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Extracellular vesicle (EV)-based liquid biopsies have been proposed to be a readily obtainable biological substrate recently for both profiling and diagnostics purposes. Development of a fast and reliable preparation protocol to enrich such small particles could accelerate the discovery of informative, disease-related biomarkers. Though multiple EV enrichment protocols are available, in terms of efficiency, reproducibility and simplicity, precipitation-based methods are most amenable to studies with large numbers of subjects. However, the selectivity of the precipitation becomes critical. Here, we present a simple plasma EV enrichment protocol based on pluronic block copolymer. The enriched plasma EV was able to be verified by multiple platforms. Our results showed that the particles enriched from plasma by the copolymer were EV size vesicles with membrane structure; proteomic profiling showed that EV-related proteins were significantly enriched, while high-abundant plasma proteins were significantly reduced in comparison to other precipitation-based enrichment methods. Next-generation sequencing confirmed the existence of various RNA species that have been observed in EVs from previous studies. Small RNA sequencing showed enriched species compared to the corresponding plasma. Moreover, plasma EVs enriched from 20 advanced breast cancer patients and 20 age-matched non-cancer controls were profiled by semi-quantitative mass spectrometry. Protein features were further screened by EV proteomic profiles generated from four breast cancer cell lines, and then selected in cross-validation models. A total of 60 protein features that highly contributed in model prediction were identified. Interestingly, a large portion of these features were associated with breast cancer aggression, metastasis as well as invasion, consistent with the advanced clinical stage of the patients. In summary, we have developed a plasma EV enrichment method with improved precipitation selectivity and it might be suitable for larger-scale discovery studies. [ABSTRACT FROM AUTHOR]

The KIAA0100 gene was identified in the human immature myeloid cell line cDNA library. Recent studies have shown that its expression is elevated in breast cancer and associated with more aggressive cancer types as well as poor outcomes. However, its cellular and molecular function is yet to be understood. Here we show that silencing KIAA0100 by siRNA in the breast cancer cell line MDA-MB-231 significantly reduced the cancer cells’ aggressive behavior, including cell aggregation, reattachment, cell metastasis and invasion. Most importantly, silencing the expression of KIAA0100 particularly sensitized the quiescent cancer cells in suspension culture to anoikis. Immunoprecipitation, mass spectrometry and immunofluorescence analysis revealed that KIAA0100 may play multiple roles in the cancer cells, including stabilizing microtubule structure as a microtubule binding protein, and contributing to MDA-MB-231 cells Anoikis resistance by the interaction with stress protein HSPA1A. Our study also implies that the interaction between KIAA0100 and HSPA1A may be targeted for new drug development to specifically induce anoikis cell death in the cancer cell. [ABSTRACT FROM AUTHOR]

Accumulating evidences indicate that long noncoding RNAs (lncRNAs) might play important roles in tumorigenesis and metastasis. EMT (epithelial-to-mesenchymal transition) is considered as a critical step in invasion and metastasis of various tumors including bladder cancer (BC). Recent researches have showed that lncRNA H19 is implicated in metastasis through regulating EMT and the reverse MET (mesenchymal-to-epithelial transition). However, underlying mechanisms remain largely unknown. Here, we screened lncRNA and mRNA expression profiles of BC with microarray assay. We found that H19 and DNMT3B displayed a higher co-expression in BC tissues and cells. Functionally, we demonstrated that H19 could increase proliferation, invasion and migration, regulate EMT as well as rearrange cytoskeleton of BC cells in vitro . Moreover, ectopic expression of H19 promoted tumorigenesis, angiogenesis and pulmonary metastasis in vivo , whereas knockdown of H19 has a contrary role in vivo and in vitro . Mechanistically, we proved that H19 could directly bind to miR-29b-3p (miR-29b) and derepress the expression of target DNMT3B. H19 and miR-29b-3p showed a co-localization. More importantly, up-regulating H19 antagonized miR-29b-3p-mediated proliferation, migration and EMT suppression in BC cells. Furthermore, H19 knockdown partially reversed the function of miR-29b-3p inhibitor on DNMT3B and facilitated miR-29b-3p-induced MET. Taken together, we demonstrated for the first time that H19 might function as ceRNA (competing endogenous RNA) for miR-29b-3p and relieve the suppression for DNMT3B, which led to EMT and metastasis of BC. Our findings highlight a novel mechanism of H19 in progression of BC and provide H19/miR-29b-3p/DNMT3B axis as a promising therapeutic target for BC. [ABSTRACT FROM AUTHOR]