Exosomal mRNA Research Articles

Comprehensive analysis of miRNA-gene regulatory network with clinical significance in human cancers.

microRNAs (miRNAs), particularly the exosomal miRNAs have been widely used as biomarkers and promising therapeutic targets in cancer. However, a comprehensive analysis of miRNA-gene regulatory network with clinical significance remains scarce. The emergence of high-throughput multi-omics data over large, well-characterized patient cohorts provides an unprecedented opportunity to address this problem. Herein, we performed a clinic-centered analysis to identify cancer-associated miRNAs, miRNA-target axis. We first calculated the correlation among miRNA, mRNA and 75 unique clinico-pathological characteristics (CPCs) in 26 cancer types, and established an online resource (4CR). Interestingly, we found that the high expression of several DNA methylation-related enzymes was associated with adverse outcomes of cancer patients, and these genes were regulated by a cluster of miRNAs. Furthermore, by integrating exosomal miRNA and mRNA databases, we identified exosomal miRNA biomarkers for non-invasive cancer surveillance and therapy monitoring. Finally, we explored the role of CPC-related miRNAs for therapeutic effect prediction of drugs based on their shared targets. Our analysis pipeline illustrated the significance of clinic-centered analysis in miRNA-gene pair identification and provided helpful clues for future cancer studies.

Down-regulation of ER-α36 mRNA in serum exosomes of the patients with hepatocellular carcinoma.

Down-regulation of ER-α36 mRNA in serum exosomes of the patients with hepatocellular carcinoma.

High-exosomal AKR1C3 mRNA expression as a marker of poor efficacy of abiraterone in metastatic PCa patients.

196 Background: Aldo-keto reductase family 1 member C3 (AKR1C3) has been proved to be an important part in the androgen biosynthesis process. Previous researches have showed that immunohistochemical (IHC) AKR1C3 expression is a prognosticator in prostate cancer (PCa) patients treated with abiraterone. This study is to find out the relationship between exosomal AKR1C3 mRNA expression and the efficacy of abiraterone in metastatic PCa patients. Methods: Blood samples of metastatic prostate cancer patients during different disease stages were collected. We isolated the exosomes and extracted the RNA for analysis of AKR1C3 by ddPCR. Absolute target mRNA concentration was measured by ddPCR as copies per milliliter (copies/20ul). Clinical data were collected for all patients. Primary study endpoint was progression-free survival (PFS). Statistical analyses were performed with SPSS 25.0. Results: Exosomal AKR1C3 mRNA expression was positive in 71.9% (41/57) patients, and high expression (defined as ≥ 20 copies/20μl) was found in 12.3% (7/57) patients. High exosomal AKR1C3 mRNA expression was not related to positive IHC AKR1C3 expression ( P=0.723). Patients with high exosomal AKR1C3 expression and negative IHC AKR1C3 expression harbored only 4.866 months’ median progression-free survival (PFS) compared with the whole cohort (9.37 months). High exosomal AKR1C3 expression is significantly associated with shorter PFS (median PFS 8.036 months, p<0.001). Multivariate Cox regression analysis revealed that high exosomal AKR1C3 expression was an independent prognosticator of abiraterone treatment efficacy (OR: 8.891, 95%CI: 1.309-61.631, P=0.026). In subsequently subgroup analyses, high exosomal AKR1C3 expression demonstrates particularly high hazard ratio in multiple subgroups (patients with baseline PSA >100 ng/mL, ALP >71 IU/L, HGB >120 g/L, and patients receiving abiraterone as first-line therapy in castration resistant prostate cancer). Conclusions: Exosomal AKR1C3 level is an independent adverse prognosticator in metastatic PCa patients receiving abiraterone treatment, especially when abiraterone was used as first-line therapy in castration resistant prostate cancer.

Exosomal TUBB3 mRNA expression level to predict prognosis for abiraterone treatment in metastatic prostate patients.

244 Background: TUBB3 (Class III β-tubulin), a dynamic component of microtubules, is considered a prognostic biomarker of tumor aggressiveness and poor survival. Previous studies have researched into its indication of resistance to chemotherapeutic drugs. However, its association with abiraterone, a next-generation androgen-axis targeting drug in prostate cancer has not been studied yet. In this study, we aimed to find out if exosomal TUBB3 expression could predict the efficacy of abiraterone in metastatic prostate cancer patients. Methods: Blood samples of metastatic prostate cancer patients during different disease stages were collected. We isolated the exosomes and extracted the RNA for analysis of TUBB3 by ddPCR. Absolute target mRNA concentration was measured by ddPCR as copies per milliliter (copies/20ul). Clinical data were collected for all patients. Primary study endpoint was progression-free survival (PFS). Statistical analyses were performed with SPSS 25.0. Results: A total of 54 metastatic prostate cancer patients who have received abiraterone treatment were enrolled. Baseline characteristics are comparable between high (≥10 copies/20ul) and low (<10 copies/20ul) TUBB3 expression groups. Univariate analyses revealed that baseline hemoglobin≤120g/L, baseline sodium concentration >140 mmol/L, baseline potassium concentration >4.39 mmol/L and high exosomal TUBB3 expression were associated with shorter PFS, while only baseline sodium concentration and exosomal TUBB3 expression were independent prognosticators in multivariate analyses. In subsequently subgroup analyses, high exosomal TUBB3 expression demonstrates particularly high hazard ratio in multiple subgroups (patients younger than 70 years old, without neuroendocrine differentiation and immunohistochemical androgen-receptor splice variant 7 expression, with baseline PSA >100 ng/mL, and patients receiving abiraterone as first-line therapy). Conclusions: Exosomal TUBB3 expression has potential in making treatment plan and predict prognosis in metastatic prostate cancer patients. High exosomal TUBB3 expression indicates poor response to abiraterone, especially as first line therapy.

Comprehensive analysis of dysregulated exosomal long non-coding RNA networks associated with arteriovenous malformations.

Arteriovenous malformations (AVMs) are congenital vascular lesions with a high tendency for aggravation and recurrence after treatment, and their genesis remains enigmatic. In this study, we investigated exosomal long non-coding RNA (lncRNA) and mRNA expression and constructed a competitive endogenous RNA regulatory network in AVMs. Ethics approval was provided, and informed written consent was given prior to the inclusion of all participants. Blood samples were obtained from patients with AVMs and healthy controls at Shanghai Ninth People's Hospital, China, from May to November 2018, and total exosomes were isolated and validated. Differentially expressed exosomal lncRNAs and mRNAs were detected by RNA-seq, analysed by bioinformatic methods and validated by qRT-PCR. A competitive endogenous RNA regulatory network was constructed. The characteristics of the captured extracellular vesicles conformed to the features of exosomes. A total of 117 dysregulated exosomal lncRNAs and 1159 dysregulated exosomal mRNAs were identified in AVMs. qRT-PCR demonstrated that the exosomal lncRNAs MIR4435-1HG, LINC00657, LOC101927854 and SEPT5-GP1BB were upregulated in AVM exosomes. The Gene Ontology (GO) terms haemopoiesis and negative regulation of neuron projection development were significantly enriched in relation to dysregulated exosomal cis lncRNAs. A total of 199 GO terms and 80 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched for the dysregulated exosomal mRNAs. In the exosomal lncRNA-miRNA-mRNA-related ceRNA regulatory network, the top 3 significant modules involved 31 dysregulated exosomal lncRNAs and 114 dysregulated exosomal mRNAs, which were enriched in the Rap 1, Ras, MAPK signalling pathways and platelet activation KEGG pathway. This study comprehensively identified dysregulated exosomal lncRNAs and mRNAs in AVMs, demonstrated the involvement of dysregulated lncRNA and mRNA patterns in AVMs and constructed an exosomal competitive endogenous RNA regulatory network.

Aberrant Expression of Cancer-Testis Antigen FBXO39 in Breast Cancer and its Clinical Significance.

Cancer/testis antigen (CTA) is a class of antigen molecules expressed only in the germinal epithelium of testis and some tumor tissues. As an important CTA molecule, the expression of F-box protein 39 (FBXO39) in breast cancer (BC) and its clinical significance remain unclear. The objective of this study is to explore the value of FBXO39 in the diagnosis, efficacy monitoring, and prognostic evaluation of BC. The expression of FBXO39 mRNA in the serum exosomes of patients with BC before and after the initial diagnosis and treatment was detected by qRT-PCR, and the corresponding ROC curve was plotted. The expression of FBXO39 protein in BC cancer tissues was detected by immunohistochemistry, along with the analysis of the correlation between FBXO39 expression and clinical pathological features as well as prognosis of BC cases. The serum-derived exosomes were successfully isolated and identified. The positive rate of FBXO39 mRNA in serum exosomes of patients with BC was up to 86%; there was a correlation between the expression level of serum exosomal FBXO39 and clinical staging, HER2, and Ki-67 expression (all with p < 0.05). The sensitivity of serum exosomal FBXO39 in distinguishing BC patients from healthy controls was 88%, with the specificity as 86%, and AUC as 0.9432. The expression change of FBXO39 in serum-sourced exosomes of patients with BC was related to their treatment situation, indicating that the level of FBXO39 decreased significantly after treatment. The expression of FBXO39 in cancer tissue was related to the clinical stage (p = 0.023) and lymphatic metastasis (p = 0.015) of the BC patients. Survival analysis showed that the expression of FBXO39 was negatively correlated with the prognosis of BC patients, with the high expression of FBXO39 indicating poor prognosis. Serum-derived exosomal FBXO39 could serve as an important indicator of BC diagnosis and efficacy evaluation; FBXO39 could be rated as an important indicator of BC prognosis evaluation.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and of their ability to penetrate physiological barriers that are impermissible to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs (mRNAs), into cell-secreted exosomes leads to low yields. Here, we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs, and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared to bulk electroporation and to other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and more than a 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic PTEN-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced tumour-growth inhibition, and increased animal survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Integrated analysis of exosomal lncRNA and mRNA expression profiles reveals the involvement of lnc-MKRN2-42:1 in the pathogenesis of Parkinson's disease.

BackgroundParkinson's disease (PD) is a common movement disorder for which diagnosis mainly depends on the medical history and clinical symptoms. Exosomes are now considered an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids, and genetic material. Long noncoding (lnc) RNA in exosomes plays a critical role in many diseases, including neurodegenerative disease.AimTo study expression differences for lncRNAs in peripheral blood exosomes of PD patients compared with healthy individuals and to look for lncRNAs that might be related to the pathogenesis of PD.Materials and MethodsWe recruited PD patients along with age‐ and sex‐matched healthy individuals as healthy controls and evaluated levels of lncRNAs extracted from exosomes in plasma samples via next‐generation sequencing and real‐time quantitative PCR. Correlation analysis was conducted for the clinical characteristics of PD patients and the expression of selected lncRNAs.ResultsWe found 15 upregulated and 24 downregulated exosomal lncRNAs in the PD group. According to bioinformatics analyses, we chose lnc‐MKRN2‐42:1 for further study. Interestingly, lnc‐MKRN2‐42:1 was positively correlated with the MDS‐UPDRS III score for PD patients.ConclusionOur study suggested that lnc‐MKRN2‐42:1 may be involved in the occurrence and development of PD.

PATH-64. PROSPECTIVE, BLINDED PLASMA BASED ANALYSIS FOR DIAGNOSIS OF NEWLY DIAGNOSED GLIOMA

Abstract INTRODUCTION In patients with newly diagnosed intracerebral lesions, gliomas are often suspected. However, other conditions such as multiple sclerosis, abscess or lymphoma are possible, as well. Furthermore, biopsy can be challenging due to eloquent and/or deep location within the brain. In this prospective, blinded study, analysis of plasma isolated cell-free DNA and exosome mRNA and miRNA from newly diagnosed glioma patients and from cancer-free volunteers was used to predict disease. METHODS Plasma was drawn from 52 patients with newly diagnosed gliomas (28 high grade glioma (HGG), 10 low grade (LGG)) and 14 patients without documented history of cancer and recent MRI brain which was negative for brain tumor. High quality DNA and RNA was isolated and sequenced using Next Generation Sequencing and Digital Droplet PCR was used for detection and verification of trace molecular artifacts. Multianalyte processing yielded data that was harmonized and interpreted through an Artificial Intelligence based algorithm to assess for possible glioma and to assign grade in a blinded fashion. EGFRvIII, TP53 and IDH1 mutations were also analyzed and compared to molecular testing from tumor specimens. RESULTS 66% (25 of 38) of glioma patients were correctly diagnosed as having a malignancy. 43% of HGG and 60% of LGG patients were correctly graded. Of the 14 normal controls, 6 were concluded to be cancer-free. IDH1, EGFRvIII, and TP53 mutation had concordance of 64% (21/33), 82% (14/17) and 36% (5/14), respectively. CONCLUSIONS Analysis of plasma cell free tumor derived DNA and RNA was relatively sensitive for detecting glioma in treatment naïve patients. In contrast, this analysis was not specific in ruling out malignancy in the normal control patients. Given this profile, in patients with newly diagnosed intracerebral lesions suspicious for glioma, this may be a useful screening test to determine the need for more invasive testing, i.e. biopsy/resection.

Integrated exosomal miRNA and transcriptome analysis of brain microvascular endothelial cells in spontaneously hypertensive rats.

Endothelial cells, which regulate arterial stiffness via endothelial-derived substances, are independently and strongly associated with hypertension. However, the exact roles of exosome miRNAs from brain endothelial cells in the development of hypertension are still not fully explored. Here, we investigated the miRNA functions systematically by examining both exosomal small RNA and mRNA of endothelial cells in Wistar Kyoto (WKY) rats versus spontaneously hypertensive rats (SHRs). Our findings revealed that miRNAs, representing ~60-70%, account for the majority of small RNAs. Moreover, we found 159 novel miRNAs in total from the unannotated reads across the diverse samples. Afterwards, 76 differentially expressed miRNAs (37 upregulated, 39 downregulated) and 1709 differentially expressed mRNAs (775 upregulated, 934 downregulated) were identified between SHRs and WKY rats, respectively. Finally, 647 genes targeted by 36 miRNAs came to our attention via identification of the target genes of those abnormal miRNAs. The differentially expressed target genes induced by miRNA changes were mapped to a number of genes involved in various gene functions and pathways. These changes lead to dysregulation of angiogenesis, axonogenesis, neuron-to-neuron synapses, focal adhesion, axon guidance, cell adhesion molecules (CAMs), adherens junction, and ECM-receptor interaction pathways. Together, our study revealed that the miRNAs are changed and contribute to the dysregulated functions and pathways of their target genes and provided more insights into their regulation mechanisms during mammalian hypertension development.

Serum Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) mRNA Protected by Exosomes as a Potential Biomarker for Gastric Cancer.

BackgroundOur previous research revealed that membrane type 1-matrix metalloproteinase (MT1-MMP) is overexpressed and plays a crucial role in gastric cancer (GC) progression. Exosomes are important for GC carcinogenesis, and the exosomal contents are ideal biomarkers. However, the expression of exosomal MT1-MMP mRNA in serum and its potential significance in GC remains unclear.Material/MethodsThe expression of exosomal MT1-MMP mRNA in serum of patients with GC, chronic gastritis, or atypical hyperplasia, and healthy controls was detected using real-time quantitative RT-PCR. Serum CEA, CA19-9, and CA72-4 were also measured by electrochemiluminescence assay.ResultsExosomes were isolated and identified in serum, and serum exosomal MT1-MMP mRNA was found to be higher in patients with GC compared with healthy controls and patients with chronic gastritis or atypical hyperplasia (all P<0.05). Serum exosomal MT1-MMP mRNA was significantly correlated with tumor diameter, differentiation, Borrmann type, invasion depth, lymphatic metastasis, distal metastasis, and TNM stage. The AUC of exosomal MT1-MMP mRNA was 0.788 (95% CI: 0.714–0.850) with 63.9% sensitivity and 87.1% specificity, and was higher than that of CEA (0.655 (95% CI: 0.573–0.730)). The combination of 2 markers gave an AUC of 0.821 (95% CI: 0.750–0.878), which was better than with the individual marker. The sensitivity, specificity, and positive and negative predictive values were 75.6%, 83.9%, 94.7%, and 47.3%, respectively. Moreover, the multiple logistic regression model showed that tumor diameter, differentiation, invasion depth, and exosomal MT1-MMP mRNA were the risk factors for lymphatic metastasis in GC.ConclusionsOur results characterized serum exosomal MT1-MMP mRNA in GC, providing a foundation for discovering serum exosomes-targeted biomarkers for GC diagnosis.

Exosomal MT1-MMP mRNA in Serum As a Potential Biomarker for Gastric Cancer

Background: Our previous results reveal that membrane type 1-matrix metalloproteinase (MT1-MMP) is overexpressed and plays a crucial roles in gastric cancer (GC) progression. Exosomes are important for GC carcinogenesis, and the exosomal contents are used as ideal biomarkers. However, the expression of exosomal MT1-MMP mRNA in serum and its potential significance in GC remains unclear. Methods: The expression of exosomal MT1-MMP mRNA in serum of GC, chronic gastritis, atypical hyperplasia, and healthy controls were detected using real-time quantitative RT-PCR. Meanwhile, serum CEA, CA19-9 and CA72-4 were also measured by electrochemiluminescence assay. Findings: Exosomes were isolated and identified in serum. Our results revealed that exosomal MT1-MMP mRNA in serum was elevated in GC, compared with healthy controls, chronic gastritis, atypical hyperplasia, respectively (all P<0.05). It was significantly correlated with tumor diameter, differentiation, borrmann type, invasion depth, lymphatic metastasis, distal metastasis, TNM stage. AUC of exosomal MT1-MMP mRNA was 0.788 (95%CI: 0.714-0.850) with sensitivity 63.9% and specificity 87.1%, and higher than that of CEA (0.655 (95%CI: 0.573-0.730)). The combination of two markers gave an AUC of 0.821 (95%CI: 0.750-0.878), better than the individual marker. The sensitivity, specificity, positive and negative predictive value were 75.6%, 83.9%, 94.7%, 47.3%, respectively. Moreover, the multiple logistic regression model showed that tumor diameter, differentiation, invasion depth and exosomal MT1-MMP mRNA were the risk factors of lymphatic metastasis in GC. Interpretation: As a pilot investigation to characterizing the serum exosomal MT1-MMP mRNA in GC, our study provided a foundation for discovering serum exosomes-targeted biomarkers for GC diagnosis. Funding Statement: National Natural Science Foundation of China (81572070, 31500741). Natural Science Foundation of Shandong Province (BS2014SW022, ZR2016HM37). Key Research and Development Project of Shandong Province (2016GSF201124, 2015GSF118152). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Our study was approved by Medical Ethics Committee of Qilu Hospital of Shandong University (KYLL-2015-097). The participants signed written informed consents.

Exosomes from high glucose–treated macrophages activate glomerular mesangial cells via TGF‐β1/Smad3 pathway in vivo and in vitro

Diabetes nephropathy (DN) is characterized by abnormal interactions between kidney-infiltrating macrophages and glomerular mesangial cells. Recently, a novel cell-cell communication mediated by exosomes has gained attention. Exosomes are membrane-bound vesicles that contain a variety of molecules such as proteins, lipids, DNA, mRNA, and microRNAs. Exosomes play an important role in the pathogenesis of DN. In this study, we show that high glucose (HG) led to increased excretion of exosomes from macrophages. Mesangial cells took up exosomes in vitro, which resulted in the activation and proliferation of mesangial cells and the secretion of extracellular matrix and inflammatory cytokines. In addition, C57BL/6 mice injected with exosomes from HG-treated macrophages showed morphologic and functional changes. We then showed that exosomes from HG-treated TGF-β1 knockdown macrophages induced less extracellular matrix and fewer inflammatory factors in mesangial cells compared with vector control. Our findings suggest that TGF-β1 mRNA in exosomes serves a role between macrophages and mesangial cells by activating the TGF-β1/ mothers against decapentaplegic homolog 3 pathway.-Zhu, Q.-J., Zhu, M., Xu, M.-X., Meng, X.-M., Wu, Y.-G. Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-β1/Smad3 pathway in vivo and in vitro.

Integrating liquid biopsy with advanced imaging analysis to improve the prediction of response to immunotherapy in patients with NSCLC.

e14054 Background: Immunotherapy has revolutionized the treatment of NSCLC. However, response rate is variable, with a substantial failure rate. Thus, the identification of predictive biomarkers of response to immunotherapy is an area of great interest. Methods: Patients with locally advanced or metastatic NSCLC treated with nivolumab or pembrolizumab were enrolled. Disease response was defined following RECIST criteria (v. 1.1). Four ml of plasma were collected for the analysis of exosomal mRNA levels of PD-L1 (e-PD-L1) and IFN-γ (e-IFN-γ) at baseline and at the time of first radiological assessment. Exosome isolation and mRNA extraction was obtained by the exoRNeasy kit (Qiagen, Valencia, CA). e-PD-L1 and e-IFN-γ were evaluated by the QX100 ddPCR (Bio-Rad, Hercules, CA) and expressed as allele frequency (%). Chest computed tomography (CT) scan at baseline was used for the radiomic analysis. Tumor segmentation was performed on DICOM-formed images taken from the picture archiving, and the regions of interest were delineated manually and analyzed using the QUIBIM SL software. Survival was calculated stratifying patients based on e-PD-L1 and e-IFN-γ median values. Results: Nivolumab was given to 17 patients as 2nd line and to 8 subjects as further line of treatment, while 13 patients received 1st line pembrolizumab. Median PFS was 11 vs 16.2 months (mos) in patients with baseline e-PD-L1 of &lt; 0.3% vs ≥0.3%, respectively (p = 0.16). e-PD-L1 significantly increased in disease progression (PD) vs partial response (PR) and disease stabilization (SD) (p = 0.01) after 2 mos of treatment. In patients with e-IFN-γ ≥4.1% vs &lt; 4.1% at baseline, median PFS was 5.6 mos vs not reached, respectively (p = 0.003). The multiparametric radiomic analysis identified the Cluster Prominence Value (CPV, p = 0.012) and the Cluster Shade Value (CSV, p = 0.034) as significantly correlated with treatment outcome. Moreover, the D2d parameter and e-IFN-γ were inversely correlated (p &lt; 0,0001). CPV and D2d reflect the intra-tumor architecture, including necrosis, cell proliferation, and angiogenesis. Conclusions: Liquid biopsy data correlate with radiomic parameters and predict response to immunotherapy.

Pantoprazole attenuates tumorigenesis via inhibition of exosomal secretion in a rat model of hepatic precancerous lesion induced by diethylnitrosamine and 2-acetamidofluorene.

The present study aimed to evaluate the potential therapeutic effect of pantoprazole, a proton-pump inhibitor, on precancerous lesion (PCL) in rats. diethylnitrosamine and 2-acetylaminofluorene were used to induce PCL in rats, in vivo. The rats were treated with three doses of pantoprazole (100, 50, and 25 mg/kg; three times weekly) during the last 4 weeks of the total 10 weeks of the experiment. Blood and liver tissue samples were collected for measurement of the exosomal abundance and exosomal competing endogenous RNA markers. Results revealed that pantoprazole administration had an ameliorating effect on liver function tests and microscopic features of the liver; and decreased exosome abundance in the liver tissue samples and sera of the rats. Meanwhile, the treatment also resulted in a dose-dependent decrease in exosomal RAB11A mRNA and long noncoding RNA RP11-513I15.6, which is an important participant in th exosomal secretion process with an increase in exosomal miRNA-1262. Based on these results, we postulated that pantoprazole has the potential to attenuate liver tumorigenesis in this rat model.

Detection of Exosomal PD-L1 RNA in Saliva of Patients With Periodontitis.

Periodontitis is the most prevalent inflammatory disease of the periodontium, and is related to oral and systemic health. Exosomes are emerging as non-invasive biomarker for liquid biopsy. We here evaluated the levels of programmed death-ligand 1 (PD-L1) mRNA in salivary exosomes from patients with periodontitis and non-periodontitis controls. The purposes of this study were to establish a procedure for isolation and detection of mRNA in exosomes from saliva of periodontitis patients, to characterize the level of salivary exosomal PD-L1, and to illustrate its clinical relevance. Bioinformatics analysis suggested that periodontitis was associated with an inflammation gene expression signature, that PD-L1 expression positively correlated with inflammation in periodontitis based on gene set enrichment analysis (GSEA) and that PD-L1 expression was remarkably elevated in periodontitis patients versus control subjects. Exosomal RNAs were successfully isolated from saliva of 61 patients and 30 controls and were subjected to qRT-PCR. Levels of PD-L1 mRNA in salivary exosomes were higher in periodontitis patients than controls (P < 0.01). Salivary exosomal PD-L1 mRNA showed significant difference between the stages of periodontitis. In summary, the protocols for isolating and detecting exosomal RNA from saliva of periodontitis patients were, for the first time, characterized. The current study suggests that assay of exosomes-based PD-L1 mRNA in saliva has potential to distinguish periodontitis from the healthy, and the levels correlate with the severity/stage of periodontitis.

GATA2 exosomal mRNA: A novel urine biomarker for the diagnosis of clinically significant prostate cancer.

18 Background: Prostate specific antigen (PSA) detection in blood is widely used to screen for prostate cancer (PCa). However, PSA has limited utility in discriminating tumors at high risk of progression from indolent-low risk tumors, which leads to PCa overtreatment and highlights the need to identify better biomarkers for the detection of clinically significant PCa (Gleason Score; GS ≥ 7). Here, we introduce a novel mRNA GATA2 exosomal biomarker detected in urine useful to diagnose clinically significant PCa. Exosomes are 50-120 nm sized vesicles shed in body fluids that contain RNA, DNA, and protein that can be used as biomarkers to interrogate the health of the originating cells. GATA2 is a pioneer transcription factor specific to PCa, increased in aggressive tumors. Methods: To evaluate the predictive value of exosomal GATA2 mRNA levels for aggressive PCa, we isolated exosomes using ultracentrifugation in non-DRE urines from 128 males with elevated PSA serum levels (&gt; 4ng/ml) and analyzed them according to their tissue biopsy result. Results: Importantly, prostate origin of GATA2 mRNA detected in urine exosomes was confirmed by observing that exosomal GATA2 mRNA levels significantly dropped after prostatectomy (p &lt; 0.05). Most remarkably, GATA2 exosomal mRNA expression was significantly increased in PCa biopsy positive patients (n=88) when compared to biopsy negative males (n=28, p &lt; 0.001). Furthermore, GATA2 levels in PCa patients were significantly associated with GS, but not with disease stage and PSA levels. Multivariable regression analysis showed that GATA2 exosomal mRNA levels are an independent factor for the diagnosis of aggressive PCa. Urine GATA2 exosome mRNA expression plus SOC (age and PSA) was associated with improved discrimination of GS ≥ 7 versus GS 6 and benign disease: AUC of SOC alone 0.59 (0.49-0.70, p value=0.083) vs SOC plus GATA2 AUC of 0.68 (95% CI, 0.58-0.78, p value=0.0004). Conclusions: The analysis of urine exosomal GATA2 mRNA in individuals with high PSA may help distinguish clinically significant PCa and reduce the number of patients that need biopsy. Future studies are focused on combining this biomarker to others, such as urine exosomal PCA3, already used in the clinic.

Glycerol kinase 5 confers gefitinib resistance through SREBP1/SCD1 signaling pathway

BackgroundDrug resistance is common in cancer chemotherapy. This study investigates the role of Glycerol kinase 5 (GK5) in mediating gefitinib resistance in NSCLC.MethodsThe exosomal mRNA of GK5 was detected using a tethered cationic lipoplex nanoparticle (TCLN) biochip. Real-time PCR and Western blot were used to examine the expression of GK5 mRNA and protein in gefitinib-sensitive and -resistant human lung adenocarcinoma cells. The cell counting kit-8, EdU assay, flow cytometry, and JC-1 dye were used to measure cell proliferation, cell cycle, and the mitochondrial membrane potential.ResultsWe found that the exosomal mRNA of GK5 in the plasma of patients with gefitinib-resistant adenocarcinoma was significantly higher compared with that of gefitinib-sensitive patients. The mRNA and protein levels of GK5 were significantly upregulated in gefitinib-resistant human lung adenocarcinoma PC9R and H1975 cells compared with gefitinib-sensitive PC9 cells. Silencing GK5 in PC9R cells induced mitochondrial damage, caspase activation, cell cycle arrest, and apoptosis via SREBP1/SCD1 signaling pathway.ConclusionsWe demonstrated that GK5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and cell cycle arrest. GK5 could be a novel therapeutic target for treatment of NSCLC with resistance to EGFR tyrosine kinase inhibitors.

178 Cellular and extracellular expression of stress response transcription factors in male and female bovine pre-implantation embryos under oxidative stress conditions

Pre-implantation embryo development is a critical stage, in which several development and stress-response related transcription factors (TF) are involved. Exposing embryos to environmental insults alter some of these stress response-related TF. However, their expression pattern in male and female embryos and their release via exosomes is still unclear. Here, we aimed to investigate the effect of culture-induced oxidative stress on development and expression pattern of stress-related TF in male and female embryos and in respective spent media coupled with exosomes. For this, bovine male and female zygotes were in vitro produced using sexed semen and cultured under 5% and 20% oxygen in exosome-depleted SOFaa medium (SOF with amino acids). Blastocysts were subjected to total RNA isolation followed by quantitative RT-PCR analysis of the selected TF (Nrf2, KLF4, NOTCH1, SREBF2, E2F1, CAT1, SOD1, and OCT4), as well as protein abundance analysis using immunofluorescence and related phenotypes analysis, including reactive oxygen species (ROS) level and total cell count. Furthermore, the spent embryo culture media were collected for exosomes isolation and expression analysis of candidate TF. The data were statistically analysed using one-way ANOVA followed by multiple pair-wise comparisons using the Tukey post hoc test. Results showed that the blastocyst rates of both male (29.9% v. 34.9%) and female (16.7% v. 26.5%) bovine embryos were significantly lower in 20% than in 5% oxygen level. Female blastocysts subjected to the higher oxygen level showed increased ROS level (37.66±1.70v. 45.32±2.05 in male and 29.42±1.44v. 45.51±2.06 in female) and significantly reduced total cell count compared with the male embryo counterpart (136.55±7.8v. 112.75±2.9 in male and 138.75±2.0v. 88.25±4.3 in female cultured in 5% and 20% oxygen levels, respectively). Consequently, the expression levels of Nrf2, KLF4, SREBF2, CAT1, SOD1, and OCT4 were significantly increased in male embryos exposed to oxidative stress compared with those cultured under the lower oxygen level. However, NOTCH1 and E2F1 were significantly increased in female embryos exposed to oxidative stress compared with the male counterparts. The mRNA level of SREBF2 was significantly increased in male embryos cultured under both 5% and 20% O2 compared with female embryos. The protein expression level of Nrf2 and KLF4 was higher in embryos cultured at 20% v. 5% O2 with greater Nrf2 abundance in male embryos. Consequently, the male embryos produced at 20% O2 released a higher number of exosomes enriched with Nrf2, SOD1, and NOTCH1 mRNA than the other groups. Interestingly, the exosomal mRNA expression level of E2F1 tended to be higher in female embryos exposed to oxidative stress than their male counterparts. Taken together, the male embryos were more tolerant to oxidative stress than female embryos via the activation Nrf2-mediated oxidative stress response and development related TF. The release of these TF via exosomes could enhance cellular homeostasis maintenance under oxidative stress.

Plasma extracellular nanovesicle (exosome)-derived biomarkers for drug metabolism pathways: a novel approach to characterize variability in drug exposure.

Demonstrate the presence of cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) proteins and mRNAs in isolated human plasma exosomes and evaluate the capacity for exosome-derived biomarkers to characterize variability in CYP3A4 activity. The presence of CYP and UGT protein and mRNA in exosomes isolated from human plasma and HepaRG cell culture medium was determined by mass spectrometry and reverse transcription-polymerase chain reaction, respectively. The concordance between exosome-derived CYP3A4 biomarkers and midazolam apparent oral clearance (CL/F) was evaluated in a small proof-of-concept study involving six genotyped (CYP3A4 *1/*1 and CYP3A5 *3/*3) Caucasian males. Exosomes isolated from human plasma contained peptides and mRNA originating from CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4 and 3A5, UGT 1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10 and 2B15, and NADPH-cytochrome P450 reductase. Mean (95% confidence interval) exosome-derived CYP3A4 protein expression pre- and post-rifampicin dosing was 0.24 (0.2-0.28) and 0.42 (0.21-0.65) ngml-1 exosome concentrate. Mean (95% confidence interval) exosome CYP3A4 mRNA expression pre- and post-rifampicin dosing was 6.0 (1.1-32.7) and 48.3 (11.3-104) × 10-11 2-ΔΔCt , respectively. R2 values for correlations of exosome-derived CYP3A4 protein expression, CYP3A4 mRNA expression, and ex vivo CYP3A4 activity with midazolam CL/F were 0.905, 0.787 and 0.832, respectively. Consistent strong concordance was observed between exosome-derived CYP3A4 biomarkers and midazolam CL/F. The significance of these results is that CYP3A4 is the drug-metabolizing enzyme of greatest clinical importance and variability in CYP3A4 activity is poorly described by existing precision dosing strategies.