Preparation and Optimization of MiR-375 Nano-Vector Using Two Novel Chitosan-Coated Nano-Structured Lipid Carriers as Gene Therapy for Hepatocellular Carcinoma.

Currently, there is still a lack of effective carriers with minimal side effects to deliver therapeutic miRNA. Thus, it is crucial to optimize novel drug delivery systems. MiR-375 has proven superior therapeutic potency in Hepatocellular carcinoma (HCC). The purpose of this study was to fabricate 2 novel and smart nano-carriers for the transportation efficiency of miR-375 in HCC cells and enhance its anti-tumor effects. We established the miR-375 construct through the pEGP- miR expression vector. Two nano-carriers of solid/liquid lipids and chitosan (CS) were strategically selected, prepared by high-speed homogenization, and optimized by varying nano-formulation factors. Thus, the two best nano-formulations were designated as F1 (0.5% CS) and F2 (1.5% CS) and were evaluated for miR-375 conjugation efficiency by gel electrophoresis and nanodrop assessment. Then, physio-chemical characteristics and stability tests for the miR-375 nano-plexes were all studied. Next, its efficiencies as replacement therapy in HepG2 cells have been assessed by fluorescence microscopy, flow cytometry, and cytotoxicity assay. The obtained data showed that two cationic nanostructured solid/liquid lipid carriers (NSLCs); F1 and F2 typically had the best physio-chemical parameters and long-term stability. Moreover, both F1 and F2 could form nano-plexes with the anionic miR-375 construct at weight ratios 250/1 and 50/1 via electrostatic interactions. In addition, these nano-plexes exhibited physical stability after three months and protected miR-375 from degradation in the presence of 50% fetal bovine serum (FBS). Furthermore, both nano-plexes could simultaneously deliver miR-375 into HepG2 cells and they ensure miR re-expression even in the presence of 50% FBS compared to free miR-375 (p-value < 0.001). Moreover, both F1 and F2 alone significantly exhibited minimal cytotoxicity in treated cells. In contrast, the nano-plexes significantly inhibited cell growth compared to free miR-375 or doxorubicin (DOX), respectively. More importantly, F2/miR-375 nano-plex exhibited more anti-proliferative activity in treated cells although its IC50 value was 55 times lower than DOX (p-value < 0.001). Collectively, our findings clearly emphasized the multifunctionality of the two CS-coated NSLCs in terms of their enhanced biocompatibility, biostability, conjugation, and transfection efficiency of therapeutic miR-375. Therefore, the NSLCs/miR-375 nano-plexes could serve as a novel and promising therapeutic strategy for HCC.

Effect of Tumor Suppressor MiR-34a Loaded on ZSM-5 Nanozeolite in Hepatocellular Carcinoma: In Vitro and In Vivo Approach.

BACKGROUND: MicroRNA modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC), however Efficient tissue-specific and safe delivery remains a major challenge. OBJECTIVE: We sought to develop an inorganic-organic hybrid vehicle for the systemic delivery of the tumor suppressor miR-34a, and to investigate the efficiency of the delivered miR-34a in the treatment of HCC in vitro and in vivo. METHODS: In the present study, pEGP-miR cloning and expression vector, expressing miR-34a, was electrostatically bound to polyethyleneimine (PEI), and then loaded onto ZSM-5 zeolite nanoparticles (ZNP). Qualitative and quantitative assessment of the transfection efficiency of miR-34a construct in HepG2 cells was applied by GFP screening and qRT-PCR, respectively. The expression of miR-34a target genes was investigated by qRT-PCR in vitro and in vivo. RESULTS: ZNP/PEI/miR-34a nano-formulation could efficiently deliver into HepG2 cells with low cytotoxicity, indicating good biocompatibility of generated nanozeolite. Furthermore, five injected doses of ZNP/PEI/miR-34a nano-formulation in HCC induced male Balb-c mice, significantly inhibited tumor growth, and demonstrated improved cell structure, in addition to a significant decrease in alphafetoprotein level and liver enzymes activities, as compared to the positive control group. Moreover, injected ZNP/PEI/miR-34a nano-formulation led to a noticeable decrease in the CD44 and c-Myc levels. Results also showed that ZNP/PEI/miR-34a nano-formulation inhibited several target oncogenes including AEG-1, and SOX-9, in vitro and in vivo. CONCLUSION: Our results suggested that miR-34a is a powerful candidate in HCC treatment and that AEG-1 and SOX-9 are novel oncotargets of miR-34a in HCC. Results also demonstrated that our nano-formulation may serve as a candidate approach for miR-34a restoration for HCC therapy, and generally for safe gene delivery.

The Promise of miRNA Replacement Therapy for Hepatocellular Carcinoma.

Hepatocellular carcinoma is a devastating tumor which accounts for death mortality rate 94% globally, and about 780,000 new cases each year. Tumor suppressor miRNAs represent a class of noncoding RNAs, which exhibit decreased or inhibited expression in the case of carcinogenesis. Therefore, the replacement of these molecules leads to post-transcriptional regulation of tens to hundreds of oncogenic targets and limiting the tumor. Interestingly, there is a group of tumor silencer miRNAs that have been highlighted in HCC and herein, our review will discuss the prominent examples of these miRs in terms of their efficient delivery using vectors, nano-delivery systems, their successful models either in vitro or in vivo and pre-clinical trials. Collectively, tumor suppressor miRNAs can act as novel therapeutics for HCC and more studies should be directed towards these promising therapeutics.

Bioinformatics functional analysis of let-7a, miR-34a, and miR-199a/b reveals novel insights into immune system pathways and cancer hallmarks for hepatocellular carcinoma.

Let-7a, miR-34a, and miR-199 a/b have gained a great attention as master regulators for cellular processes. In particular, these three micro-RNAs act as potential onco-suppressors for hepatocellular carcinoma. Bioinformatics can reveal the functionality of these micro-RNAs through target prediction and functional annotation analysis. In the current study, in silico analysis using innovative servers (miRror Suite, DAVID, miRGator V3.0, GeneTrail) has demonstrated the combinatorial and the individual target genes of these micro-RNAs and further explored their roles in hepatocellular carcinoma progression. There were 87 common target messenger RNAs (p ≤ 0.05) that were predicted to be regulated by the three micro-RNAs using miRror 2.0 target prediction tool. In addition, the functional enrichment analysis of these targets that was performed by DAVID functional annotation and REACTOME tools revealed two major immune-related pathways, eight hepatocellular carcinoma hallmarks-linked pathways, and two pathways that mediate interconnected processes between immune system and hepatocellular carcinoma hallmarks. Moreover, protein-protein interaction network for the predicted common targets was obtained by using STRING database. The individual analysis of target genes and pathways for the three micro-RNAs of interest using miRGator V3.0 and GeneTrail servers revealed some novel predicted target oncogenes such as SOX4, which we validated experimentally, in addition to some regulated pathways of immune system and hepatocarcinogenesis such as insulin signaling pathway and adipocytokine signaling pathway. In general, our results demonstrate that let-7a, miR-34a, and miR-199 a/b have novel interactions in different immune system pathways and major hepatocellular carcinoma hallmarks. Thus, our findings shed more light on the roles of these miRNAs as cancer silencers.