Exosomes, as cell–cell communicators with an endosomal origin, are involved in the progression of various diseases. RAB5A, a member of the small Rab GTPases family, which is well known as a key regulator of cellular endocytosis, is expected to be involved in exosome secretion. Here, we found the impact of RAB5A on exosome secretion from human hepatocellular carcinoma cell line using a rapid yet reliable bioinformatics approach followed by experimental analysis. Initially, RAB5A and exosome secretion‐related genes were gathered from bioinformatics tools, namely, CTD, COREMINE, and GeneMANIA; and published papers. Protein–protein interaction (PPI) was then constructed by the Search Tool for Retrieval of Interacting Genes (STRING) database. Among them, several genes with different combined scores were validated by the real‐time quantitative polymerase chain reaction (RT‐qPCR) in stable RAB5A knockdown cells. Thereafter, to validate the bioinformatics results functionally, the impact of RAB5A knockdown on exosome secretion was evaluated. Bioinformatics analysis showed that RAB5A interacts with 37 genes involved in exosome secretion regulatory pathways. Validation by RT‐qPCR confirmed the association of RAB5A with candidate interacted genes and interestingly showed that even medium to low combined scores of the STRING database could be experimentally valid. Moreover, the functional analysis demonstrated that the stable silencing of RAB5A could experimentally decrease exosome secretion. In conclusion, we suggest RAB5A as a regulator of exosome secretion based on our bioinformatics approach and experimental analysis. Also, we propose the usage of PPI‐derived from the STRING database regardless of their combined scores in advanced bioinformatics analysis.

Accurate relative gene expression analysis by reverse transcription‐quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well‐known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes‐isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK‐BR‐3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes‐isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

Background: MicroRNAs (miRNA) are small non-coding RNAs that act as one of the main regulators of gene expression. They are involved in maintaining a proper balance of diverse processes including differentiation, proliferation and cell death in normal cells. Cancer biology can also be affected by these molecules by modulating the expression of oncogenes or tumor suppressor genes. Thus, miRNA based anticancer therapy is currently being developed either alone or in combination with chemotherapy agents used in cancer management, aiming at promoting tumor regression and increasing cure rate. Access to large quantities of RNA agents can facilitate RNA research and development. In addition to currently used in vitro methods, fermentation-based approaches have recently been developed which can cost‐effectively produce biological RNA agents with proper folding needed for the development of RNA-based therapeutics. Nevertheless, a major challenge in translating preclinical studies to clinical for miRNA-based cancer therapy is the efficient delivery of these agents to target cells. Targeting of miRNAs/antimiRNAs using antibodies and/or peptides can minimize cellular and systemic toxicity. Conclusion: Here, we provide a brief review of miRNA in the following aspects: biogenesis and mechanism of action of miRNAs, the role of miRNAs in cancer as tumor suppressors or oncogenes, the potential of using miRNAs as novel and promising therapeutics, miRNA-mediated chemo-sensitization, and currently utilized methods for the in vitro and in vivo production of RNA agents. Finally, an update on the viral and non-viral delivery systems is addressed.

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