الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma (HCC) is a lethal disease. The most notable risk factors for the development of HCC are chronic diseases of the liver and cirrhosis. Identifying the molecular pathogenesis of HCC is poor and unsatisfactory so far. Therefore, to create an efficient therapeutic strategy, manipulating a more exhaustive analysis of HCC is a request. Newly, examinations concentrated on targeted therapies, because of the aggressiveness of the tumor.
MiRNAs are short non-coding RNAs composed of 20-24 nucleotides, their abnormal expression is ordinary in cancer. They can simulate different processes linked to cancer onset and advancement, and could function as posttranscriptional regulators by targeting mRNAs. The upregulation of oncogenic miRNAs stimulates tumorigenesis by limiting mRNAs translation of tumor suppressor. On the opposite, the expression of tumor suppressor miRNAs is diminished in different cancers and stops tumors growth by repressing oncogenes.
The miR-181 family is four members (miR-181a, miR-181b, miR-181c, and miR-181d) in humans and mice encoded in three distinct transcripts located on three distinct chromosomes that can regulate numerous biological activities. The miR-181 family members have a critical role in cancer by acting as oncogenic or tumor suppressor miRNAs. The miR-181 family has a crucial role in hepatocarcinogenesis as its downregulation reduced the migration of hepatic cancer cells. Different investigations have documented that miR-181c had aberrant expression in various tumors.
MiR-181c was downregulated and worked as a tumor suppressor in metastatic neuroblastoma, breast cancer, glioblastoma, oral cancers, and gastric cancer. On the contrary, miR-181c acts as a potent oncomiR in inflammatory breast cancer and pancreatic cancer.
In HCC, it was documented that the mature miR-181 family members were increased significantly in HCC patients, HCC stem cells, and progenitors, on the other hand, other analyses showed that miR-181c was downregulated, and suppressed the HCC growth and metastasis.
The bioinformatics analysis of the expression data of miR-181c-5p in HCC was determined using several databases to shed the light on its involvement in HCC development.
Nanoparticles (NPs) possess extraordinary properties regarding gene delivery when utilized for in vivo therapy. As a consequence of the NP’s smaller size, and their minor vulnerability to clearance via the RES, they can target and penetrate specific tissues or cells.
Polyamidoamine (PAMAM) polymers were reviewed extensively for nucleic acids delivery, can be manufactured in a defined configuration, at which its cationic charges high density displays electrostatic interactions with nucleic acids as a proton sponge. It can condense polyanionic nucleic acids, create dendriplexes to stop the nucleic acids degradation, facilitate cellular uptake on various cell lines, and permit endo-lysosome localization, due to the “proton sponge” effect which prefers the endosomal escape.
Lactobionic acid (LA) is a ligand at which the galactosyl moiety binds the ASGP-R that is overexpressed on HCC tissue to allow efficient targeted delivery to HCC cells. Since HCC involved a high risk of recurrence and poor prognosis, discovering more facts about its molecular pathogenesis is very challenging.
Due to the inconsistent data concerning the miR-181c role in HCC, identification of the miR-181c role in HCC may give more indications for HCC therapeutic strategies. In this study, the role of miR-181c expression using LA-PAMAM targeted delivery was examined in vitro and in vivo. The miR-181c-5p expression status results was presented from the databases of miRNA disease association that are widely used in the literature; dbDEMC, HMDD, miR2Disease, and miRcancer. Moreover, survival analysis of miR-181c-5p was performed. MiR-181c-5p sequence, expression pattern, and its predicted target was obtained using miRBase, phenomiR, and miRWalk v2.0, respectively. Only those target genes whose downregulated in HCC were selected to choose the predicted target gene of this current study. MiR-181c-5p was amplified from genomic DNA using PCR.
The amplified MiR-181c-5p was cloned in pEGP-miR cloning and expression vector to construct pmiR-181c-5p and the cloned constructs were thereafter transformed into Escherichia coli TOP10 cells and verified by sequencing. Hyperbranched PAMAM was synthesized, decorated with LA, and the LA-PAMAM-pmiR-181c and LA-PAMAM-pNull polyplexes were prepared.
The expression of miR-181c-5p in vitro was examined by transfection of LA-PAMAM-pmiR-181c-5p in HepG2 cells. Subsequently, the transfection efficiency was qualitatively examined by green fluorescent protein (GFP screening) expression in comparison to untransfected cells. HepG2 cells’ viability was confirmed by MTT analysis. In addition qRT-PCR was performed in HepG2 cells to determine the expression level of miR-181c-5p.
In vivo animal experiments, mice were divided randomly into 2 groups: the negative control group and the HCC group. Briefly, liver cancer was induced by i.p. single-dose injection of mice by DEN, followed by 21 oral gavage doses of CCl4. After 20 weeks, HCC was proved in the HCC group by liver gross examination and histopathological analysis. Then the HCC group was divided into two subgroups, LA-PAMAM-pmiR-181c-5p treated group, and LA-PAMAM-pNull treated group.
BWG of all mice was documented at the beginning and end of the experiment. Blood samples were collected from each mice group for further biochemical analysis of serum ALT and AST as well as AFP. The liver tissues were collected and weighed to get the RLW. BWC and RLW were calculated for every single mouse. Liver tumors were determined at sacrifice by investigating the liver macroscopically, then the liver tissue was prepared for molecular and histopathological examinations.
Using QIAzol reagent, total RNA was extracted from liver tissues, the concentration and quality of the extracted RNA were measured and the RNA was used to synthesize the first strand of cDNA. The expression levels of miR-181c-5p, was quantified by PCR. GO and KEGG functional enrichment analysis was accomplished using Enrichr to detect the biological processes and implicated signaling pathways of the obtained downregulated miR-181c-5p targets in HCC.
FBXL3 is an F-box protein that its overexpression represses cell proliferation, promotes cell apoptosis, arrests cell cycle, and suppresses cell migration and invasion in an efficient manner. Fbxl3, is an example for miR-181c-5p potential targets that has a tumor suppression effect, its downregulation and correlation with miR-181c-5p were validated by qPCR.
The current study results indicated the significant upregulation of miR-181c-5p in vitro and in vivo that stimulates the advancement of HCC. The liver characteristics were agreeing with this fact, as the LA-PAMAM-pmiR-181c-5p treated group exhibited an increased number of HCC nodules, extreme malignant histological transformations, and an increased level of the liver function enzymes ALT and AST in serum, compared to LA-PAMAM-pNull group. The Fbxl3 was predicted as a strong target for miR-181c-5p via bioinformatics analysis and its expression was inversely correlated with miR-181c-5p. The miR-181c-5p was proposed here to exert an oncogenic role in HCC in vivo resulting in HCC progression stimulation. Moreover miR-181c-5p downregulated targets were found to be significantly enriched to repress numerous pathways and biological processes implicated in HCC advancement. Accordingly, miR-181c-5p inhibition may be a promising therapeutic technique for HCC patients hereafter by repressing all these pathways and biological processes.