Exosomal miRNA Expression Research Articles

WITHDRAWN: Lack of miR-216b-5p, miR-33a-5p or miR-122-5p in MSCs exosomes promotes breast cancer glycolysis by targeting HK2, LDHA and PKM2

// Jiyi Xia 1, * , Xiaolan Yu 2, * , Xiguang Mao 3 , Hongyuan An 4 , Xiaoping Tang 5 , Xiaoyan Wang 5 , Tao He 1 and Yong Liu 6 1 Cancer Research Institute of SouthWest Medical University, Luzhou, Sichuan, China 2 Department of Obstetrics and Gynecology, Affiliated TCM Hospital of SouthWest Medical University, Luzhou, Sichuan, China 3 Department of Obstetrics and Gynecology, Affiliated Hospital of SouthWest Medical University, Luzhou, Sichuan, China 4 The Second Ward of Surgery (Breast Surgery), Luzhou People’s Hospital, Luzhou, Sichuan, China 5 Experimental Medicine Center, The Affiliated Hospital of SouthWest Medical University, Luzhou, Sichuan, China 6 Department of Pathology, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China * These authors have contributed equally to this work Correspondence to: Tao He, email: hetaipeace@163.com Yong Liu, email: liuyong_lz@126.com Keywords: MSC; exosomes; breast cancer; miRNA Received: April 27, 2017 Accepted: October 28, 2017 Published: January 02, 2018 ABSTRACT Exosomes contain many important components including miRNAs for cancer-stromal communication. Mesenchymal stromal cells support tumor development, however, the low expression of miRNAs in exosomes and their regulation roles from mesenchymal stromal cells are still unclear. In this study, we hypothesized that mesenchymal stromal cells-derived exosomes promote breast cancer cell glycolysis. Our results showed that mesenchymal stromal cells-derived exosomes promoted cell proliferation and glycolysis of breast cancer cells. Furthermore, sequencing of exosomal RNAs revealed miR-216b-5p, miR-33a-5p and miR-122-5p were lack of expression in exosomes from mesenchymal stromal cells. MiR-216b-5p, miR-33a-5p and miR-122-5p overexpression in breast cancer cells suppressed glycolysis by targeting HK2, LDHA and PKM2 respectively. These results provided valuable insights and mechanism of mesenchymal stromal cells-secreted exosomes on the glycolysis of breast cancer.

MiRNA profiling of uveal melanoma exosomes as a metastatic risk biomarker

PurposeUveal melanoma (UM) is the most common primary intraocular malignancy in adults. It is a highly aggressive cancer in which nearly 50% of patients die from liver metastasis. UM patients can be divided into 3 groups based on their genetic profile and disease free survival; the low risk, intermediate risk and high risk tumours. Recently, we have shown that high risk patients can be identified based on the expression of five miRNAs. Since tumour tissue is not always available and biopsies are not without risk, it is important to develop a method that can identify high risk patients in a non‐invasive manner. Exosomal miRNAs are an excellent candidate for this application.MethodsExosomes were isolated from the cell culture medium of a non‐metastatic and high risk metastatic UM cell line by ultracentrifugation and were characterized by western blot, electron microscopy and Nanosight Tracking Analysis (NTA). RNA was isolated from exosomes by the Qiagen RNeasy micro kit and quantified by an Agilent bioanalyzer. Subsequently, miRNA expression was measured by Taqman miRNA qPCR assays.ResultsAll exosome samples isolated from cell medium showed expression of CD81. Bioanalyzer confirmed the absence of ribosomal RNA and an abundance of small RNAs. qPCR analysis shows changes in the expression of some classifier miRNAs in exosomes extracted from cell lines. Showing that the classifier miRNA signature in UM cells partially overlaps with the miRNA signature in exosomes secreted by UM cells.ConclusionsThese exosomal miRNAs show great promise as a reliable predictor of disease free survival in UM. Next step is to detect these exosomal markers in blood of UM patients as this will enable us to provide all UM patients with a prognosis in a non‐invasive manner.

Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients.

Chronic kidney disease (CKD) is a severe disorder with an increasing incidence worldwide. An early detection may help to prevent its progression and to minimize the risk of cardiovascular diseases as one of the major comorbidities. Recently, extracellular miRNAs like urinary exosomal miRNAs became of great interest as non-invasive biomarkers which can be determined by RT-qPCR. But until now, there is no consensus regarding the normalization of miRNAs isolated from body fluids. The present study analyzed the miRNAs miR-16, miR-92a, miR-21, miR-124a and the small nuclear RNA RNU6B for their applicability as an endogenous reference gene in expression studies of exosomal miRNAs isolated from CKD patients. For this purpose, miRNA expression levels were determined by RT-qPCR after the isolation of urinary exosomes from 33 CKD patients and from 5 healthy controls. Expression data was analyzed with the normalization determination software NormFinder, BestKeeper, GeNorm and DeltaCt. Our results revealed an abundant expression of the four candidate miRNAs in urinary exosomes and no detectable expression of RNU6B. We identified miR-16 as the most stable endogenous reference gene in our data set, making it a suitable endogenous reference gene for miRNA studies of urinary exosomes derived from CKD patients.

Plasma exosome miR-196a and miR-1246 are potential indicators of localized pancreatic cancer.

Patients with localized pancreatic cancer (stage I and stage IIA) have a much higher survival rate than those presenting at later stages, yet early detection remains a challenge to this malignancy. The aim of this study was to evaluate whether exosome miRNA signatures are indicative of localized pancreatic cancer. Exosomes were collected from the conditioned media of pancreatic cancer cell lines and plasma samples of localized pancreatic cancer patients (Stage I-IIA, n=15), and healthy subjects (n=15). Cellular and exosome miRNAs from pancreatic cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome miRNA expression was analyzed by qRT-PCR. We found that certain miRNAs, such as miR-196a and miR-1246, are highly enriched in pancreatic cancer exosomes. Consistently, plasma exosome miR-196a and miR-1246 levels were significantly elevated in pancreatic cancer patients as compared to healthy subjects. An analysis of the cancer subtypes indicated that plasma exosome miR-196a is a better indicator of pancreatic ductal adenocarcinoma (PDAC), whereas plasma exosome miR-1246 is significantly elevated in patients with intraductal papillary mucinous neoplasms (IPMN). In contrast, there were no differences in the plasma exosome miR-196a and miR-1246 levels between patients with pancreatic neuroendocrine tumors (NET) and healthy subjects. In conclusion, we demonstrate that certain miRNA species, such as miR-196a and miR-1246, are highly enriched in pancreatic cancer exosomes and elevated in plasma exosomes of patients with localized pancreatic cancer.

Differential expression of exosomal miRNAs between breast cancer patients with and without recurrence.

BackgroundExosomal microRNAs (miRNAs) are promising candidate biomarkers for diagnosis or prognosis for breast cancer. We investigated the prognostic role of exosomal miRNAs in serum samples derived from patients with breast cancer and compared miRNA expression between serum and tumor tissues.MethodsThe miRNA profile derived from exosome between breast cancer patients with recurrence (n = 16) and without recurrence (n = 16) were compared by miRNA PCR array. Further, we examined the expression of miRNAs derived from tissues in the patients with breast cancer with (n = 35) and without recurrence (n = 39) by qRT-PCR.ResultsOf 384 miRNAs, three miRNAs (miR-338-3p, miR-340-5p, and miR-124-3p) were significantly upregulated and eight (miR-29b-3p, miR-20b-5p, miR-17-5p, miR-130a-3p, miR-18a-5p, miR-195-5p, miR-486-5p, and miR-93-5p) were significantly downregulated in the patients with recurrence. We evaluated the expression of the miRNAs in tumor tissues. The patients with recurrence had higher levels of miR-340 at their primary site as well as in the serum. In contrast, miR-195-5p, miR-17-5p, miR-93-5p, and miR-130a-3p, derived from tumor tissues that were downregulated in the serum from patients with recurrence, were higher in the patients with recurrence than in those with no recurrence. In logistic regression analysis, miR-340-5p, miR-17-5p, miR-130a-3p, and miR-93-5p were significantly associated with recurrence.ConclusionsSeveral exosomal miRNAs may be useful biomarkers to predict breast cancer recurrence. We show the different expression patterns of miRNAs between tumor tissues and serum. These findings may suggest selective mechanism of release of exosomal miRNAs by cancer cells to regulate their progression.

Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration Type 1 diabetes

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.

Abstract 5441: Circulating exosomal microRNA-203 and microRNA-373 as non-invasive biomarkers for predicting prognosis and metastasis in human hepatocellular carcinoma

Abstract Background and Aim Hepatocellular carcinoma (HCC) is the fifth most common cancer and the most leading cause of cancer-related death worldwide. Although considerable progress has been made in treatment of HCC, early detection is still highly considered the key to improved survival. Thus, for the earlier diagnosis and accurate prediction of HCC, identification of non-invasive molecular biomarkers for HCC patients is an imperative need. Recently, cancer cell-derived extracellular vesicles (EVs) have been known to contain various intracellular biomolecules including microRNAs (miRNAs). The aim of this study was to evaluate whether exosomal miRNAs can serve as a serum-based biomarker in HCC. Materials and Methods We isolated exosome from serum samples from HCC patients as well as normal healthy controls using ultracentrifugation, and it was confirmed by expression of exosome markers (CD9, CD63, ALIX, and TSG101) based on immunoblotting. Next, the expression of 6 miRNAs (miRNA-24, -130a, -182, -203, -373, and -423) was analyzed in the exosome samples. We also investigated expression status of the 6 miRNAs in matched HCC tissues and corresponding normal liver tissues. MiRNAs expression was determined by quantitative real-time PCR (qRT-PCR), and miRNAs expression was normalized relative to cel-miR-39 and RNU6B expression for serum and tissue samples, respectively. Results We successfully purified exosome from serum clinical samples and detected miRNAs in the exosome. We observed that a subset of miRNAs from serum exosome, including miRNA-24, -130a, -182, -203, and -373 were enhanced in HCC patients than normal healthy controls. In further comparison between early stage and advanced stage of HCC patients, serum exosomal miRNA-203 (P<0.05) and miRNA-373 (P<0.05) were significantly up-regulated in advanced HCC patients. While no significant changes in the expression of other serum exosomal miRNAs (miRNA-24, -130a, -182, and -423) according to HCC progression. More interestingly, high serum exosomal miRNA-203 and miRNA-373 was associated with HCC progression (P<0.01) as well as prognosis (P<0.05) of HCC patients. Conclusions We provided the novel evidence for usefulness of serum circulating exosomal miR-203 and miR-373 expressions as strong potential biomarkers for predicting prognosis and metastasis of HCC patients. Citation Format: Gyeonghwa Kim, Eunhye Lee, Se Young Jang, Won Young Tak, Young Oh Kweon, Soo Young Park, Keun Hur. Circulating exosomal microRNA-203 and microRNA-373 as non-invasive biomarkers for predicting prognosis and metastasis in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5441. doi:10.1158/1538-7445.AM2017-5441

Gender-specific differential expression of exosomal miRNA in synovial fluid of patients with osteoarthritis

The pathogenesis of osteoarthritis (OA) is poorly understood, and therapeutic approaches are limited to preventing progression of the disease. Recent studies have shown that exosomes play a vital role in cell-to-cell communication, and pathogenesis of many age-related diseases. Molecular profiling of synovial fluid derived exosomal miRNAs may increase our understanding of OA progression and may lead to the discovery of novel biomarkers and therapeutic targets. In this article we report the first characterization of exosomes miRNAs from human synovial fluid. The synovial fluid exosomes share similar characteristics (size, surface marker, miRNA content) with previously described exosomes in other body fluids. MiRNA microarray analysis showed OA specific exosomal miRNA of male and female OA. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified gender-specific target genes/signaling pathways. These pathway analyses showed that female OA specific miRNAs are estrogen responsive and target TLR (toll-like receptor) signaling pathways. Furthermore, articular chondrocytes treated with OA derived extracellular vesicles had decreased expression of anabolic genes and elevated expression of catabolic and inflammatory genes. In conclusion, synovial fluid exosomal miRNA content is altered in patients with OA and these changes are gender specific.

Increased Circulating Exosomal miRNA-223 Is Associated with Acute Ischemic Stroke.

Recent studies have demonstrated that exosomal microRNAs (miRNAs) are novel biomarkers and therapeutic targets for various diseases including vascular disease. However, specific exosomal miRNAs expression in stroke patients has not been reported yet. Here, we explored whether circulating exosomal miRNAs can serve as potential biomarkers for the diagnosis of acute ischemic stroke and discussed the potential for clinical application. Blood samples were collected from acute ischemic stroke patients within the first 72 h (n = 50). Circulating exosomes were exacted by Exoquick exosome isolation kit and characterized by transmission electron microscopy. Western blot was performed to assess the expression of exosomal protein makers. Exosomal miRNA-223 (miR-223) was detected by RT-PCR assay. The relationship between the expression levels of miR-223 and National Institutes of Health Stroke Scale (NIHSS) scores, brain infarct volume, and neurological outcomes were analyzed. Circulating exosomes were isolated and the size of vesicles ranged between 30 and 100 nm. The identification of exosomes was further confirmed by the detection of specific exosomal protein markers CD9, CD63, and Tsg101. Exosomal miR-223 in acute ischemic stroke patients was significantly upregulated compared to control group (p < 0.001). Exosomal miR-223 level was positively correlated with NIHSS scores (r = 0.31, p = 0.03). Exosomal miR-223 expression in stroke patients with poor outcomes was higher than those with good outcomes (p < 0.05). Increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes. Future studies with large sample are needed to assess the clinical application of exosomal miR-223 as a novel biomarker for ischemic stroke diagnosis.

MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.

Abstract P1-02-13: Differential expression of exosomal miRNAs between breast cancer patients with recurrence and no-recurrence

Abstract Background In recent years, there has been a concerted effort to identify biomarkers derived from body fluid in various cancers. Exosomal microRNA (miRNA) has been used as one of useful diagnostic or prognostic biomarkers. We aimed to investigate the prognostic role of the exosomal miRNAs in serum samples derived from patients with primary breast cancer. Additionally, we evaluated whether the exosomal miRNA in serum reflect the origin of primary tumor by means of comparing their expression levels between in serum and tumors. Patients and Methods Exosomes in serum sample (500uL) were extracted using ExoQuick (System Biosciences) and miRNAs were isolated using SeraMirTM Exosome RNA Amplification Kit (SBI). We compared the miRNA profile derived from exosome between the patients with breast cancer with recurrence (n=16) and without recurrence (n=16) by miRNA PCR array. Further, we examined the expression of miRNA derived from tumor tissues in the patients with breast cancer recurrence (n=35) and without recurrence (n=39) by qRT-PCR. All samples were collected before treatment and surgery. Results Of the 384 miRNAs, 11 miRNAs were significantly expressed; three miRNAs (miR-338-3p, miR-340-5p, and miR-124-3p) were significantly up-regulated and eight (miR-29b-3p, miR-20b-5p, miR-17-5p, miR-130a-3p, miR-18a-5p, miR-195-5p, miR-486-5p, and miR-93a-5p) were significantly down-regulated in the patients with recurrence compared to those without recurrence. Next, we evaluated expression of the above miRNAs in tumor tissues. The patients with recurrence have higher levels of miR-340 at their primary site as well as in the serum. On the contrary, miR-195-5p, miR-17-5p, miR-93-5p, and miR-130a-3p derived from tumor tissues that were down-regulated in the patients with recurrence in serum, were highly expressed in the patients with recurrence than those with no-recurrence. In logistic regression analysis, tumor size, miR-340-5p, miR-17-5p, miR-130a-3p, and miR-93-5p were significantly associated with breast cancer recurrence (each P &amp;lt; 0.05). Conclusions Several exosomal miRNAs at diagnosis may be useful biomarker to predict the breast cancer recurrence. Moreover, we showed the different expression pattern of miRNAs between tumor tissues and serum. These findings may suggest selective mechanism of release of exosomal miRNA by cancer cells to regulate their progression. Further studies to confirm our results are needed. Citation Format: Sueta A, Yamamoto Y, Tomiguchi M, Takeshita T, Ibusuki M, Iwase H. Differential expression of exosomal miRNAs between breast cancer patients with recurrence and no-recurrence [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-13.

A comparison of serum miRNAs influencing metastatic growth of EMT6 vs 4THM tumor cells in wild-type and CD200R1KO mice

We investigated whether miRNAs in exosomes from EMT6 or 4THM tumor-bearing mice played a role in regulating inflammatory cytokine expression and/or metastasis in WT mice injected with EMT6 and/or 4THM tumor cells. EMT6 tumors in BALB/c CD200R1KO mice resolve following surgical resection of localized tumor and immunization with irradiated EMT6 cells along with CpG as adjuvant. Wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20days of surgery. DLNs from CD200R1KO mice contain no tumor cells detectable at limiting dilution. In contrast, 4THM tumor cells injected into CD200R1KO show increased metastasis compared with WT mice. Transfer of serum exosomes from 4THM tumor-bearing mice to WT animals increased metastasis of EMT6 tumors, an effect attenuated by anti-IL-6 antibody. We compared miRNA expression in exosomes from the serum of 4THM/EMT6 WT or CD200R1KO tumor-bearing mice, and the effects of antagomirs to miRNAs on tumor growth. Complex changes in miRNA expression were observed in the isolated exosomes. Some miRNAs, including miR155, have been reported to potentiate inflammatory responses and augment inflammatory cytokine expression. Expression of miR155 increased in exosomes from 4THM relative to EMT6 tumor bearers, and antagomirs to miR155 attenuated tumor growth and metastasis, and improved survival, following infusion into WT mice. Antagomirs to the miR205 family were thought to affect metastasis by targeting epithelial-to-mesenchymal transition (EMT), increased growth and metastasis in both 4THM and EMT6 tumor-bearing mice, and decreased survival, with some modulation of inflammatory cytokine production. Multiple pathways are implicated in differential metastasis of EMT6/4THM, and targeting these may have clinical utility in human breast cancer.

Serum Exosomal miRNAs Expression as Novel Biomarkers for Detection of Esophageal Adenocarcinoma

Serum Exosomal miRNAs Expression as Novel Biomarkers for Detection of Esophageal Adenocarcinoma

Abstract 3162: Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer

Abstract Introduction &amp; objectives: In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC. Materials &amp; methods: Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS. Results: We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers. Conclusion: These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing. Citation Format: Joana Heinzelmann, Sophie Baumgart, Sebastian Hoelters, Martin Janssen, Michael Stöckle, Kerstin Junker. Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3162.

Human papillomavirus E6 and E7 oncoproteins affect the expression of cancer-related microRNAs: additional evidence in HPV-induced tumorigenesis.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer and are also associated with other types of cancers. HPVs can modulate microRNAs (miRNAs) expressed by infected cells. The production of extracellular vesicles is deregulated in cancer, and their cargo delivered to the microenvironment can promote tumorigenesis. The involvement of HPV oncoproteins on miRNA expression in cells and exosomes was analyzed in keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38). MiRNAs were investigated through the TaqMan Array Human MicroRNA Cards, followed by real-time RT-PCR assay for specific miRNAs. Selected miRNA targets were analyzed by Western blot and correlated to the HPV oncoproteins by specifically silencing E6 and E7 expression. Exosomes, isolated from K16 and K38 supernatants by differential centrifugations, were quantified through the vesicle-associated acetylcholinesterase activity. MiRNAs deregulated in K16 and K38 cells were identified. HPV-16 and/or HPV-38 E6 and E7 single proteins can modify the expression of selected miRNAs involved in the tumorigenesis, in particular miR-18a, -19a, -34a and -590-5p. The analysis of the content of exosomes isolated from HPV-positive cells revealed the presence of E6 and E7 mRNAs and few miRNAs. MiR-222, a key miRNA deregulated in many cancers, was identified in exosomes from K16 cells. HPV E6 and/or E7 oncoprotein expression can induce the deregulation of some miRNAs. Through the production and function of exosomes, HPV oncogenes as well as HPV-deregulated miRNAs can potentiate the virus oncogenic effects in the tumor cell microenvironment.

MP78-07 BLOOD BASED EXOSOMAL MIRNAS AS BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF CLEAR CELL RENAL CELL CANCER

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging II1 Apr 2016MP78-07 BLOOD BASED EXOSOMAL MIRNAS AS BIOMARKERS FOR DIAGNOSIS AND PROGNOSIS OF CLEAR CELL RENAL CELL CANCER Joana Heinzelmann, Sophie Baumgart, Seastian Hölters, Martin Janssen, Michael Stöckle, and Kerstin Junker Joana HeinzelmannJoana Heinzelmann More articles by this author , Sophie BaumgartSophie Baumgart More articles by this author , Seastian HöltersSeastian Hölters More articles by this author , Martin JanssenMartin Janssen More articles by this author , Michael StöckleMichael Stöckle More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1963AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC. METHODS Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS. RESULTS We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers. CONCLUSIONS These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1026-e1027 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Joana Heinzelmann More articles by this author Sophie Baumgart More articles by this author Seastian Hölters More articles by this author Martin Janssen More articles by this author Michael Stöckle More articles by this author Kerstin Junker More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro.

Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson’s theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-016-2358-1) contains supplementary material, which is available to authorized users.

Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche.

The different prostate cancer (PCa) cell populations (bulk and cancer stem cells, CSCs) release exosomes that contain miRNAs that could modify the local or premetastatic niche. The analysis of the differential expression of miRNAs in exosomes allows evaluating the differential biological effect of both populations on the niche, and the identification of potential biomarkers and therapeutic targets. Five PCa primary cell cultures were established to originate bulk and CSCs cultures. From them, exosomes were purified by precipitation for miRNAs extraction to perform a comparative profile of miRNAs by next generation sequencing in an Illumina platform. 1839 miRNAs were identified in the exosomes. Of these 990 were known miRNAs, from which only 19 were significantly differentially expressed: 6 were overexpressed in CSCs and 13 in bulk cells exosomes. miR-100-5p and miR-21-5p were the most abundant miRNAs. Bioinformatics analysis indicated that differentially expressed miRNAs are highly related with PCa carcinogenesis, fibroblast proliferation, differentiation and migration, and angiogenesis. Besides, miRNAs from bulk cells affects osteoblast differentiation. Later, their effect was evaluated in normal prostate fibroblasts (WPMY-1) where transfection with miR-100-5p, miR-21-5p and miR-139-5p increased the expression of metalloproteinases (MMPs) -2, -9 and -13 and RANKL and fibroblast migration. The higher effect was achieved with miR21 transfection. As conclusion, miRNAs have a differential pattern between PCa bulk and CSCs exosomes that act collaboratively in PCa progression and metastasis. The most abundant miRNAs in PCa exosomes are interesting potential biomarkers and therapeutic targets.

An Exploration of Molecular Correlates Relevant to Radiation Combined Skin-Burn Trauma.

BackgroundExposure to high dose radiation in combination with physical injuries such as burn or wound trauma can produce a more harmful set of medical complications requiring specialist interventions. Currently these interventions are unavailable as are the precise biomarkers needed to help both accurately assess and treat such conditions. In the present study, we tried to identify and explore the possible role of serum exosome microRNA (miRNA) signatures as potential biomarkers for radiation combined burn injury (RCBI).MethodologyFemale B6D2F1/J mice were assigned to four experimental groups (n = 6): sham control (SHAM), burn injury (BURN), radiation injury (RI) and combined radiation skin burn injury (CI). We performed serum multiplex cytokine analysis and serum exosome miRNA expression profiling to determine novel miRNA signatures and important biological pathways associated with radiation combined skin-burn trauma.Principal FindingsSerum cytokines, IL-5 and MCP-1, were significantly induced only in CI mice (p<0.05). From 890 differentially expressed miRNAs identified, microarray analysis showed 47 distinct miRNA seed sequences significantly associated with CI mice compared to SHAM control mice (fold change ≥ 1.2, p<0.05). Furthermore, only two major miRNA seed sequences (miR-690 and miR-223) were validated to be differentially expressed for CI mice specifically (fold change ≥ 1.5, p<0.05).ConclusionsSerum exosome miRNA signature data of adult mice, following RCBI, provides new insights into the molecular and biochemical pathways associated with radiation combined skin-burn trauma in vivo.

Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer

Peritoneal metastasis is the most frequent type of recurrence in patients with gastric cancer (GC) and is associated with poor prognosis. Peritoneal lavage cytology, used to evaluate the risk of peritoneal metastasis, has low sensitivity. Here, we assessed the diagnostic potential of exosomal miRNA profiles in peritoneal fluid for the prediction of peritoneal dissemination in GC. Total RNA was extracted from exosomes isolated from six gastric malignant ascites (MA) samples, 24 peritoneal lavage fluid (PLF) samples, and culture supernatants (CM) of two human gastric carcinoma cell lines that differ in their potential for peritoneal metastasis. Expression of exosomal miRNAs was evaluated with Agilent Human miRNA microarrays and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microarray analysis indicated a low variability in the number and signal intensity of miRNAs detected among the samples. In the six MA fluids, miR-21 showed the highest signal intensity. We identified five miRNAs (miR-1225-5p, miR-320c, miR-1202, miR-1207-5p, and miR-4270) with high expression in MA samples, the PLF of serosa-invasive GC, and the CM of a highly metastatic GC cell line; these candidate miRNA species appear to be related to peritoneal dissemination. Differential expression of miR-21, miR-320c, and miR-1225-5p was validated in the PLF of serosa-invasive and non-invasive GC by qRT-PCR and miR-21 and miR-1225-5p were confirmed to be associated with serosal invasion in GC. PLF can be used to profile the expression of exosomal miRNAs. Our findings suggest that miR-21 and miR-1225-5p may serve as biomarkers of peritoneal recurrence after curative GC resection, thus providing a novel approach to early diagnosis of peritoneal dissemination of GC.