الفهرس 
Hepatocellular carcinomaOnly 14 pages are availabe for public view
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Abstract
Several therapeutic approaches are used nowadays for the treatment of patients with HCC. One possible and promising solution is gene therapy that can treat cellular abnormalities by counteracting or correcting the malfunctioning genes within a cancerous cell.
MiRNAs are small ( 20–22 nucleotide long ), endogenous, single stranded, noncoding RNA molecules that have emerged as key posttranscriptional modulators of gene expression. Analogous to coding genes, miRNAs are comprised of subsets that can promote tumorigenesis (”Onco-miRs”), or those that inhibit neoplastic transformation (tumor suppressors ”TSG-miRs”). Restoration of tumor suppressive miRNAs leads to cell cycle block, increased apoptosis, and reduced tumor angiogenesis and metastasis by inhibiting migration and invasion. Among these miRNAs, miR-34a appears to be particularly important in HCC.
Efficient tissue-specific delivery of therapeutic miRNAs is a major challenge for miRNA-based therapy. To circumvent these challenges, several approaches for miRNA delivery have been explored.
In the present study, a novel delivery system for miR-34a delivery was prepared. The delivery system composed of branched PEI which electrostaticaly binding to the negatively charged miR-34a construct and adsorbed onto surface-functionalized ZSM-5 zeolite nanoparticles through electrostatic interaction and hydrogen bonding, for the targeted treatment of HCC.
Aim of the work
The aim of the present study is to assess the effect of tumor suppressor micro-RNA loaded on nano – zeolites in hepatocellular carcinoma induced mice.
Methods
Preparation of ZNP and nano-formulations of miR-34a construct
Amine functionalized ZNP was synthesized and characterized by X-ray powder diffraction (XRD), Transmission Electron Microscope (TEM). The ZNP size and the zeta potential were measured by Zetasizer Nano ZS instrument. Gel retardation assay was performed for the examination of the complex formation.
In vitro studies
In vitro cytotoxic activities and qualitative transfection efficiency of the ZNP and their complexes were investigated in HepG2 cells MTT assay and GFP screening, respectively. The expression of miR-34a and mRNA of target genes (SOX-9 and AEG-1), were measured by qRT-PCR. The prediction of the miR-34a-targeted genes was performed using miRGator v3.0, and miRWalk v2.0.
In vivo studies
Male wild-type balb-c mice (n=75) were randomly subdivided into three groups (n=25 each): control untreated healthy mice (negative control group), DEN/CCl4-treated mice (positive control group) to induce HCC (22 weeks), and DEN/CCl4 (22 weeks) plus intravenously injection of ZNP/PEI/miR-34a (20 μg/mouse, twice weekly for a total of five doses (miR-34a-treated group).
Parameters to be investigated
• Serum ALT and AST activities
• Tissue alpha-fetoprotein (AFP)
• CD44 and c-Myc protein levels in liver tissues (by indirect ELISA)
• Target genes mRNA (AEG-1, and SOX-9), by qRT-PCR
• Macroscopic and histological examinations
Results
Results of the present study showed that:
• Mean size of ZNP and prepared nano-formulations were ranged between 170 and 265 nm with PDI bellow 0.4.
• ZNP/PEI/miR-34a nano-formulation could efficiently delivered in vitro and in vivo with low cytotoxicity.
• ZNP/PEI/miR-34a nano-formulation in HCC induced mice significantly inhibited tumor growth and demonstrated improved cell structure.
• ZNP/PEI/miR-34a nano-formulation led to a significant decrease in alpha-fetoprotein level and liver enzymes activities in vivo.
• ZNP/PEI/miR-34a nano-formulation led to a noticeable decrease in the CD44 and c-Myc levels in vivo.
• ZNP/PEI/miR-34a nano-formulation inhibited several target oncogenes including AEG-1, and SOX-9, in vitro and in vivo.