The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes ~3 weeks.

Background: Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens.

Extracellular vesicles (EVs) play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. Production and function of microRNAs require coordinated processing by proteins of the microRNA machinery. Dicer and Drosha (RNase III endonucleases) are essential components of the microRNA machinery. Recently, the ribosome anti-association factor eIF6 has also been found to have a role in microRNA-mediated post-transcriptional silencing. We characterized the alterations in the expression of genes encoding proteins of microRNA machinery in ovarian serous carcinoma. Protein expression of eIF6 and Dicer was quantified in a tissue microarray of 66 ovarian serous carcinomas. Dicer, Drosha and eIF6 mRNA expression was analysed using quantitative reverse transcription-PCR on an independent set of 50 formalin-fixed, paraffin-embedded ovarian serous carcinoma samples. Expression profiles of eIF6 and Dicer were correlated with clinicopathological and patient survival data. We provide definitive evidence that eIF6 and Dicer are both upregulated in a significant proportion of ovarian serous carcinomas and are associated with specific clinicopathological features, most notably low eIF6 expression being associated with reduced disease-free survival. The status of eIF6 and proteins of the microRNA machinery may help predict toxicity and susceptibility to future interfering RNA-based therapy.

BackgroundM2-polarized macrophages are tumor-associated-macrophages (TAMs), which are important contents of tumor-infiltrating immune cells. Toll-like receptor 4 (TLR4) is a molecular biomarker of tumor aggressiveness and poor prognosis. Toll-like receptors (TLRs) have important roles in the immune system and M2-polarized macrophages. However, the effects of TLR4 on M2-polarized macrophages in hepatocellular carcinoma (HCC) are unknown. Here, TLR4 expressed on HCC cells mediates the pro-tumor effects and mechanisms of M2-polarized macrophages.MethodsTHP-1 cells were induced to differentiate into M2-like macrophages through treatments with IL-4, IL-13, and phorbol myristate acetate (PMA). We used the HCC cell lines SMMC-7721 and MHCC97-H cultured in conditioned medium from M2-like macrophages (M2-CM) to investigate the migration potential of HCC cells and epithelial-mesenchymal transition (EMT)-associated molecular genetics. Signaling pathways that mediated M2-CM-promoted HCC migration were detected using western blotting.ResultsHCC cells cultured with M2-CM displayed a fibroblast-like morphology, an increased metastatic capability, and expression of EMT markers. TLR4 expression was markedly increased in M2-CM-treated HCC cells. TLR4 overexpression promoted HCC cell migration, and a TLR4-neutralizing antibody markedly inhibited HCC EMT in cells cultured with M2-CM. Furthermore, the TLR4/(signal transducer and activator of transcription 3 (STAT3) signaling pathway contributed to the effects of M2-CM on HCC cells.ConclusionsTaken together, M2-polarized macrophages facilitated the migration and EMT of HCC cells via the TLR4/STAT3 signaling pathway, suggesting that TLR4 may be a novel therapeutic target. These results improve our understanding of M2-polarized macrophages.Electronic supplementary materialThe online version of this article (10.1186/s12957-018-1312-y) contains supplementary material, which is available to authorized users.