In Silico and In Vivo Evaluation of microRNA-181c-5p’s Role in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a fatal disease, accounting for 75–85% of primary liver cancers. The conclusive research on miR-181c-5p’s role in hepatocarcinogenesis, whether it has oncogenic effects or acts as a tumor repressor, is limited and fluctuating. Therefore, the current study aimed to elucidate the role of miR-181c-5p in HCC in silico and in vivo. The bioinformatics analysis of miR-181c-5p expression data in HCC using several databases strongly shed light on its involvement in HCC development, but also confirmed the fluctuating data around its role. miR-181c-5p was proven here to have an oncogenic role by increasing HepG2 cells’ viability as confirmed by MTT analysis. In addition, miR-181c-5p was upregulated in the HCC positive control group and progressed the HCC development and malignant features by its forced expression in an HCC mouse model by targeted delivery using a LA-PAMAM polyplex. This is indicated by the cancerous gross and histological features, and the significant increase in liver function biomarkers. The functional enrichment bioinformatics analyses of miR-181c-5p-downregulated targets in HCC indicated that miR-181c-5p targets were significantly enriched in multiple pathways and biological processes involved in HCC development. Fbxl3, an example for miR-181c-5p potential targets, downregulation and its correlation with miR-181c-5p were validated by qPCR. In conclusion, miR-181c-5p is upregulated in HCC and has an oncogenic role enhancing HCC progression.


Introduction
Hepatocellular carcinoma (HCC) is a lethal disease; it is the fifth most frequent cancer [1] and the fourth largest contributor to cancer deaths worldwide [2]. It represents the fourth most widespread cancer in Egypt [3,4]. The most remarkable risk factors for the emergence of HCC are chronic liver diseases and cirrhosis [5]. Recognizing the molecular pathogenesis of HCC is insufficient and inconclusive so far. Consequently, to develop an efficient therapeutic strategy, conducting a more inclusive analysis of HCC is demand [6]. Lately, studies focused on targeted therapies, due to the aggressiveness of the tumor nature [7].

Database Analysis of the Expression of miR-181c-5p
The human miRNA tissue atlas is available at (https://ccb-web.cs.uni-saarland.de/ tissueatlas/) (accessed on 2 October 2021) and was used to demonstrate miR-181c-5p organ specificity, as miRNA research showed distinct sets of miRNAs that expressed in varied tissues and types of cells. Using dbDEMC2 software (http://www.picb.ac.cn/dbDEMC) (accessed on 4 October 2021), the expression status of miR-181c-5p in normal and tumor tissues, including HCC, was determined. An integrated database called DbDEMC (database of Differentially Expressed MiRNAs in Human Cancers) holds high throughput data on how miRNAs are expressed differentially in human cancers [44]. Other miRNA disease association databases, HMDD [45], miR2Disease [46], and miRcancer [47], that are widely used in the literature were utilized to clarify the differential expression status of miR-181c-5p in HCC. StarBase v2.0 (http://starbase.sysu.edu.cn/) (accessed on 4 October 2021) was used to perform the survival analyses for miR-181c-5p sourced from TCGA [48].

Recombinant pmiR-181c-5p Construct Synthesis
The sequence of pri-miR-181c-5p was amplified from the genomic DNA of human Peripheral Blood Mononuclear Cells (PBMCs), extracted by QIAamp genomic DNA kit (QI-AGEN, USA), using Thermo Scientific DreamTaq Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA) and forward primer: 5 -TCGA-GGATCC-ACTTAAGGAGCG-GGCTTGAG-3 and reverse primer: 5 -TCGA-GCTAGC-TCACAACCCACCGACAACA-3 , according to the manufacturer's instruction. The PCR for pri-miR-181c-5p amplification was carried out as follows: 95 • C for 5 min; and 35 cycles of 94 • C for 15 s, 54 • C for 30 s, and 72 • C for 30 s. The amplified product was subsequently cloned in a pEGP-miR vector to form pmiR-181c-5p with the aid of Thermo Scientific T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA). The cloned constructs were transformed subsequently into the cells of Escherichia coli TOP10 and confirmed by sequencing. Following the manufacturer's guidelines, the verified clone was purified by an endotoxin-free GeneJET Plasmid Maxiprep Kit from Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The Thermo Fisher Scientific Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the concentration and purity of the pmiR-181c-5p construct. The pEGP-miR null vector (pNull) was considered a control vector.

Polyplex Formulation of pEGP-miR-181c-5p
The LA-PAMAM-pmiR-181c-5p and LA-PAMAM-pNull polyplexes were prepared directly before usage, according to the previous method [43] by diluting LA-PAMAM and pmiR-181c-5p or pNull separately in phosphate buffer saline (PBS; pH 7.4) to accomplish equal volumes of the required concentrations. Then, both solutions were set to equilibrate at room temperature for five minutes. The LA-PAMAM solution was added to the pmiR-181c-5p or pNull solution and vortexed gently for 5 s. The weight ratio of LA-PAMAM to plasmid was 2:1. The formed polyplexes of LA-PAMAM-pmiR-181c-5p and LA-PAMAM-pNull afterward were incubated at room temperature for 30 min to be ready to use.

Cell Viability Study
The MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed to evaluate the viability of the cells after treatment with various concentrations of LA-PAMAM-pmiR-181c-5p. In 96-well plates, 2 × 10 4 HepG2 cells per well were seeded and then maintained for 24 h at 37 • C with 5% CO 2 in a humidified environment. The transfection mixture was prepared by adding LA-PAMAM-pNull or LA-PAMAM-pmiR-181c-5p (20 µL) (in different concentrations {0.5-2 µg} plasmid DNA {the ratio of LA-PAMAM to plasmid was 2:1}) to the pre-warmed media (180 µL). This mixture was let to sit for 5 min at room temperature. Afterward, the HepG2 cells were transfected with the transfection mixture (200 µL) directly and incubated for 48 h. After transfection, 40 µL of 5 g/L MTT per well was added and incubated for 4 h at 37 • C with cells, then the media was discarded and the acidified isopropanol solution was added. Assessment of cell viability by measuring the absorbance value for each well at 570 nm on a microplate spectrophotometer was carried out.

Animal Experiment
The experiments on animals were carried out as stated by the guidelines of the National Research Center Animal Care Committee and approved according to ethics (Approval Number 10431). Male Balb-c mice (8 weeks in age) (body weights 24 g ± 4) (n = 80) were retained in a temperature-controlled atmosphere (12 h light/dark cycle at 24 • C, drinking water and feed ad libitum). Mice were adapted to the laboratory environment one week before the experiment's start. Mice were divided randomly into 2 groups: the negative control group (n = 20) and the HCC group (n = 60). HCC induction and treatment were performed based on the modified protocol from Salah et al. (2019). Briefly, liver cancer was induced by intraperitoneal (i.p.) injection of mice by a single dose of DEN (freshly diluted in sodium chloride saline solution with a sterility level of 0.9%), followed by 20 doses of CCl4 (corn oil dissolved 1:2 v/v) received by oral gavage (once/week). HCC was confirmed in the HCC group by liver gross examination and histopathological investigation until the prevalence of liver tumors is expected to become 100% (Supplementary Figure S1). Then, the group of HCC was split into two subgroups treated with DNA-polyplexes, into a LA-PAMAM-pmiR-181c-5p treated group (n = 30), and a LA-PAMAM-pNull treated group (n = 30; as HCC positive control). The polyplexes treated groups were injected intravenously (i.v.) via tail vein with DNA-polyplexes in a ratio 2:1 polymer: DNA for five doses following Salah et al. 2019. A single i.p. dose of saline given to the negative control group and after 2 weeks corn oil was received by oral gavage for 20 weeks, then PBS for the following 5 weeks. The detailed timeline and doses of treatment are shown in Figure 1. Ultimately, 7 days following the last injection, the mice were sacrificed.

Blood and Tissue Sampling
Body weight Change (BWC) % was calculated by recording the body weight of all mice at the experiment's beginning and end. The liver tissue organs were collected, washed with PBS, and weighed to obtain the relative liver weight (RLW). BWC and RLW were calculated for every single mouse. Hepatic tissue specimens were collected and liver tumors were identified during sacrifice by macroscopic examination of the liver. Then, the liver tissue was prepared for histopathological examinations. For further biochemical and molecular analysis, blood samples were collected from each mouse group and hepatic tissue specimens were excised and directly frozen at −80 • C.
Genes 2022, 13, x FOR PEER REVIEW 5 of 18 negative control group and after 2 weeks corn oil was received by oral gavage for 20 weeks, then PBS for the following 5 weeks. The detailed timeline and doses of treatment are shown in Figure 1. Ultimately, 7 days following the last injection, the mice were sacrificed.

Blood and Tissue Sampling
Body weight Change (BWC) % was calculated by recording the body weight of all mice at the experiment's beginning and end. The liver tissue organs were collected, washed with PBS, and weighed to obtain the relative liver weight (RLW). BWC and RLW were calculated for every single mouse. Hepatic tissue specimens were collected and liver tumors were identified during sacrifice by macroscopic examination of the liver. Then, the liver tissue was prepared for histopathological examinations. For further biochemical and molecular analysis, blood samples were collected from each mouse group and hepatic tissue specimens were excised and directly frozen at −80 °C.

Biochemical Analysis
Colorimetric assays (BIOLABS, Paris, France) were used to measure the serum enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Using a mouse α-fetoprotein ELISA kit from Elabscience Biotechnology Co., following the directions from the manufacturer (Houston, TX, USA), α-fetoprotein (AFP) was assessed.

Histopathological Analysis
Hepatic tissue specimens (including grossly visible tumors) were excised in sections of 3 to 5 mm thick, followed by fixation in neutral buffered formalin 10%. The hepatic tissue specimens were paraffin wax embedded, sections were cut at a thickness of 5 μm and hematoxylin and eosin (H&E) stained, following the previous method [49]. The sections were examined using an Olympus digital camera mounted on an Olympus microscope with a 1/2× power adaptor. The 4-scale Edmondson and Steiner system was used to classify HCC lesions [50].

Prediction of the Targets of miR-181c-5p
MiR-181c-5p's sequence and expression pattern was obtained using the miRBase database (http://www.miRbase.org/) (accessed on 8 October 2021) and phenomiR database (http://mips.helmholtz-muenchen.de/phenomiR/) (accessed on 8 October 2021), respectively. To predict a miR-181c-5p target, the miRWalk v2.0 database was used [51]. The MiRWalk server offers the targets of miRNA that have been generated by the intersection of various prediction algorithms. In the current analysis, the following algorithmic target

Biochemical Analysis
Colorimetric assays (BIOLABS, Paris, France) were used to measure the serum enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Using a mouse α-fetoprotein ELISA kit from Elabscience Biotechnology Co., following the directions from the manufacturer (Houston, TX, USA), α-fetoprotein (AFP) was assessed.

Histopathological Analysis
Hepatic tissue specimens (including grossly visible tumors) were excised in sections of 3 to 5 mm thick, followed by fixation in neutral buffered formalin 10%. The hepatic tissue specimens were paraffin wax embedded, sections were cut at a thickness of 5 µm and hematoxylin and eosin (H&E) stained, following the previous method [49]. The sections were examined using an Olympus digital camera mounted on an Olympus microscope with a 1/2× power adaptor. The 4-scale Edmondson and Steiner system was used to classify HCC lesions [50].

Prediction of the Targets of miR-181c-5p
MiR-181c-5p's sequence and expression pattern was obtained using the miRBase database (http://www.miRbase.org/) (accessed on 8 October 2021) and phenomiR database (http://mips.helmholtz-muenchen.de/phenomiR/) (accessed on 8 October 2021), respectively. To predict a miR-181c-5p target, the miRWalk v2.0 database was used [51]. The MiRWalk server offers the targets of miRNA that have been generated by the intersection of various prediction algorithms. In the current analysis, the following algorithmic target prediction was chosen: miRWalk, TargetScan v7.0, MiRmap, MiRanda, Pictar, and RNA22 to attain the common predicted targets with a cut-off p-value < 0.05. We selected only those target genes downregulated in HCC to pick the predicted target gene of the present study [52].

Functional Enrichment Analysis
The predicted and validated downregulated targets of miR-181c-5p obtained from the miRWalk tool were subsequently used in the functional enrichment analysis. To detect the biological processes as well as the implicated signaling pathways of the obtained downregulated miR-181c-5p targets in HCC, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis was performed through Enrichr (https://maayanlab.cloud/Enrichr/) (accessed on 25 January 2022). Only <0.05 p-value results were c regarded as statistically significant.

RNA Extraction and Quantitative Real Time PCR (qRT-PCR)
Following the manufacturer's directions, hepatic tissue specimens' and HepG2 cells' total RNA extraction was carried out using Trizol reagent (Qiagen, Hilden, Germany). The concentration and quality of the extracted RNA were measured using a Nanodrop 2000 spectrophotometer. The first-strand cDNA was created using the miScript II RT Kit (Qiagen, Hilden, Germany) with one µg of total RNA. The miR-181c-5p, as well as F-Box and Leucine-Rich Repeat Protein 3 (Fbxl3) expression levels, were quantified using miScript SYBR Green PCR Kit with miR-181c-5p miScript Primer Assay (Qiagen, Hilden, Germany) and gene-specific primers (Table 1), respectively. Relative expression was normalized using endogenous housekeeping control U6 small nuclear RNA (snRNA) for miR-181c-5p and GAPDH for Fbxl3 quantification. The qRT-PCR for miR-181c-5p expression level was established as follows: 95 • C for 15 min; and 40 cycles of 94 • C for 15 s, 55 • C for 30 s, and 72 • C for 30 s, while the qRT-PCR conditions for quantification of Fbxl3 were as follows: 95 • C for 15 min; and 40 cycles of 95 • C for 15 s, 60 • C for 30 s. All quantitative PCR reactions were performed on the Applied Biosystems 7500 Real-Time PCR system. A triplicate sample run was performed. The ∆∆Ct method was used to determine the value of the expression fold change. Table 1. QRT-PCR primer sequences list.

Gene
Forward Sequence Reverse Sequence mGAPDH mFbxl3

Statistical Analysis
The standard error (SE) was used to represent the data as mean ±. Version 21.0 of the statistical software program SPSS (SPSS Inc., Chicago, IL, USA) was carried out to perform multiple comparisons after the one-way ANOVA or Student's t-test was used to identify the statistical differences. Data were approved to be statistically significant when values of p < 0.05. Pearson's coefficient correlation (2-tailed) was used to investigate the correlation between miR-181c-5p and its predicted targets Fbxl3 to assess the function of miR-181c-5p.

MiR-181c-5p Expression Data in HCC
Understanding how miR-181c-5p is expressed and distributed in various tissues is important to understanding the normal development of the disease of respective tissues. To evaluate the miR-181c-5p expression in liver tissue, the human miRNA tissue atlas data were used. These results revealed that miR-181c-5p has a low expression level in the normal liver. The overall miR-181c-5p tissue-specific profile is presented in (Figure 2a). The results obtained from literature as well as databases about the miR-181c-5p expression in HCC, whether it is up-or downregulated is fluctuating, as shown in Table 2. In addition, miR-181c gives out relatively different expression statuses in multiple human malignancies, which includes HCC, as calculated using dbDEMC2 software (http://www.picb.ac.cn/dbDEMC) (accessed on 4 October 2021) (Figure 2b). Moreover, the survival analysis was obtained from the starBase v2.0 database and the Kaplan-Meier analysis curves p-value indicated that no significant difference statistically between the population survival curves for the low and high expression (Figure 2c p = 0.23).
Genes 2022, 13, x FOR PEER REVIEW 7 o normal liver. The overall miR-181c-5p tissue-specific profile is presented in (Figure 2a). results obtained from literature as well as databases about the miR-181c-5p expressio HCC, whether it is up-or downregulated is fluctuating, as shown in Table 2. In addit miR-181c gives out relatively different expression statuses in multiple hum malignancies, which includes HCC, as calculated using dbDEMC2 softw (http://www.picb.ac.cn/dbDEMC) (accessed on 4 October 2021) (Figure 2b). Moreover, survival analysis was obtained from the starBase v2.0 database and the Kaplan-M analysis curves p-value indicated that no significant difference statistically between population survival curves for the low and high expression (Figure 2c p = 0.23).    0.05). To investigate the impact of LA-PAMAM-pmiR-181c-5p on HCC cell proliferation, the transfection of HepG2 cells with different concentrations from 0.5 μg to 2 μg of LA-PAMAM-pmiR-181c-5p or LA-PAMAM-pNull as control were applied. MTT analysis indicated that miR-181c-5p significantly promoted the proliferation when the concentration of LA-PAMAM-pmiR-181c-5p is increased in HepG2 cells after 48 h of transfection, compared with the pNull transfected control groups (Figure 3c, p < 0.05).

MiR-181c-5p Overexpression Contributes to HCC Tumor Progression
To verify the oncogenic role of miR-181c-5p on mice with chemically developed HCC, the effect of miR-181c-5p-forced expression specifically delivered by the LA-PA-MAM (LA-PAMAM-pmiR-181c-5p) was examined on the development of HCC. The liver of all groups was examined macroscopically (Figure 4a). The liver in the negative control group was dark red in color showing sharp edges, a smooth surface, and medium texture.

MiR-181c-5p Overexpression Contributes to HCC Tumor Progression
To verify the oncogenic role of miR-181c-5p on mice with chemically developed HCC, the effect of miR-181c-5p-forced expression specifically delivered by the LA-PAMAM (LA-PAMAM-pmiR-181c-5p) was examined on the development of HCC. The liver of all groups was examined macroscopically (Figure 4a). The liver in the negative control group was dark red in color showing sharp edges, a smooth surface, and medium texture. The pmiR-181c-5p treatment showed an apparently increased number of nodules compared to pNull treatment. The LA-PAMAM-pmiR-181c-5p treated group, besides presenting a pale cirrhotic appearance with a granular surface, showed massive nodule formation more than has been apparent in the LA-PAMAM-pNull treated group. In addition, paraffin sections of hepatic tissue of all groups were histologically examined by the light microscope ( Figure 4b). The negative control group showed that hepatic tissue specimens have normal architecture formed of distinct hexagonal hepatic lobules. The hepatic sinusoids appear as narrow spaces, which take the same direction as hepatic lobules. Areas of the portal tract appear at the angles of the periphery of the hepatic lobule. Hepatocytes showed central, rounded, vesicular nuclei and acidophilic granular cytoplasm and some cells are binucleated. Portal spaces were also normal, with no observed inflammatory infiltration or fatty degeneration. The liver tissue of the LA-PAMAM-pNull treated group had an appearance that is cirrhotic with a granular surface to malignant changes accompanied by a nuclear atypia, heightened nuclear:cytoplasmic ratio, and clearly visible mitotic activity. In addition, the LA-PAMAM-pNull treated group had nodules that were dysplastic and showed changes such as fatty and focal nodular hyperplasia. On the other hand, the liver of the LA-PAMAM-pmiR-181c-5p treated group exhibited malignant progression to HCC, liver lobular structure alterations, and hepatic degeneration with regenerative nodules typical of cirrhotic liver. Also, proliferative tumor lesions were present, which are accompanied with atypical mitosis as well as dysplastic aspects resembling HCCs. The LA-PAMAM-pmiR-181-5p treated liver showed different grades of HCC, ranging from dysplastic nodules, up to a well-differentiated HCC, which was significantly worse compared with that of the LA-PAMAM-pNull treated group. gression to HCC, liver lobular structure alterations, and hepatic degeneration with regenerative nodules typical of cirrhotic liver. Also, proliferative tumor lesions were present, which are accompanied with atypical mitosis as well as dysplastic aspects resembling HCCs. The LA-PAMAM-pmiR-181-5p treated liver showed different grades of HCC, ranging from dysplastic nodules, up to a well-differentiated HCC, which was significantly worse compared with that of the LA-PAMAM-pNull treated group.

MiR-181c-5p Overexpression Deteriorates Liver Functions in the HCC Mouse Model
The BWC % and RLW in all groups were examined. The weight changes showed that LA-PAMAM-pmiR-181c-5p treatment did not significantly affect the BWC % compared to the LA-PAMAM-pNull treated group; however, both groups manifested a significant decrease (p < 0.05) in BWC % in comparison to the negative group. In addition, there was no observed significant change in RLW among all groups (Figure 5a,b). To check the miR-181c-5p effect on liver functions, the activities of serum liver enzymes (ALT and AST), as well as serum AFP, were measured. Results demonstrate a significant increase in ALT and AST of the LA-PAMAM-pmiR-181c-5p treated group, as compared with the LA-PAMAM-pNull treated group (p < 0.05). Although LA-PAMAM-pmiR-181c-5p treatment did not significantly affect the AFP compared to the LA-PAMAM-pNull treated group, AFP showed a significant increase in LA-PAMAM-pmiR-181c-5p and LA-PAMAM-pNull treated groups, as compared with negative control (p < 0.05) (Figure 5c,d).

Bioinformatics Functional Analysis of MiR-181c-5p Downregulated Targets
To examine the oncogenic role of the upregulated miR-181c-5p in HCC development, it was necessary to determine miR-181c-5p downregulated targets in HCC and their functions. Therefore, the targets of miR181c-5p that are mutual between the selected bioinformatics target prediction tools and the significantly downregulated genes in HCC were compiled as previously described in the Methods section. GO and KEGG terms were obtained to determine the most significantly enriched biological process and pathways of the downregulated overlapping target genes (p < 0.05). The results revealed that the miR-181c-5p downregulated target genes in HCC were significantly enriched in 123 biological process terms, such as cellular response to cytokine stimulus, steroid metabolic process, retinoic acid metabolic process, positive regulation of pri-miRNA transcription by RNA polymerase II, and regulation of pri-miRNA transcription by RNA polymerase II (Supplementary Table S1, Figure 6a). Enriched KEGG pathways indicated that the miR-181c-5p downregulated target genes were categorized primarily into 13 statistically significant cancer-related pathways, which are associated with HCC (Supplementary Table S1, Figure 6b). KEGG pathway analysis revealed that the downregulated genes were primarily enriched in tryptophan metabolism, retinol metabolism, chemical carcinogenesis, and parathyroid hormone synthesis, secretion, and action.
Genes 2022, 13, x FOR PEER REVIEW 10 of 18 The BWC % and RLW in all groups were examined. The weight changes showed that LA-PAMAM-pmiR-181c-5p treatment did not significantly affect the BWC % compared to the LA-PAMAM-pNull treated group; however, both groups manifested a significant decrease (p < 0.05) in BWC % in comparison to the negative group. In addition, there was no observed significant change in RLW among all groups (Figure 5a,b). To check the miR-181c-5p effect on liver functions, the activities of serum liver enzymes (ALT and AST), as well as serum AFP, were measured. Results demonstrate a significant increase in ALT and AST of the LA-PAMAM-pmiR-181c-5p treated group, as compared with the LA-PA-MAM-pNull treated group (p < 0.05). Although LA-PAMAM-pmiR-181c-5p treatment did not significantly affect the AFP compared to the LA-PAMAM-pNull treated group, AFP showed a significant increase in LA-PAMAM-pmiR-181c-5p and LA-PAMAM-pNull treated groups, as compared with negative control (p < 0.05) (Figure 5c,d).

Bioinformatics Functional Analysis of MiR-181c-5p Downregulated Targets
To examine the oncogenic role of the upregulated miR-181c-5p in HCC development, it was necessary to determine miR-181c-5p downregulated targets in HCC and their functions. Therefore, the targets of miR181c-5p that are mutual between the selected bioinformatics target prediction tools and the significantly downregulated genes in HCC were compiled as previously described in the Methods section. GO and KEGG terms were obtained to determine the most significantly enriched biological process and pathways of the downregulated overlapping target genes (p < 0.05). The results revealed that the miR-181c-5p downregulated target genes in HCC were significantly enriched in 123 biological process terms, such as cellular response to cytokine stimulus, steroid metabolic process, retinoic acid metabolic process, positive regulation of pri-miRNA transcription by RNA polymerase II, and regulation of pri-miRNA transcription by RNA polymerase II (Supplementary Table S1, Figure 6a). Enriched KEGG pathways indicated that the miR-181c-5p downregulated target genes were categorized primarily into 13 statistically significant cancer-related pathways, which are associated with HCC (Supplementary Table S1, Figure 6b). KEGG pathway analysis revealed that the downregulated genes were primarily enriched in tryptophan metabolism, retinol metabolism, chemical carcinogenesis, and parathyroid hormone synthesis, secretion, and action.

Fbxl3 as a Target for MiR-181c-5p Involved in HCC
To prove the oncogenic effect of miR-181c-5p in vivo, the qRT-PCR assay was performed. Results revealed that the miR-181c-5p expression level was significantly upregulated in the HCC tissue of the LA-PAMAM-pNull treated group (3.5-fold) compared to

Fbxl3 as a Target for MiR-181c-5p Involved in HCC
To prove the oncogenic effect of miR-181c-5p in vivo, the qRT-PCR assay was performed. Results revealed that the miR-181c-5p expression level was significantly upregulated in the HCC tissue of the LA-PAMAM-pNull treated group (3.5-fold) compared to the negative control group (p < 0.05) and the miR-181c-5p expression level was significantly upregulated in HCC tissue of the LA-PAMAM-pmiR-181c-5p treated group (7.5-fold) compared to the LA-PAMAM-pNull treated group (Figure 7a p < 0.05). The miRWalk v2.0 manifested that "Fbxl3" is a novel predicted target for miR-181c-5p. MiRanda and Tar-getScan miRNA-target prediction tools were utilized to confirm this result (Figure 7b). To check the effect of the miR-181c-5p forced expression on its predicted target, the qRT-PCR analysis was carried out to assess the Fbxl3 mRNA expression levels in all mice groups as an example of miR-181c-5p potential targets significantly downregulated in HCC [52,55]. Results revealed that the LA-PAMAM-pmiR-181c-5p group showed significantly low expression (p < 0.05) of Fbxl3 mRNA (0.26 folds), compared to the LA-PAMAM-pNull treated group (0.64 folds) (Figure 7c). Moreover, significant downregulation of the Fbxl3 mRNA expression level in the LA-PAMAM-pmiR-181c-5p and LA-PAMAM-pNull treated groups were observed as compared with negative control (p < 0.05).

Discussion
The molecular mode of the miR-181c-5p, whether to stimulate or inhibit the H progression is controversial and not fully elucidated yet. The miR-181c-5p expression tus results obtained from the miRNA disease association databases that are mostly u in the literature; dbDEMC [44], HMDD [45], miR2Disease [46], and miRcancer [47], survival analysis confirmed this fluctuation and provides strong bases for its involvem in HCC development. In this research, the role of miR-181c-5p in HCC progression w unraveled by its specific and targeted delivery using LA-PAMAM and forced express

Discussion
The molecular mode of the miR-181c-5p, whether to stimulate or inhibit the HCC progression is controversial and not fully elucidated yet. The miR-181c-5p expression status results obtained from the miRNA disease association databases that are mostly used in the literature; dbDEMC [44], HMDD [45], miR2Disease [46], and miRcancer [47], and survival analysis confirmed this fluctuation and provides strong bases for its involvement in HCC development. In this research, the role of miR-181c-5p in HCC progression was unraveled by its specific and targeted delivery using LA-PAMAM and forced expression in HepG2 and mouse liver with chemically induced HCC. The present results revealed a significantly elevated miR-181c-5p expression level in the HCC tissue of the LA-PAMAM-pNull treated mice group when compared to the negative control mice group. This result is in agreement with the previous research which demonstrated that mature miR-181 family members increased significantly in twenty cases of HCC, HCC stem cells, and progenitors [21,31].
In a recent study, we selectively delivered and restored miR-218 expression in HCC by constructing a biocompatible hyperbranched polyamidoamine with lactobionic acid (LA-PAMAM) decoration. The LA moieties were reported by our group to enhance the cellular uptake and efficient delivery of LA-PAMAM-pmiR-218 to HepG2 and HCC tissue in mice by targeting ASGP-Rs that are highly expressed on HCC cells. That study revealed that ASGPR expression was upregulated significantly in the tissue of HCC compared with the tissue of the normal liver and the receptor competition assay confirmed that LA-PAMAM-pmiR-218 was captured by endocytosis, mediated prevalently by ASGPR. Additionally, LA-PAMAM-pmiR-218 cellular uptake was increased compared with the naked pmiR-218 and PAMAM-pmiR-218. Moreover, the cytotoxicity of LA-PAMAM is very low in comparison to PAMAM and PEI on HepG2 cells. LA moiety conjunction could shield the cationic charges on the PAMAM polymer, which enhances its biocompatibility and reduces cytotoxicity [43]. In agreement with the previous study, transfection of LA-PAMAM-pmiR-181c-5p to HepG2 cells in the current study showed successful overexpression of pmiR-181c-5p. Here, the increased miR-181c-5p expression enhanced the viability of HepG2 cells. Furthermore, overexpression of miR-181c-5p could promote the development of HCC. Our results are in agreement with several studies that suggested that the family of miR-181, including miR-181c, could activate hepatic progenitor cells and HCC by blocking HCC cell differentiation and enhancing HCC development and progression. These roles are mediated through the Wnt/β-catenin signaling pathway [31,53]. It was revealed, on the other hand, that miR-181c could repress cell cycle, apoptosis, and metastasis in HCC through targeting oncogenic secreted phosphoprotein 1 (SPP1) [33].
The features of the liver were consistent with this fact, wherein the LA-PAMAM-pmiR-181c-5p treated group showed an increased number of HCC nodules, severe malignant histological changes, and upregulation in liver function enzymes (ALT and AST) in the serum, in correspondence with the malignant features shown by T. Uehara et al., 2014 [56], which were evident in 100% of all mice compared to the LA-PAMAM-pNull group, indicating that miR-181c-5p might be correlated with the progression of HCC. miR-181c-5p downregulated targets in HCC were enriched significantly in different pathways and biological processes associated with HCC. The twenty miR-181c-5p targets significantly predicted in this study, "PLAC8, KBTBD11, CXCL12, FOS, MME, FBXL3, KMO, EGR1, DCN, BCHE, LIFR, CYP26A1, HSD11B1, CYP2C8, TMEM27, ITLN1, GPM6A, CNDP1, GYS2, and INMT" were downregulated in HCC [52]. For example, one of these targets is Decorin (DCN), an effective protein involved in the transforming growth factor-β (TGF-β) signaling pathway, which decreased TGF-β bioavailability [57]. In addition, DCN deficiency promoted hepatic carcinogenesis, and Decorin null (Dcn−/−) mice developed increased tumors after treatment with DEN [58]. The current analysis showed the proposed impact of miR-181c-5p to act as oncomiR and to control many significantly enriched pathways and biological processes related to HCC, such as immune system and metabolic pathways, cellular response to cytokine stimulus, cytokine-cytokine receptor interaction, and the intestinal immune network for IgA production, as well as tryptophan metabolism, retinol metabolism, arginine and proline metabolism, chemical carcinogenesis, and parathyroid hormone synthesis, secretion, and action. Involvement of these pathways in our chemically induced HCC model is highly probable because DEN is a hepatotoxic chemical and can lead to hepatocytes' necrotic cell death, which contain pre-made interleukin (IL)-1α. This cytokine release can trigger an inflammatory chain reaction which ultimately leads to high expression of tumor necrosis factor (TNF), IL-6, and hepatocyte growth factor, which can act to start the carcinogenesis of hepatocytes [59]. Another study analyzed the chemical carcinogenesis pathway-specific process and reported HCC pathogenesis and progression through the downregulation of Hydroxysteroid 11-β Dehydrogenase 1 (HSD11B1) [60]. It has been established that the cytokine-cytokine receptor interaction pathway may be a key pathway associated with the development of HCC [61]. Additionally, activation of the intestinal immune network for the IgA production signaling pathway contributes to HCC cell proliferation and migration [62]. Further, metabolism appeared to be closely related to cancer epigenetics and altered metabolism stimulates tumor proliferation and metastasis [63]. Amino acids are vital nutrients and energy sources for tumor cells associated with lipid, glucose, and nucleotide metabolism, which are significant for the invasion, proliferation, and metastasis of tumor [64]. Tryptophan metabolism has an essential role in the HCC occurrence and development. It was revealed that the tryptophan side-chain oxidase (TSO) enzyme, which can break down tryptophan, had a restrictive effect on the HCC cell lines' proliferation, invasion, and migration [65]. It was indicated that argininosuccinate synthase 1 (ASS1), which is a rate-limiting enzyme for arginine biosynthesis, inhibited the metastasis of HCC by suppressing the STAT3 signaling pathway [66]. An altered retinol metabolism pathway is implicated in HCC [67]. The associations between retinoids and hepatic disease have been demonstrated, involving retinoid activity loss in HCC cell lines and reduced retinoid reserves in the liver, as well as the transformed retinoid signaling in cirrhosis and HCC patients [68]. The hepatic retinoid signaling loss has been correlated with more prompt progression of the development of liver disease emerging from reactive oxygen species [69]. Acyclic retinoid (ACR) is a synthetic retinoid that is reported to suppress liver tumors induced chemically, as well as spontaneous HCC development in rodents by stimulating apoptosis and inhibiting cellular proliferation in HCC [70]. It was determined that ACR represses Ras/MAP kinase signal transduction and preserves the function of retinoid X receptor α (RXRα), which is a substantial nuclear receptor involved in the process of hepatocarcinogenesis, as its ligand represses HCC development [71]. Furthermore, it has been determined that ACR regulates the growth of HCC via repressing the expression of transforming growth factor α (TGFα) [72] through the modulation of fibroblast growth factor signaling [73] and platelet-derived growth factor signaling [74]. The level of cytochrome P450 members, such as CYP2C8 and CYP26A1, was downregulated in consistence with the progression of HCC in patients. Both CYP2C8 and CYP26A1 are involved in retinol metabolism [75].
Fbxl3 overexpression suppresses cell proliferation, stimulates cell apoptosis, arrests cell cycle, and prohibits cell invasion and migration efficiently, and has tumor inhibition prospects. In addition, Fbxl3 was reported to be downregulated in HCC [55,76,77]. A previous study demonstrated that miR-181d can act as an oncomiR that is upregulated in colorectal cancer (CRC) tissues and identified Fbxl3 as a direct target of miR-181d [78]. The miR-181c and miR-181d clusters are located similarly on chromosome 19 and share identical 8 base seed sequences [79], which serve as the main factor in the base complementation process and confirms the results of the bioinformatics analysis, which predicted Fbxl3 as a strong target for miR-181c-5p. The possible molecular effect of miR-181c-5p-forced expression on the expression level of its predicted target "Fbxl3" in HCC liver tissues was investigated and the results revealed that the miR-181c-5p expression was inversely correlated with Fbxl3 mRNA levels, which strongly suggested Fbxl3 as a possible target for miR-181c-5p in the HCC model.

Conclusions
In brief, the current study confirmed that the upregulation of miR-181c-5p is oncogenic in HCC in vitro and in vivo. As a step to identify the involved molecular mechanism, a novel correlation at which miR-181c-5p downregulates the expression of the Fbxl3 gene was proposed here. According to the miR-181c-5p biological mechanisms proposed here, it is valuable to further validate these molecular mechanisms for regulating HCC progression and to investigate their therapeutic significance. The inhibition of miR-181c-5p may represent a promising therapeutic strategy for HCC patients in the future by inhibiting all these pathways and biological processes. We are also proposing a future study on HCC patients to determine the correlation between mir-181c-5p and Fbxl3 in the diagnosis and prognosis of the HCC cases.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Conflicts of Interest:
The authors declare no conflict of interests.