Dysregulation of miRISC Regulatory Network Promotes Hepatocellular Carcinoma by Targeting PI3K/Akt Signaling Pathway

Hepatocellular carcinoma (HCC) remains the third leading malignancy worldwide, causing high mortality in adults and children. The neuropathology-associated gene AEG-1 functions as a scaffold protein to correctly assemble the RNA-induced silencing complex (RISC) and optimize or increase its activity. The overexpression of oncogenic miRNAs periodically degrades the target tumor suppressor genes. Oncogenic miR-221 plays a seminal role in the carcinogenesis of HCC. Hence, the exact molecular and biological functions of the oncogene clusters miR-221/AEG-1 axis have not yet been examined widely in HCC. Here, we explored the expression of both miR-221 and AEG-1 and their target/associate genes by qRT-PCR and western blot. In addition, the role of the miR-221/AEG-1 axis was studied in the HCC by flow cytometry analysis. The expression level of the AEG-1 did not change in the miR-221 mimic, and miR-221-transfected HCC cells, on the other hand, decreased the miR-221 expression in AEG-1 siRNA-transfected HCC cells. The miR-221/AEG-1 axis silencing induces apoptosis and G2/M phase arrest and inhibits cellular proliferation and angiogenesis by upregulating p57, p53, RB, and PTEN and downregulating LSF, LC3A, Bcl-2, OPN, MMP9, PI3K, and Akt in HCC cells.


Introduction
Hepatocellular carcinoma (HCC) is the most common cancer and the third leading cancer-related death with a poor prediction [1]. HCC is the fastest rising cancers, following others, and has the highest incidence in developing countries, such as India [2]. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major risk factors in developing human HCC, with approximately 50-80% of cases from HBV and 10 to 25% from HCV infections, respectively [3]. Non-alcoholic fatty liver disease (NAFLD) has a role in developing HCC caused by the accumulation of fat exceeding 5% of liver weight in the absence of alcohol. NAFLD is a non-viral risk factor for developing HCC worldwide that majorly causes liver damage that leads to HCC from liver cirrhosis [4,5]. Surgery is considered the primary treatment for HCC, and recent studies have reported that chemotherapy is the most suitable treatment for HCC [6]. Nevertheless, the overall survival time of drug-treated patients is merely 2-5 years [7]. The molecular studies and targeted therapies for the regulatory protein networks of the apoptosis, cell cycle, and angiogenesis progression could be promising for treating HCC.
2.3. miR-221/AEG-1 Axis Regulates Apoptosis, Cell Cycle, Angiogenesis, and Autophagy Mechanism by the Activation of Regulatory Genes in HCC Cells In Vitro Next, we analyzed the AEG-1 and miR-221 roles in their regulatory network, which regulates cell cycle, apoptosis, angiogenesis, and autophagy in miR-221 mimic-, miR-221 inhibitor-, AEG-1 siRNA-, and their corresponding controls-transfected HCC panel by qRT-PCR. We observed the decreased expression of LSF, MMP9, OPN, Bcl2, LC3A, and PI3K/Akt and increased the PTEN, p57, p53, and RB expressions (Figures 3 and 4) in the AEG-1 siRNA-and miR-221 inhibitor-transfected groups in HCC cells. The outcomes revealed that AEG-1 and miR-221 could play an essential role in the HCC regulatory networks.

Knockdown of miR-221 and AEG-1 Inhibits Invasion, Migration, and Cellular Proliferation In Vitro
Next, we examined the effects of AEG-1 and miR-221 in the invasion, migration, and cellular proliferation activity by transwell assays, wound healing, and cell viability assays

Downregulation of miR-221 and AEG-1 Promotes Apoptosis and Cell Cycle Arrest in HCC Cells In Vitro
Furthermore, we performed flow cytometry analysis to confirm the miR-221 and AEG-1 regulation of cell cycle and apoptosis in HCC cells. HCC cells were treated with miR-221 mimic/inhibitor and AEG-1 siRNA and performed cell cycle analysis by PI staining and apoptosis assay by Alexa Fluor-conjugated Annexin V-FITC/PI dual-staining. The apoptosis ( Figure 6A) and cell cycle ( Figure 6B) results showed that the percentage of the apoptotic cells increased in G0-G1 and induced cell cycle arrest in sub-G1 and G2/M compared with miR-221 mimic and their corresponding control groups. These results proved that miR-221 and AEG-1 were involved and could control cell cycle regulation and apoptosis.

Downregulation of miR-221 and AEG-1 Inhibits Angiogenesis and Enhances Apoptosis and Cell Cycle Arrest by Modulating Regulatory Proteins In Vitro
We analyzed the AEG-1 protein expression HCC panel and confirmed that the relative protein expression of AEG-1 was overexpressed significantly in HepG2, Huh-7, and Hep3B compared to THLE-2 cells ( Figure 7A). Following, the HCC cells were transfected with miR-221 mimic, miR-221 inhibitor, and AEG-1 siRNA, with corresponding controls to analyze the relative AEG-1 expression. The results showed that miR-221 mimic, miR-221 inhibitor, and their corresponding controls did not alter AEG-1 expression and significantly decreased in the AEG-1 siRNA-transfected group when compared to the control ( Figure 7B).           , and Hep3B (C) cells using β-actin as an internal control. Error bars are represented as mean ± s.d. and * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the control group. ns: non-significance.

Discussion
Recently, cancer has been the main challenge globally with limited treatment. Chemotherapy is considered a potential treatment that improves the survival of the patients. However, early cancer detection and prevention remain challenged because it is the only disease affecting the normal cell directly without any injury. In recent days, targeted therapies have been considered an advanced treatment for early detection and cancer metastasis [30]. HCC is one of the aberrant cancers in growing countries such as India. HBV, HCV, and NAFLD are the common risk factors for causing HCC [31], which stimulates cell proliferation by regulating several oncogenes or oncogenic miRNAs, especially AEG-1 [32] and miR-221 [33]. Astrocyte-elevated genes are one of the targets of the tumor-associated antigen (TAA), which also is a component of RISC that leads to regulating the miRNAs [20,21,34]. TAA is a class of tumor antigens that are overexpressed in cancer cells and observed in a lower expression in normal cells. TAA promotes tumorigenesis by regulating cell proliferation, chemoresistance, metastasis, and apoptosis in cancers like HCC [28]. These findings revealed that AEG-1 is frequently overexpressed in human cancers and associated with several hallmarks of HCC. We affirmed the previous findings that AEG-1 is overexpressed in HCC cells, which indicates that AEG-1 is a therapeutic hallmark for HCC.
The advanced evidence showed the miRNAs that contributes in several human pathological process, including cancers. The oncogenic or tumor suppressor miRNAs dysregulate the specific regulatory mRNAs, which are mainly involved in the malignant activities including metastasis, survival, proliferation, chemoresistance, and apoptosis in various cancers, including HCC. miR-221 is one of the onco-miRNAs that is significantly overexpressed in cancers and regulates cell proliferation and metastasis. Our findings revealed the aberrant expression of miR-221 and its regulation in HCC. These findings prove that miR-221 plays a crucial role in liver carcinogenesis.
Earlier studies confirmed that AEG-1 was targeted by some tumor suppressor miR-NAs, especially miR-375 and miR-195. Also, it showed lower expression of these tumor suppressor miRNAs during carcinogenesis. The ectopic expression of miR-375 and miR-195 inhibited cell proliferation, angiogenesis, and cancer metastasis by targeting and inhibiting the AEG-1 expression in HCC [8,[27][28][29] and cervical cancers [11]. However, the ectopic expression of onco-miR-221 enhanced cellular proliferation and metastasis in HCC through the dysregulation of their targeted oncogenes and tumor suppressor mRNAs [24][25][26]. Moreover, miR-221 enhanced the angiogenesis activity in HCC by upregulation of SND1 oncogene [35]. These findings confirmed that AEG-1 and miR-221 play major oncogenic roles and regulate human carcinogenesis, including HCC. The silencing of AEG-1 and miR-221 inhibits angiogenesis activity and cell proliferation in several cancers, including HCC. Therefore, we figured that the silencing of AEG-1 and miR-221 inhibits angiogenesis and cellular proliferation by AEG-1 siRNA and anti-miR-221 in HCC cells. The miRNAs play a vital role in cell cycle regulation and apoptosis and induce cell cycle regulation by targeting the regulatory mRNA during carcinogenesis. The ectopic expression of tumor suppressor miRNAs miR-875-5p and miR-375 enhanced G0 phase apoptosis by G1 Phase arrest. It confirmed that the upregulation of miR-875-5p [36] and miR-375 [9] induce apoptosis and cell cycle arrest through the silencing of AEG-1 in cervical cancer and HCC. Our results confirmed that the silencing of AEG-1 and miR-221 induces apoptosis in sub-G0-G1 and G2-M phase arrest and inhibits angiogenesis and cellular proliferation in HCC cells.
Previous studies revealed the onco-miR-oncogene cluster regulations and proved that the upregulation of onco-miR-26a and the CENTG1 oncogene enhances cell proliferation and metastasis in Glioma cancer cells [37]. However, the molecular mechanisms of the onco-miR/oncogene cluster and their correlations are still unclear and limited in human cancers, including HCC, especially the miR-221 and AEG-1 cluster. Therefore, we focused on the onco-miR-oncogene regulation and correlations in HCC, especially the miR-221 and AEG-1 axis. Also, we analyzed the miR-221 and AEG-1 expressions, correlations, and effects of this cluster on their regulatory network proteins, which involve apoptosis, cell proliferation, invasion, autophagy, and angiogenesis in HCC.
Several findings showed that oncogenes or onco-miRNAs target several oncogenes and tumor suppressor regulatory genes directly, or association through pathways. One of these oncogenes is the LSF/TFCP2 (transcription factor LSF) involved in the angiogenesis regulations and is overexpressed in human cancers, including HCC [38]. The LSF targets some downstream signal genes and different regulatory mRNAs, including OPN [39] and MMP9 [40]. Osteopontin (OPN) is a phospho-glycoprotein involved in tumor metastasis and cell death. The matrix metalloproteinase 9 (MMP9) is also responsible for cell growth and cancer metastasis. It helps tumor growth by enhancing angiogenesis regulation during carcinogenesis. The overexpression of LSF enhances the angiogenesis activity, cell invasion, and migration in HCC cells. In addition, it was previously identified that the transcription factor LSF is a direct downstream target of AEG-1, and the LSF mRNA levels significantly increased during the overexpression of AEG-1 in HCC cells [41].
Interestingly, miR-221 regulates OPN in osteoblasts and induces the OPN protein levels during carcinogenesis [42]. On the other hand, the AEG-1 targets MMP2/9 in thyroid cancer cells and enhances cell invasion and migration by inducing the MMP2/9 [43]. Moreover, the upregulation of miR-221/222 regulates and induces the MMP-9 protein expression in pancreatic cancers [44]. Our outcomes confirmed that the LSF, OPN, MMP-9 mRNA, and protein levels decreased while silencing the miR-221 and AEG-1 in HCC cells. The miRNAs or oncogenes regulate their downstream target proteins through direct targets or signaling pathways. The signaling pathways playing critical roles and promoting carcinogenesis include tumor initiation and progression. NF-κB is an essential transcription factor, and several pathological and physiological processes regulate this pathway in human cancers, including cancer development and metastasis. The NF-kB is an aberrant expression in tumors by the miRNAs or some oncogenes through different molecular mechanisms [45], including AEG-1 [46] and miR-221 [47].
miRNAs play a crucial role in cell proliferation that controls or promotes cell differentiation by dysregulating the cell cycle or apoptosis regulatory proteins such as PTEN, p27, and p57 in human cancers. The PTEN is a tumor suppressor protein that helps prevent cell proliferation and induces cell cycle arrest to promote apoptosis in normal conditions (48,49). The cyclin-dependent kinase inhibitor 1B (P27) and cyclin-dependent kinase inhibitor 1C (p57) work as inhibitors of the cyclin/cyclin-dependent kinase (CDK) complexes in the different phases of the cycle to prevent cell differentiation and induce the cell cycle arrest. The ectopic expression of miR-221 regulates PTEN, inhibiting its expression in lung cancer [48] and inhibiting the p57 and p27 protein levels in HCC. During knockdown of the miRNA-221, the p57 and p27 expression level increases, resulting in induced cell death in HCC cells [49]. On the other hand, some findings showed that the AEG-1 oncogene targets PTEN and p57 and regulates their protein level in HCC [24]. Our results also prove that the PTEN, p27, and p57 expressions significantly increase while silencing the AEG-1 and miR-221 in HCC cells.
The activation of the NF-kB signaling pathway alters several regulatory proteins, including PTEN. The overexpression of NF-kB inhibits the transcription level of PTEN by the activation of anti-apoptotic protein Bcl-2 and promotes cell proliferation [50]. NF-kB inhibits cell death and promotes cell proliferation in prostate cancer by the overexpression of Bcl-2 [51]. Some previous studies confirmed that AEG-1 regulates Bcl-2 and significantly downregulates the Bcl-2 protein levels while silencing the AEG-1 in HCC [52]. Moreover, the miR-221 regulates Bcl-2 and BAX proteins, enhances the BAX protein level, and inhibits Bcl-2 while silencing the miR-221 in bladder cancer [53]. The PI3K/Akt is another essential signaling pathway that involves and regulates cancer activation and metastasis. The upregulation of PI3K/Akt induces MDM2-mediated p53 degradation by silencing PTEN protein levels in gastric cancer [54].
Based on the earlier studies, both AEG-1 and miR-221 regulate several cancers, including HCC, by direct targeting or through signaling pathways. Hence, the interaction between the AEG-1 and miR-221 in HCC is not yet known. We identified a part of this, such as AEG-1 and miR-221 regulating cooperatively in HCC tumorigenesis.

miRNA and siRNA Transfection
HCC cells were cultured in a 6-well plate (1 × 10 6 ) and transfected with miR-221 mimic, miR-221 inhibitor, and their control miRNAs (5 µM), small interfering RNA (siRNA) negative control, and AEG-1 siRNA (sense: 5 -GACACUGGAGAUGCUAAUAUU-3 , antisense: 5 -UAUUAGCAUCUCCAGUGUCUU-3 ) (2 nM) as per the manufacture protocols using Lipofectamine 2000 and RNAi MAX reagents (Invitrogen, USA) in Opti-MEM medium (Thermo Fisher, Waltham, MA, USA). The mock control group was treated with transfection reagent alone in Opti-MEM (Without miRNAs and siRNAs). The transfection complex was prepared, and the cells were added directly to the transfection complex and incubated at 37 • C for 48 h. The transfection complex was removed in 6-8 h incubation and replaced with the fresh culture medium. The cells were collected and analyzed for the effects of miRNA and siRNA on the treated groups after 48 h transfection by quantitative real-time PCR (qRT-PCR) (Life Technologies, Burlington, Ontario, Canada).

Quantitative Real-Time PCR
Total RNA was isolated from the RNAi-transfected HCC cells using TRIZOL (Invitrogen, CA, USA). The 2 µg of total RNA was collected and randomly primed to synthesize the complementary DNA (cDNA) using the SuperScript First Strand cDNA Synthesis kit (K1622) (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. Following, we performed the qRT-PCR using Step One plus qRT-PCR system (Life Technologies, Burlington, ON, Canada) with SYBR™ Green Master Mix-Real-Time PCR Master Mix (Applied Biosystems, Waltham, MA, USA) to analyze the miR-221, AEG-1, LSF, MMP9, p57, p53, RB, OPN, PTEN, Bcl-2, PI3K, Akt, and LC3A mRNA expressions. The primers were purchased from Eurofins Genomics (Louisville, KY, USA) and listed in Supplementary Table S1. The U6 snRNA and GAPDH were internal controls for miR-221 and AEG-1. The data was collected, and the specificity of the primers was verified by melt curve analysis. The fold changes of miR-221 and mRNAs were calculated using the 2 −∆∆Ct method.

Transwell Migration and Invasion Assay
The transwell chamber (Corning) with the Matrigel-coated insert (2 mg/mL) was used for the invasion assay, and the Matrigel-free inserts were used for the migration assay. For invasion assay, cells were transfected with miR-inhibitor, miR-221 mimic, AEG-1 siRNA, and their corresponding controls for 48 h. Following the incubation, cells were collected and seeded (1 × 10 5 ) in the upper chamber of the 8 µm transwell inserts with a serum-free DMEM. FBS (10%) containing DMEM was filled in the lower chamber and incubated for 48 h at 37 • C. After, the upper chamber was removed from the plate and fixed with methanol for 20 min at RT and stained with crystal violet (0.1 mg/mL). Following the staining, the non-invading cells were removed from the surface of the Matrigel, a cotton swab was used for removal, and the invading cells on the bottom of the upper chamber were imaged and counted using a light microscope with 20× magnification (Carl Zeiss, North York, ON, Canada). The same method was performed on the Matrigel-free membrane inserts.

Annexin V-FITC/PI Double Staining of Cell Apoptosis
The Annexin dual staining assay was performed to detect the apoptosis activity in HCC cells using Annexin V-FITC and propidium iodide (PI) (Invitrogen, Carlsbad, CA, USA). The cells were collected after 48 h incubation and washed twice with ice-cold phosphate buffer saline (PBS), and then the cells were resuspended in 100 µL of binding buffer containing Annexin V-FITC (5 µL) and PI (1 µL) (1 mg/mL) and kept in the dark (RT) for 15 min. Following the incubation, 400 µL of the binding buffer was added to each tube, and the cells were mixed well and analyzed within an hour using BD Accuri C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Flowjo TM software (v.10.0.6) (Becton, Dickinson and Company, Ashland, OR, USA) was used for data analysis.

Propidium Iodide Staining of Cell Cycle Analysis
The flow cytometry analysis was performed to analyze the cell cycle using propidium iodide staining. After 48 h transfection, cells were collected and washed with ice-cold PBS twice and fixed with 70% ice-cold ethanol at −20 • C for 24 h. Following the incubation, cells were stained with 250 µL of propidium iodide (PI) containing RNAs staining solution (50 mg mL/1 mg) (MP Biomedicals, Santa Ana, CA, USA) and incubated at 4 • C in the dark for 30 min. Following the incubation, samples were mixed well and analyzed using FACS Calibur flow cytometry (BD FACS). FlowJo™ Software v.10.0.6 (Becton, Dickinson and Company, Ashland, OR, USA) was used for data analysis.

Wound-Healing Assay
The HCC cells were seeded in 12-well plates (1 × 10 5 ) and transfected with RNAi. After the 48-h incubation, a wound was created on the surface of the monolayer using a 200 µL tip, and images were captured at different time intervals (0, 12, and 24 h) using Floid Cell Imaging Station (Life Technologies, Burlington, ON, Canada). The wound gap and width were measured by using ImageJ (v.1.46r) (National Institutes of Health, Bethesda, ML, USA).

Western Blotting Analysis
HCC cells were treated with the miR-221 inhibitor, miR-221 mimic, AEG-1 siRNA, and the corresponding controls. Following the 48 h, the cell lysate was collected and homogenized using RIPA (Radio Immunoprecipitation Assay) lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA). Lowry's method was performed to estimate the protein.

Statistical Analysis
GraphPad Prism (v 7.0) (GraphPad Software, San Diego, CA, USA) was used for statistical analyses, and one-way ANOVA was used for comparison. p < 0.05 was considered statistically significant, and all the data were performed and presented in at least three separate experiments.

Conclusions
We investigated and proved that miR-221 and AEG-1 were overexpressed in HCC and enhanced the HCC tumorigenesis by the regulatory network and signaling pathways. Recent studies considered targeted gene silencing by oncogenic or tumor suppressor miRNAs for diagnosis and therapy. We showed both onco-miR-221/AEG-1 oncogene axis regulations and their role in HCC. Our results indicated that the miR-221 expression level decreased in AEG-1 siRNA-transfected group, but the miR-221 did not alter the AEG-1 expression level in HCC cells. Our results showed that the miR-221 interaction with AEG-1 through RISC activation of miR-221 may be associated with AEG-1 during RISC complex formation and regulation by optimizing or enhancing the miR-221 activity. This complex leads to the dysregulation of their target/associated genes in HCC tumorigenesis.
Moreover, the silencing of both miR-221 and AEG-1 significantly increased the p53, p57, RB, and PTEN expression levels and decreased BCL2, LSF, MMP9, LC3A, PI3K, and p-Akt mRNA and protein levels in HCC cells. These findings revealed that the AEG-1/miR-221 plays a crucial role during HCC tumorigenesis by direct targeting or through PTEN/PI3K/Akt signaling pathways. Thus, the AEG-1 and miR-221 describe a novel therapeutic strategy to treat HCC. However, the exact interaction between miR-221 and AEG-1 in RISC and their molecular mechanism remains unclear. Further studies on the relationship between miR-221 and AEG-1 are essential to confirm their regulation in vivo. Further, our future study will explore the miR-221 and AEG-1 effects on RISC in NAFLD or liver cirrhosis-associated HCC.