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An experimental strategy unveiling exosomal microRNAs 486-5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer.

ABSTRACT: Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2) or hypoxia (0.2% O2) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo-electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line-specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate-circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen-sensitive miRNAs 486-5p, 181a-5p and 30d-5p, which were retrieved as circulating markers of high-risk LARC.

SUBMITTER: Bjornetro T

PROVIDER: S-EPMC6352936 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundRadiotherapy resistance is a major obstacle in the treatment of oesophageal squamous cell carcinoma (OSCC). Hypoxia is a critical cause of radioresistance. However, the communication between hypoxic cells and aerobic cells via exosomes during the transfer of radiation resistance remains unclear.MethodsExo-miR-340-5p levels were analysed by RNA-seq and qRT-PCR. We co-cultured OSCC cells with isolated normoxic and hypoxic exosomes to study their impact on radiosensitivity. We used a specific exo-miR-340-5p mimic and knock-down retrovirus to explore the role of this miRNA in the transfer of radioresistance from hypoxic to normoxic cells. Dual-luciferase reporter and RIP assays were used to verify KLF10 as a putative target of miR-340-5p. Several in vitro assays were conducted and xenograft models were established to investigate the effect of exo-miR-340-5p on OSCC radiosensitivity. The plasma exo-miR-340-5p levels in OSCC patients were analysed to study the clinical value of this parameter.ResultsHypoxic exosomes alleviated radiation-induced apoptosis and accelerated DNA damage repair. miR-340-5p was highly expressed in hypoxic exosomes and was transferred into normoxic cells, where it induced radioresistance. Overexpression of miR-340-5p in normoxic OSCC cells mimicked the radioresistance of cells co-cultured with hypoxic exosomes. Knockdown of miR-340-5p in hypoxic exosomes reversed the radioresistance effect, indicating that exo-miR-340-5p is critical for hypoxic EV-transferred radioresistance. KLF10 was identified as the direct target of miR-340-5p. Moreover, metformin was found to increase the expression of KLF10 and enhance the radiosensitivity of OSCC. Higher levels of miR-340-5p in the plasma exosomes from OSCC patients are related to a poorer radiotherapy response and prognosis.ConclusionsHypoxic tumour cell-derived exosomal miR-340-5p confers radioresistance in OSCC by targeting KLF10/UVRAG, suggesting that miR-340-5p could be a potential biomarker and therapeutic target for the enhancement of radiosensitivity in OSCC. Metformin can increase KLF10 expression, which ameliorates the radioresistance induced by exo-miR-340-5p transfer. Therefore, metformin could be further investigated as a therapeutic option for the treatment of OSCC.

Project description:Exosomal microRNAs (EXO-miRNAs) are promising non-invasive diagnostic biomarkers for cardiovascular disease. Heart failure with preserved ejection fraction (HFpEF) is a poorly understood cardiovascular complication of diabetes mellitus (DM). Little is known about whether EXO-miRNAs can be used as biomarkers for HFpEF in DM. We aimed to investigate the relationship between EXO-miRNAs and HFpEF in STZ-induced diabetic rats. We prepared STZ-induced diabetic rats exhibiting a type 1 DM phenotype with low body weight, hyperglycemia, hyperlipidemia and hypoinsulinemia. Histological sections confirmed atrophy and fibrosis of the heart, with collagen accumulation representing diabetic cardiomyopathy. Significant decreases in end-diastolic volume, stroke volume, stroke work, end-systolic elastance and cardiac output indicated impaired cardiac contractility, as well as mRNA conversion of two isoforms of myosin heavy chain (α-MHC and β-MHC) and increased atrial natriuretic factor (ANF) mRNA indicating heart failure, were consistent with the features of HFpEF. In diabetic HFpEF rats, we examined a selected panel of 12 circulating miRNAs associated with HF (miR-1-3p, miR-21-5p, miR-29a-5p, miR-30d-5p, miR-34a-5p, miR-126a-5p, miR-143-3p, miR-145-5p, miR-195-5p, miR-206-3p, miR-320-3p and miR-378-3p). Although they were all expressed at significantly lower levels in the heart compared to non-diabetic controls, only six miRNAs (miR-21-5p, miR-30d-5p, miR-126a-5p, miR-206-3p, miR-320-3p and miR-378-3p) were also reduced in exosomal content, while one miRNA (miR-34a-5p) was upregulated. Similarly, although all miRNAs were correlated with reduced cardiac output as a measure of cardiovascular performance, only three miRNAs (miR-30d-5p, miR-126a-5p and miR-378-3p) were correlated in exosomal content. We found that miR-30d-5p and miR-126a-5p remained consistently correlated with significant reductions in exosomal expression, cardiac expression and cardiac output. Our findings support their release from the heart and association with diabetic HFpEF. We propose that these two EXO-miRNAs may be important for the development of diagnostic tools for diabetic HFpEF.

Project description:BackgroundPolymorphonuclear neutrophils (PMNs) play an important role in sepsis-related acute lung injury (ALI). Accumulating evidence suggests PMN-derived exosomes as a new subcellular entity acting as a fundamental link between PMN-driven inflammation and tissue damage. However, the role of PMN-derived exosomes in sepsis-related ALI and the underlying mechanisms remains unclear.MethodsTumor necrosis factor-α (TNF-α), a key regulator of innate immunity in sepsis-related ALI, was used to stimulate PMNs from healthy C57BL/6J mice in vitro. Exosomes isolated from the supernatant were injected to C57BL/6J wild-type mice intraperitoneally (i.p.) and then examined for lung inflammation, macrophage (Mϕ) polarization and pyroptosis. In vitro co-culture system was applied where the mouse Raw264.7 macrophages or bone marrow-derived macrophages (BMDMs) were co-cultured with PMN-derived exosomes to further confirm the results of in vivo animal study and explore the potential mechanisms involved.ResultsExosomes released by TNF-α-stimulated PMNs (TNF-Exo) promoted M1 macrophage activation after in vivo i.p. injection or in vitro co-culture. In addition, TNF-Exo primed macrophage for pyroptosis by upregulating NOD-like receptor 3 (NLRP3) inflammasome expression through nuclear factor κB (NF-κB) signaling pathway. Mechanistic studies demonstrated that miR-30d-5p mediated the function of TNF-Exo by targeting suppressor of cytokine signaling (SOCS-1) and sirtuin 1 (SIRT1) in macrophages. Furthermore, intravenous administration of miR-30d-5p inhibitors significantly decreased TNF-Exo or cecal ligation and puncture (CLP)-induced M1 macrophage activation and macrophage death in the lung, as well as the histological lesions.ConclusionsThe present study demonstrated that exosomal miR-30d-5p from PMNs contributed to sepsis-related ALI by inducing M1 macrophage polarization and priming macrophage pyroptosis through activating NF-κB signaling. These findings suggest a novel mechanism of PMN-Mϕ interaction in sepsis-related ALI, which may provide new therapeutic strategies in sepsis patients.

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An experimental strategy unveiling exosomal microRNAs 486-5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer.

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