MiR-29a Curbs Hepatocellular Carcinoma Incidence via Targeting of HIF-1α and ANGPT2

A high-fat diet is responsible for hepatic fat accumulation that sustains chronic liver damage and increases the risks of steatosis and hepatocellular carcinoma (HCC). MicroRNA-29a (miR-29a), a key regulator of cellular behaviors, is present in anti-fibrosis and modulator tumorigenesis. However, the increased transparency of the correlation between miR-29a and the progression of human HCC is still further investigated. In this study, we predicted HIF-1α and ANGPT2 as regulators of HCC by the OncoMir cancer database and showed a strong positive correlation with HIF-1α and ANGPT2 gene expression in HCC patients. Mice fed the western diet (WD) while administered CCl4 for 25 weeks induced chronic liver damage and higher HCC incidence than without fed WD mice. HCC section staining revealed signaling upregulation in ki67, severe fibrosis, and steatosis in WD and CCl4 mice and detected Col3a1 gene expressions. HCC tissues significantly attenuated miR-29a but increased in HIF-1α, ANGPT2, Lox, Loxl2, and VEGFA expression. Luciferase activity analysis confirms that miR-29a specific binding 3′UTR of HIF-1α and ANGPT2 to repress expression. In summary, miR-29a control HIF-1α and ANGPT2 signaling in HCC formation. This study insight into a novel molecular pathway by which miR-29a targeting HIF-1α and ANGPT2 counteracts the incidence of HCC development.


Introduction
Hepatocellular carcinoma (HCC) is considered the sixth most common cancer and the second principal detriment of cancer-related deaths worldwide, accounting for approximately 841,000 new cases and 782,000 deaths every year [1,2]. Risk factors include viral hepatitis B, hepatitis C, alcoholic fatty liver, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) are ultimately the causes of liver fibrosis and cirrhosis, and nearly 70-80% will convert into HCC patients [3,4]. Solid tumors have a feature on in HCC patients and matched normal data to evaluate their roles in HCC progression. HIF1A and ANGPT2 gene expression were characterized by managing the bioinformatics web-based platform UALCAN. The mRNA expression data were obtained from RNA-seq profiles and generated from the TCGA (The Cancer Genome Atlas) datasets. The results showed that, compared with normal clinical patients, the expression of HIF1A and ANGPT2 in HCC was upregulated (Figure 2A,B), and was correlated with decreased HCC patient survival rate by the Kaplan-Meier method analysis.

Western Diet (WD) Combine Carbon Tetrachloride (CCl4) Treated Promote Chronic Liver Disease and Cancer Formation
To generate a clinical comparative animal model and accelerated progress of HCC, we followed administration procedures described by Tsuchida et al. [25], which consists of multiple treated CCl4 with Western diet feed as the WD/CCl4 group ( Figure 3A). Feed WD was shown to enhance and accelerate the roughness of fur, but there was no significant difference in body weight compared with the normal diet (ND) plus CCl4 group (Figure 3B,C). A significant discrepancy in liver pathology was observed between ND/CCl4 and WD/CCl4 groups. After 25 weeks of WD/CCl4 treatment, nodules were obtained in the liver tissue of mice, indicating that hepatocellular carcinoma (HCC) was induced and

Western Diet (WD) Combine Carbon Tetrachloride (CCl4) Treated Promote Chronic Liver Disease and Cancer Formation
To generate a clinical comparative animal model and accelerated progress of HCC, we followed administration procedures described by Tsuchida et al. [25], which consists of multiple treated CCl4 with Western diet feed as the WD/CCl4 group ( Figure 3A). Feed WD was shown to enhance and accelerate the roughness of fur, but there was no significant difference in body weight compared with the normal diet (ND) plus CCl4 group ( Figure 3B,C). A significant discrepancy in liver pathology was observed between ND/CCl4 and WD/CCl4 groups. After 25 weeks of WD/CCl4 treatment, nodules were obtained in the liver tissue of mice, indicating that hepatocellular carcinoma (HCC) was induced and successfully established an animal disease model ( Figure 3D). To test whether cancer-related gene ki67 involve in HCC growth and proliferation. We performed immunohistological analysis, demonstrated that liver tissue from the WD/CCl4 group displayed strong signaling of ki67 and quantified positively stained cells ( Figure 3E). Masson staining showed that the WD/CCl4 group also resulted in severe liver fibrosis and infiltration of inflammatory cells under the disordered lobular structure ( Figure 3F). In addition, we further performed qRT-PCR to detect the expression level of Col3al, which confirmed that the liver tissue of the WD/CCL4 mice had excessive collagen deposition in liver tissue compared to ND/CCl4 mice ( Figure 3G).
successfully established an animal disease model ( Figure 3D). To test whether cancer-related gene ki67 involve in HCC growth and proliferation. We performed immunohistological analysis, demonstrated that liver tissue from the WD/CCl4 group displayed strong signaling of ki67 and quantified positively stained cells ( Figure 3E). Masson staining showed that the WD/CCl4 group also resulted in severe liver fibrosis and infiltration of inflammatory cells under the disordered lobular structure ( Figure 3F). In addition, we further performed qRT-PCR to detect the expression level of Col3al, which confirmed that the liver tissue of the WD/CCL4 mice had excessive collagen deposition in liver tissue compared to ND/CCl4 mice ( Figure 3G).

WD/CCl4 Treated Intervention miR-29a Expression and Promoted Tumorigenesis Signaling in Liver Tissue
Furthermore, miR-29a decreased expression in high-risk HCC using WD/CCl4treated mice ( Figure 4A). Expanding evidence has revealed that miR-29a is indispensable in chronic liver disease [26]. We uncovered that liver tissue of mice treated with WD/CCl4 upregulated the expression of HIF-1α and Angpt2 ( Figure 4B,C). This finding is consistent with liver cancer patients' bioinformatics analysis and regulatory network, whereas Lox, Loxl2, and Vegfα mRNA expression also increased in WD/CCl4 mice rather than ND/CCl4 mice ( Figure 4D-F).

WD/CCl4 Treated Intervention miR-29a Expression and Promoted Tumorigenesis Signaling in Liver Tissue
Furthermore, miR-29a decreased expression in high-risk HCC using WD/CCl4-treated mice ( Figure 4A). Expanding evidence has revealed that miR-29a is indispensable in chronic liver disease [26]. We uncovered that liver tissue of mice treated with WD/CCl4 upregulated the expression of HIF-1α and Angpt2 ( Figure 4B,C). This finding is consistent with liver cancer patients' bioinformatics analysis and regulatory network, whereas Lox, Loxl2, and Vegfα mRNA expression also increased in WD/CCl4 mice rather than ND/CCl4 mice ( Figure 4D-F). The expression of miR-29a in the WD/CCl4 treatment group was significantly reduced, and (B-F) five related genes were significantly increased. Data per group are expressed as mean ± SEM calculated from five to six mice. * p-value < 0.05; ** p-value < 0.001 between groups.

miR-29a Targeted the 3′-UTR of HIF-1a and ANGPT2
As predicted by bioinformatics (miRBase 22.1) show HIF-1a and ANGPT2 putative target of miR-29a. We constructed luciferase reporters for control, and the 3-base pair mutated 3′-UTR of HIF-1a and ANGPT2 ( Figure 5A,B) that deciphered how miR-29a affected specific areas gene expression in HepG2 cells. Notably, increased miR-29a significantly decreased luciferase reporter activity of 3′-UTR of HIF-1a and ANGPT2 ( Figure 5C,D), whereas miR-29a-mimic also attenuated protein expression of HIF-1a and ANGPT2 (  The expression of miR-29a in the WD/CCl4 treatment group was significantly reduced, and (B-F) five related genes were significantly increased. Data per group are expressed as mean ± SEM calculated from five to six mice. * p-value < 0.05; ** p-value < 0.001 between groups.

Discussion
Hepatocellular carcinoma (HCC) is a common malignant liver disease, which accounts for one of the leading cause of mortality globally [2]. Clinical diagnosis of various liver diseases, including chronic viral and C, alcoholic liver disease, NAFLD and NASH, etc., all increase the probability of HCC [27,28]. While accumulating studies reveal that dysfunctional hepatic cells, oxidative stress, and immune dysregulation accelerate HCC development [29,30], little is known about how chronic liver disorders shift into HCC. Clarifying the underlying molecular mechanisms of the progression of HCC is essential for determining novel therapeutic targets for HCC. As HCC diagnosis and treatment status is not promising, many clinical trials looking for more ideal tools are underway. One of these tools is miRNA, which can be considered a promising HCC diagnostic and prognostic tool. The importance of miRNA dysregulation and expression has been confirmed in many cancers [31,32]. Chaotic miRNA expression is related to HCC tumorigenesis, and plasma miRNA expression has also been mentioned as a potential regulator of HCC [33].
Our previous study confirmed that miR-29a targets multiple biological function impacts, including attenuated shoulder stiffness fibrosis [34], liver fibrosis [8,12,35], and mitigated NAFLD [14,36], as well as influenced progression of HCC formation [26]. Similar findings have been confirmed by Song et al., who showed miR-29a downregulated Bcl-2 expression to ameliorate liver tumorigenesis [35]. In our previous study, we utilized and combined multiple public databases that perceived miR-29a expression was significantly decreased in HCC patients [16]. Of note, research establishes HIF-1α dominated roles in HCC patients that co-expression of HIF-1α and PD-L1 has significantly increased the risk of recurrence [37]. In this study, we also provide direct evidence that the two prognostic markers HIF-1α and ANGPT2 are upregulated in HCC patients and HCC animal models. In addition, our research also confirmed that miR-29a has a broader epigenetic effect on tumorigenesis-related gene expression and could regulate the expression of HIF-1α and ANGPT2 in HepG2 cells.
It is well-known that the microenvironment of tumorigenesis increased hypoxia status to promote neovasculogenesis for additional growth [38]. The significant role of HIF-1α in tumor development has been demonstrated in various tumor types and is responsible for tumor initiation, progression, and drug resistance [22]. Along with the crucial role of HCC growth under hypoxia conditions, the accumulation of reactive oxygen species (ROS) leads to the electron transport chain of mitochondrial obscurely and oxidative stress [39]. In this reaction, HIF-1α co-work with Notch signaling to modulate mitochondrial biogenesis metabolism and cross-reacted with HEY1 and PINK1 gene expression [39]. Furthermore, HIF-1α was also shown to strongly correlate with a higher rate of lymph node metastasis and vasculogenic mimicry [38].
Again, ANGPT2 (angiopoietin-2) as a prognostic marker plays a pivotal role in liver cancer and concomitantly with ANGPT1 (angiopoietin-1) and VEGF (vascular endothelial growth factor) promoted activity hypervascularity [17]. In steatohepatitis, the severity of pathogenesis was positively correlative with serum concentration of ANGPT2 and indispensable found, for which ANGPT2 excess of the HCC organization can be used as a predictor in HCC patients in recurrent or de novo rate. At the same time, the ANGPT2 showed a positive relationship with liver stiffness [40]. Evolving evidence suggests that ANGPT2 may utilize two molecular pathways to influence the growth of HCC and that ANGPT2 could secrete via exosomes and exist on the surface of HCC to promote epithelialto-mesenchymal transition (EMT) activity [41]. On the other hand, the ANGPT2 and TIE2-expressing monocytes (TEMs) co-excess pathway has also been confirmed to have participated in metastatic and recurrent HCC [42].
Advanced HCC implicated multiple cellular pathways and played central roles in tumor metastasis and recurrence. Activity signaling cascade in HCC, including Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR, which promotes transcription of genes involved in tumor proliferation [43]. Sorafenib, a TKI (tyrosine kinases inhibitor) drug, was the first multikinase inhibitor authorized for the medicine of HCC. The efficacy of Sorafenib has been considered in numerous clinical trials and demonstrated affects many kinases, not only RAF and MEK, but other kinases, such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and others. However, emerging studies found that advanced HCC patients who exhibit a B-RAS mutation or DCP (des-γcarboxyl prothrombin) are more likely to be multifocal, aggressive, and resistant to TKI therapies [44,45]. Thus, finding more signaling pathways such as epigenetic moderators include Long non-coding RNA (lncRNA) [44], siRNA [46], miR agonists/antagonists [47], and small molecules [48], could help develop promising therapeutic strategies.
Trans-arterial chemoembolization (TACE) is a first-line treatment for patients with hepatocellular carcinoma (HCC) in Barcelona Clinic Liver Cancer stage B (BCLC-B) [2]. However, TACE puts hypoxic and chemotherapeutic stress on HCC, and some surviving tumors frequently transform into more aggressive and TACE-resistant tumor tissues [49,50]. As a result, hypoxia-induced by TACE can stimulate VEGF production by the residual tumor cells, promoting angiogenesis and ultimately tumor progression following TACE [51,52]. AMG386, a peptibody (first-in-class peptide-Fc fusion protein) that impedes ANGPT1/2 signaling, was exploited to develop trebananib for clinical studies with human patients [53,54]. Furthermore, HIF-1 activity is required to express some lysyl oxidase (LOX) family members, including Lox, Loxl2, and Loxl4 [55][56][57]. LOX family members are characterized by their catalytic activity contributing to structural integrity and increased tensile strength, acting to remodel the cross-linking of the structural extracellular matrix (ECM) of such fibrotic organs as the liver [58,59], as well as cancer microenvironments [58,60]. More recently, mounting evidence has recognized the emerging role of Lox and Loxl2/4 in fostering the corrupt microenvironment of HCC via angiogenesis promotion, epithelial-mesenchymal transition (EMT) program, and formation of pre-metastatic sites [55,[61][62][63]. While the study delineates the contribution of miR-29a in the HCC development, considering the initial liver disease and the different stages of long-term development, which have increased the complexity of studying HCC. However, we are also aware of the experiment's limitations that lack identification of miR-29a expression on human liver biopsies of various stages of HCC and engineering virus vector-mediated miR-29a to extend gene therapy strategies for HCC mice to explanation mechanisms on angiogenesis. In addition, the immune response is also the focus of future investigation. For instance, HIFs and miR-29a participated with liver tissue innate immunity [9,64]. In conclusion, profound evidence revealed that miR-29a loss, HIF-1α, and Angpt2 increase were correlated and pronounced influences with HCC development while contributing a clearly identifiable molecular mechanism in chronic liver disease.

miR-29a Interacted Cancer Gene Sets Platform Interpretation and Genes Differential Expression in HCC Patients Outcome Correlation
Bioinformatic predictive analysis of miR-29a regulate gene network in HCC patients, and candidate genes identification were performed public web analysis platforms, including OMCD (OncoMiR Cancer Database), UALCAN, GSCALite, TCGA (Cancer Genome Atlas), and TargetScan database. The detailed analysis procedure refers to the description of the previously published manuscript [15].

The HCC Mice Model Generative
The animal operative protocol, experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Chang Gung Memorial Hospital (KCGMH, Affidavit No. 2020121109). Age 8 weeks C57BL/6N male mice (25-30 g) were purchased BioLASCO (Taipei, Taiwan), and all experimental animals were housed and followed IACUC use committee guidelines. Mice were randomized divided into two groups, which were fed with a regular chow diet (ND) or western diet (WD) containing 21.1% fat, 41% sucrose, and 1.25% cholesterol (TD. 120528, Teklad diets). Group of ND and WD were intraperitoneally given CCl4 (0.32 µg/g of body weight, 289116, Sigma-Aldrich, St. Louis, MO, USA) once a week for 25 weeks [65]. The animal measured body weight every four weeks until euthanasia and harvested the liver tissues for section and staining.

Liver Tissue Section and Staining
Tissue samples from at least two representative fragments of each liver lobes were taken and fixed in 10% paraformaldehyde for 36 h and, then, embedded in paraffin wax. Continuous 5 mm slices underwent Masson's trichrome staining (Polysciences, NY, USA) in accordance with the manufacturer's standard protocol and previously described [66]. For immunohistochemistry, tissue slides were dewaxed and conducted epitope retrieval (Thermal scientific at 95 • C for 30 min). Primary antibodies against Ki67 (ab15580, abcam, Cambridge, UK) were immunoreaction in sections to probed ki67 and reaction color use BioGenex detection kits (BioGenex, Fremont, CA, USA). Histological images were captured utilizing the digital slide scanner (Pannoramic MID), and images were randomly selected for quantification under constant magnification by ImageJ (V1.48) from three fields of each section and two sections of each liver specimen.

Cell Culture and Transfection
Human HCC cell line HepG2 purchased from American Type Tissue Collection (ATCC) and cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamax, and antibiotic-antimycotic at 37 • C in a humidified incubator with 5% CO 2 . HepG2 were seeded into 6-cm dishes (1.5 × 10 6 cells/dish) overnight and then transfected with miR-29a precursor (a miR-29a mimic, GE Healthcare Dharmacon, Inc., Lafayette, CO, USA) or miR negative control (GE Healthcare Dharmacon, Inc.) for 24 h by using the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. The complete cell extracts are subjected to Western Blot analysis.

Quantitative RT-PCR
Total RNA in liver tissue was extracted by using TRIzol ® reagent (Invitrogen, CA, USA) and conducted reverse transcription of 1 µg total RNA to yield cDNA. The qPCR reaction was undertaken using 2× SYBR Green PCR Master Mix (04887352001, Roche Molecular Systems, Pleasanton, CA, USA) on LightCycler480 ® (Roche). The specific primers for detected genes expression and detailed sequences of mouse genes are shown in Table 1. Table 1. Sequence of primers pairs.

Gene Name Forward Primers (5
Tissue samples from at least two representative fragments of each liver lobes were taken and fixed in 10% paraformaldehyde for 36 hours and, then, embedded in paraffin wax. Continuous 5 mm slices underwent Masson's trichrome staining (Polysciences, NY, USA) in accordance with the manufacturer's standard protocol and previously described [66]. For immunohistochemistry, tissue slides were dewaxed and conducted epitope retrieval (Thermal scientific at 95 °C for 30 min). Primary antibodies against Ki67 (ab15580, abcam, Cambridge, UK) were immunoreaction in sections to probed ki67 and reaction color use BioGenex detection kits (BioGenex, Fremont, CA, USA). Histological images were captured utilizing the digital slide scanner (Pannoramic MID), and images were randomly selected for quantification under constant magnification by ImageJ (V1.48) from three fields of each section and two sections of each liver specimen.

Cell Culture and Transfection
Human HCC cell line HepG2 purchased from American Type Tissue Collection (ATCC) and cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamax, and antibiotic-antimycotic at 37 °C in a humidified incubator with 5% CO2. HepG2 were seeded into 6-cm dishes (1.5 × 10 6 cells/dish) overnight and then transfected with miR-29a precursor (a miR-29a mimic, GE Healthcare Dharmacon, Inc., Lafayette, CO, USA) or miR negative control (GE Healthcare Dharmacon, Inc.) for 24 h by using the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. The complete cell extracts are subjected to Western Blot analysis.

Quantitative RT-PCR
Total RNA in liver tissue was extracted by using TRIzol ® reagent (Invitrogen, CA, USA) and conducted reverse transcription of 1 μg total RNA to yield cDNA. The qPCR reaction was undertaken using 2X SYBR Green PCR Master Mix (04887352001, Roche Molecular Systems, CA, USA) on LightCycler480 ® (Roche). The specific primers for detected genes expression and detailed sequences of mouse genes are shown in Table 1.

) Reverse Primers (5
Tissue samples from at least two representative fragm taken and fixed in 10% paraformaldehyde for 36 hours and wax. Continuous 5 mm slices underwent Masson's trichrom USA) in accordance with the manufacturer's standard proto [66]. For immunohistochemistry, tissue slides were dewaxe trieval (Thermal scientific at 95 °C for 30 min). Primary anti abcam, Cambridge, UK) were immunoreaction in sections color use BioGenex detection kits (BioGenex, Fremont, CA were captured utilizing the digital slide scanner (Pannorami domly selected for quantification under constant magnifica three fields of each section and two sections of each liver spe

Cell Culture and Transfection
Human HCC cell line HepG2 purchased from Amer (ATCC) and cultured in DMEM medium supplemented wi bovine serum (FBS), glutamax, and antibiotic-antimycotic a bator with 5% CO2. HepG2 were seeded into 6-cm dishes (1 and then transfected with miR-29a precursor (a miR-29a mi con, Inc., Lafayette, CO, USA) or miR negative control (GE for 24 h by using the Lipofectamine™ RNAiMAX Transfectio bad, CA, USA), following the manufacturer's instructions. T subjected to Western Blot analysis.

Quantitative RT-PCR
Total RNA in liver tissue was extracted by using TRIz USA) and conducted reverse transcription of 1 μg total RN reaction was undertaken using 2X SYBR Green PCR Master M lecular Systems, CA, USA) on LightCycler480 ® (Roche). The genes expression and detailed sequences of mouse genes are

Luciferase Reporter Activity Assay
The oligonucleotides that contained the HIF-1α or ANGPT 3 UTR target sequence were annealed and cloned into the pMIR-REPORTTM miRNA Expression Reporter Vector (Applied Biosystems, Waltham, MA, USA) to generate pMIR-HIF-1α luciferase plasmid or pMIR-ANGPT2 luciferase plasmid. The sequences in which the miR-29a binding site was replaced with the mutant site were annealed and cloned into the pMIR-REPORTM reporter vector to generate the pMIR-HIF-1α Mut luciferase plasmid or pMIR-ANGPT2-Mut luciferase plasmid. We then purified the plasmids using the EasyPrep EndoFree Maxi Plasmid Extraction Kit (BIOTOOLS, Ltd., Taipei, Taiwan). HepG2 cells were seeded at 3 × 10 6 cells in a 10-cm dish for 18 h and transfected with a 6 µg reporter plasmid using Turbofect transfection reagent (Thermo Fisher Scientific, Rockford, IL, USA). After 18 h, the culture medium changed to a fresh medium and placed for 6 h. Post-transfection 24h, cells were trypsinized cells and seeded on a 6-cm dish with a density of 1.6 × 10 6 cells/dish overnight. The cells were transfected with miR-29a precursor (a miR-29a mimic, GE Healthcare Dharmacon, Inc., Lafayette, CO, USA) or MIR negative control (GE Healthcare Dharmacon, Inc.) for 24 h using the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 48 h transfection, cells were lysed for the detection of luciferase signal with Neolite Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA) [67].

Statistical Analysis
The experiment results of transfected cells were repeated six times, and animal tissues as well and were presented as mean ± sem. Between two-groups compare was utilized t-test with unpaired two-tailed. Ordinary one-way ANOVA and Tukey's multiple comparisons test with a single pooled variance were significantly analyzed for multiple groups. Statistical all values for significantly different as the p values were set at <0.05.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author.