

AGO2 Regulated Expression of Survivin, Vimentin, and
Snail. To explore the downstream pathways of AGO2 involved in HCC tumorigenesis, we performed microarray profiling of gene expression. We initially evaluated the global transcriptomic changes associated with depletion of AGO2 in SMMC-7721 cells. Heatmaps for gene expression exhibited a list of 2,327 genes showing >2-fold differential expression (Figure 4(a)). Gene ontology (GO) enrichment analysis revealed that these differentially expressed genes (DEG) were mainly enriched in the pathways that control cell behaviors, including cell death and survival, cellular growth, and proliferation, as well as cellular movement (Figures 4(b) and 4(c)).  It has been reported that Survivin, Vimentin, and Snail play an important role in cell proliferation and metastasis of hepatocellular carcinoma [16,17]. A western blot test confirmed that Survivin, Vimentin, and Snail expression dramatically declined in SMMC-7721 AGO2 -/cells when compared with control cells (Figures 5(a)-5(d)). Especially, Vimentin was decreased by around 60% and Snail was decreased by 90% due to knockout of AGO2 ( Figure 5(d)). Moreover, expression of Survivin, Vimentin, and Snail proteins were   Color key -4 -2 0 2 4 Column Z-score

Discussion
AGO2 was first identified as an oncogene in renal cell carcinoma. It has been reported that the single-nucleotide polymorphism (SNP) of AGO2 was related to the tumorigenesis of renal cell carcinoma [18]. Abnormal overexpression of AGO2 has been found in several human tumors, including breast cancer [19], urothelial carcinoma of the bladder [11], glioma [12], and nasopharyngeal carcinoma [10]. Accumulation of AGO2 is generally associated with higher tumor grading and poorer prognosis [3,20]. The present study showed that AGO2 expressed at high levels in the tumors isolated from HCC patients, which is consistent with previous studies [13]. Our study also showed that AGO2 was associated with lower overall survival of the HCC patients, suggesting that AGO2 could serve as an independent predictor for worse clinical outcomes. It has been well established that AGO2 is important for the developmental processes, including neural tube closure, cardiac failure, and embryonic lethality [6,21]. Recently, increasing evidences have demonstrated that AGO2 also plays multifunctional roles in tumorigenesis and progression. In this study, we generated AGO2 knockout SMMC-7721 cells by using CRISPR/Cas9-based gene editing technology. The depletion of AGO2 obviously reduced cell proliferation and migration, as previously reported [9,13]. Parallel results were observed from in vivo experiments. Mice bearing AGO2 -/xenografts showed decreased tumor sizes and delayed tumor progression. These data confirmed that AGO2 could be a promising indicator for prognostic prediction of HCC.
With nuclease and ribonuclease activity [22,23], AGO2 generally affects tumorigenesis by regulating miRNA processing and maturation. Despite AGO2 being an essential mediator of miRNA function, recent studies have also revealed that AGO2 could directly interact with mRNA of famous oncogenes via miRNA-independent ways [20]. For instance, it has been proved that AGO2 could bind to the promoter of FAK (focal adhesion kinase), leading to the improvement of HCC metastasis [13]. Aberrant expression of multiple genes might contribute to HCC development and progression [24,25]. Therefore, we further determined the AGO2-associated gene expression profiling by performing mRNAs microarray analysis using native and gene-  The bioinformatics analysis showed a potential role of AGO2 in regulating a series of mRNA transcription, especially of which related to the cell proliferation and migration. Knockout of AGO2 conducted by CRISPR/Cas-9 system significantly reduced the expression of Survivin, Vimentin, and Snail in both mRNA and protein levels. Survivin was found crucial in mediating cell proliferation and survival of hepatocellular carcinoma [16,26]. Vimentin and Snail are typical mesenchymal biomarker proteins that could promote HCC metastasis [27]. Our results suggested the participation of these molecules in AGO2 promoting HCC progression. A future work could further illustrate the molecular mechanisms how AGO2 regulates the expression of Survivin, Vimentin, and Snail.

Conclusions
In conclusion, high expression of AGO2 is a potential indicator for prognostic prediction of HCC. Disruption of AGO2 gene expression led to inhibition of HCC tumor proliferation and metastasis in vitro and in vivo. The depletion of AGO2 could remarkably decrease the expression of two clusters of genes involved in cell proliferation (e.g., Survivin) and cell metastasis (e.g., Snail, Vimentin), respectively. This study not only explored the role of AGO2 in the tumorigenesis and progression in HCC but also revealed that AGO2 might facilitate HCC tumorigenesis through modulating expression of Survivin, Vimentin, and Snail.