miR Profiles Research Articles

FAK signaling in cancer-associated fibroblasts promotes breast cancer cell migration and metastasis by exosomal miRNAs-mediated intercellular communication.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the major components of tumor microenvironment (TME) and play crucial roles in tumor development and metastasis. Here, we generated fibroblast-specific inducible focal adhesion kinase (FAK) knockout (cKO) mice in a breast cancer model to study potential role and mechanisms of FAK signaling in CAF to promote breast cancer metastasis in vivo. While not affecting primary tumor development and growth, FAK deletion significantly suppressed breast cancer metastasis in vivo. Analyses of CAFs derived from cKO mice as well as human CAFs showed that FAK is required for their activity to promote mammary tumor cell migration. We further showed that FAK ablation in CAFs decreased their exosome amount and functions to promote tumor cell migration and other activities, which could contribute to the reduced metastasis observed in cKO mice. Lastly, profiling of miRs from CAF exosomes showed alterations of several exosomal miRs in FAK-null CAFs, and further analysis suggested that miR-16 and miR-148a enriched in exosomes from FAK-null CAFs contribute to the reduced tumor cell activities and metastasis. Together, these results identify a new role for FAK signaling in CAFs that regulate their intercellular communication with tumor cells to promote breast cancer metastasis.

P096 Specific plasma micro-RNA profiles for prediction of recurrence in Crohn’s disease in pre-surgery and established recurrence scenarios

Abstract Background Crohn’s disease (CD) patients who undergo ileocolonic resection are at high risk of postoperative recurrence (POR). Both environmental and genetic factors are known to influence CD pathogenesis. MicroRNAs (miRs) are important epigenetic regulators in CD, but their role in the progression to POR is still unknown. Thus, we aimed to identify differential miRs profiles and their potential use to predict POR in CD patients. Methods A cohort of 33 CD patients who underwent a resection was consecutively and prospectively followed for at least 1 year. Patients were classified according to the presence or absence of POR during the follow-up. POR was assessed by ileocolonoscopy (Rutgeerts’ index ≥ i2b) or MRI enterography within 6–12 months after surgery. Patients received postoperative therapy to prevent POR (thiopurines 47%; anti-TNFα 25%; mesalamine 9%; combination therapy 3%; none 16%). Peripheral blood samples were taken at fixed times. Total RNA was obtained from miRNeasy Serum/Plasma Advanced Kit (Qiagen) and run on the miRCURY LNAtm Universal RT microRNA PCR with a miRs panel selection (32) based on previous experiments. The predictive accuracy of the possible predictors was assessed by using receiver operating characteristic curves with the R software. Results MicroRNAs profiles were studied at two different times: presurgery (PS) and at the time of the morphological POR or, in those who remained in remission, 1 year after surgery (1 year). The miRs whose fluorescent signal did not differ from background noise were excluded from the analysis [8 in PS (25%); 5 in 1 year (16%)]. Using elastic net regression, five miRs (hsa.miR.191.5p, hsa.miR.15b.5p, hsa.miR.106b.5p, hsa.miR.451a and hsa.miR.93.5p) were selected for discriminating the two patient groups at the time pre-surgery. Seven miRs (hsa.miR.15b.5p, hsa.miR.126.3p, hsa.miR.451a, hsa.let.7b.5p, hsa.miR.93.5p, hsa.miR.423.5p and hsa.miR.125b.5p) were selected for discriminating the two patient groups at the time of 1 year. Using a predictive model with these miRs, an overall AUC of 0.86 (95% confidence interval, 0.75–0.97) was achieved for PS, and 0.96 (0.92–1.0) for 1 year. Conclusion This study shows that plasma microRNAs are useful biomarkers in the POR scenario. When recurrence is already established, a group of plasma miRs is also specifically expressed, thus assessing them could prevent patients from receiving and endoscopic procedure for POR evaluation. Furthermore, microRNA assessment PS could predict which patients will develop recurrence within 1 year of surgery. This could also have an impact on clinical management, selecting patients who can avoid immunosuppressive treatment and the ones who may need more intensive therapeutic strategies.

Selection of the Most Stable Endogenous Control Genes for Microrna Quantitation in Chicken Ovarian Follicles

Abstract MicroRNAs (miRNAs or miRs) belong to a class of small non-coding RNAs of 19 to 24 nucleotides long that act as negative gene regulators at the post-transcriptional level. Quantitative PCR (q-PCR) is a commonly used technique in the profiling of miRs, and identification of reliable endogenous controls is crucial for proper data normalisation. To date, no study has been performed on reference miRs for the normalisation of miR expression in chicken ovarian tissues. Therefore, the aim of the present study was to experimentally identify the most stable reference mirs for normalisation of miR q-PCR expression data in the chicken ovary. Relying on high-throughput sequencing, five putative reference miR (let-7a-3p, miR-140a-3p, miR-22-5p, miR-33-5p, miR-99a-3p) were identified and subsequently analysed in a total of 66 tissue samples. The stability of candidate endogenous controls validated by the most widely used algorithms, geNorm, NormFinder, and BestKeeper, showed that let-7a-3p, miR-140a-3p, and miR-22-5p are the most appropriate choice of reference genes. Application of different normalisation approaches to the relative quantitation of randomly chosen miR-1552-5p in chicken ovarian follicles indicated the impact of the selected reference genes on miR expression. Further, the results revealed a downregulation of miR-1552-5p. In summary, the three identified endogenous reference miRs are suitable for profiling the miR expression in ovarian tissues of laying hens. Our findings provide valuable information for future miR expression studies in the avian ovary.

Effects of the anti-oxidant PDTC in combination with a single bout of treadmill running on murine skeletal muscle

ABSTRACT Objectives: Skeletal muscle adaptation to physical activity is dependent on various factors. Important signaling mediators are reactive oxygen species (ROS). However, recent research suggests that ROS have both beneficial and deleterious effects on exercise adaptation, dependent on training intensity and training status, so that the question of whether anti-oxidants should be taken in connection with exercise cannot easily be answered. Thus, it is important to gain more insight into the complex roles of ROS in regulating training adaptation. Methods: The effects of ROS inhibition on skeletal muscle training adaptation were analyzed by applying the anti-oxidant PDTC, which is also an inhibitor of the ROS-activated transcription factor nuclear factor kappa B (NFκB), to juvenile mice in connection with a single bout of treadmill running. Results: We found that PDTC inhibits exercise-mediated induction of specific stress- and inflammation-associated genes. Other genes, specifically those encoding metabolic and mitochondrial factors, were affected to a lesser extent and there appeared to be little effect on the microRNA (miR) profile. Discussion: Our data suggest that anti-oxidants regulate distinct sets of adaptation-relevant genes, which might have important implications for the design of exercise-based preventive and therapeutic approaches.

MicroRNAs in Asthma and Respiratory Infections: Identifying Common Pathways.

MicroRNAs (miRs) are single-stranded RNAs of 18-25 nucleotides. These molecules regulate gene expression at the post-transcriptional level; several of these are differentially expressed in asthma as well as in viral acute respiratory infections (ARIs), the main triggers of acute asthma exacerbations. In recent years, miRs have been studied in order to discover drug targets as well as biomarkers for diagnosis, disease severity and prognosis. We describe recent findings on miR expression and function in asthma and their role in the regulation of viral ARIs, according to cell tissue specificity and asthma severity. By combining the above information, we identify miRs that may be important in virus-induced asthma exacerbations. This is the first attempt to link miR profiles of asthmatic patients and ARI-induced miRs, addressing the question of whether there might be a specific miR deficit in asthmatic subjects that make them more susceptible and/or reactive to infection.

Dietary delphinidin inhibits human colorectal cancer metastasis associating with upregulation of miR-204-3p and suppression of the integrin/FAK axis

Delphinidin is a flavonoid belonging to dietary anthocyanidin family that has been reported to possess diverse anti-tumoral activities. However, the effects of delphinidin on colorectal cancer (CRC) cells and the underlying mechanisms are not fully understood. Thus, we aimed to investigate the anti-cancer activity of delphinidin in CRC cells and the underlying molecular mechanisms. The effects of delphinidin on the viability, metastatic characteristics, signaling, and microRNA (miR) profile of human CRC cell lines used were analyzed. In vivo metastasis was also evaluated using xenograft animal models. Our findings showed that delphinidin (<100 μM) inhibited the colony formation of DLD-1, SW480, and SW620 cells, but non-significantly affected cell viability. Delphinidin also suppressed the migratory ability and invasiveness of the tested CRC cell lines, downregulated integrin αV/β3 expression, inhibited focal adhesion kinase (FAK)/Src/paxillin signaling, and interfered with cytoskeletal construction. Analysis of the miR expression profile revealed a number of miRs, particularly miR-204-3p, that were significantly upregulated and downregulated by delphinidin. Abolishing the expression of one upregulated miR, miR-204-3p, with an antagomir restored delphinidin-mediated inhibition of cell migration and invasiveness in DLD-1 cells as well as the αV/β3-integrin/FAK/Src axis. Delphinidin also inhibited the lung metastasis of DLD-1 cells in the xenograft animal model. Collectively, these results indicate that the migration and invasion of CRC cells are inhibited by delphinidin, and the mechanism may involve the upregulation of miR-204-3p and consequent suppression of the αV/β3-integrin/FAK axis. These findings suggest that delphinidin exerts anti-metastatic effects in CRC cells by inhibiting integrin/FAK signaling and indicate that miR-204-3p may play an important role in CRC metastasis.

Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Next-Generation RNA Sequencing-Based Analysis Identifies a Novel Set of Prognostic Micrornas (miRs) in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML)

Circulating miR-652-3p as a biomarker of drug resistance in metastatic colorectal cancer patients

Abstract Background MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis, carcinogenesis and control multiple oncogenic pathways. Numerous miRs deregulation is associated with clinical outcome. Chemo-refractory metastatic CRC (mCRC) patients are often treated with regorafenib. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient selection and identification of mechanisms of resistance. Here we aimed to identify circulatory miRs that might be exploited for the upfront selection of patients’ to regorafenib treatment. Methods We ran a translational phase II trial of regorafenib in chemo-refractory mCRC patients. Serum, plasma and tissue biopsies were obtained at baseline (BL), every four weeks and at disease progression (PD). Patient Derived Organoids (PDOs) and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. MiR profiling was performed by NanoString nCounter platform of 800 genes and validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Progression Free Survival (PFS) and Overall Survival (OS) was measured. Further validation was performed in 97 patients from an independent patient’s cohort. Results MiR expression was tested in 43 BL sera and dysregulation in 28 miRs was associated with PFS and OS. Up-regulation of miR-652-3p was associated with worse PFS and OS. These results were validated by ddPCR on the same serum samples, and matching plasmas. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. Validation in an independent patient’s cohort confirmed direct association of miR-652-3p dysregulation with OS. Functional experiments to define miR652-3p mediated resistance have shown that there is a decrease of tumour growth and migration in miR-652-3p inhibition in cell lines and PDOs. Conclusions We provided initial evidence suggesting that circulating miR-652-3p might work as a predictive biomarker for the upfront selection of patients’ candidate to regorafenib treatment. Legal entity responsible for the study Academic. Funding Royal Marsden. Disclosure All authors have declared no conflicts of interest.

P4460microRNA-17-5p and microRNA-20a-5p downregulation by atorvastatin in Hepg2 cells: a new mechanism involved in the lipid-lowering therapy

Abstract Background Hypercholesterolemia increases the risk of coronary artery disease and its pharmacological treatment has demonstrated, besides reducing cholesterol levels, a decrease in the incidence and mortality from coronary events. The treatment of hypercholesterolemia is mainly driven by using statins. However, the response to pharmacological therapy shows high inter-individual variability, resulting in a variable effect in both lipid lowering and risk reduction. Thus, a better understanding of lipid-lowering mechanism and response variability at molecular level is required. Previously, we demonstrated a deregulation of microRNA (miR) profile in HepG2 cells after atorvastatin treatment, including the downregulation of miR-17-5p and miR-20a-5p which potentially targets the LDL receptor gene (LDLR), suggesting that it might be involved in allowing the LDLR overexpression on the surface of hepatic cells to subsequently capture circulating LDL and to reach the expected lipid-lowering effect. Purpose To determine the role of miR-17-5p and miR-20a-5p on the regulation of LDLR gene expression in HepG2 cells. Methods Cells HepG2 were treated with atorvastatin 10μM for 24 hours. RNA extraction and enrichment of smallRNAs were performed. The gene expression of miR-17-5p, miR-20a-5p, miR-24-3p, miR-93-5p, miR-106a-5p and LDLR were evaluated. To evaluate the effect of miR-17-5p and miR-20a-5p on LDLR gene expression, both miRs were overexpressed or repressed by transfection of mimics or inhibitors respectively into HepG2 cells for 24, 48 and 72 Hours. The gene expression of LDLR was quantified by real time PCR using RPL27 gene as reference gene. The protein expression of LDLR and beta actin were evaluated using western blot and quantified using the ImageJ software. Results Our data showed that atorvastatin significantly repressed the expression of miR-17-5p (P&lt;0.0001) and miR-20a-5p (P=0.0456) in HepG2 cells. In silico studies showed that miR-17-5p interact with the 3'-UTR region of the LDLR. Consistently, when miR-17-5p or miR-20a-5p were overexpressed by using mimics, we observed that gene and protein expression of LDLR decreased significantly (P&lt;0.0001 and P&lt;0.05 respectively). Consistently, when miR-17-5p or miR-20a-5p were repressed by the use inhibitors, we observed that the gene and protein expression of LDLR increases significantly (P&lt;0.005). Conclusions In conclusion, we demonstrate that atorvastatin induces a significant down-regulation of the miR-17-5p and miR-20a-5p in HepG2 cells. The overexpression or repression regulate the gene and protein expression of LDLR. Acknowledgement/Funding FONDECYT N°3160567, FONDECYT N°1171765 and DIUFRO DI19-0094

Silver, titanium dioxide, and zinc oxide nanoparticles trigger miRNA/isomiR expression changes in THP-1 cells that are proportional to their health hazard potential

After over a decade of nanosafety research, it is indisputable that the vast majority of nano-sized particles induce a plethora of adverse cellular responses – the severity of which is linked to the material’s physicochemical properties. Differentiated THP-1 cells were previously exposed for 6 h and 24 h to silver, titanium dioxide, and zinc oxide nanoparticles at the maximum molar concentration at which no more than 15% cellular cytotoxicity was observed. All three nanoparticles differed in extent of induction of biological pathways corresponding to immune response signaling and metal ion homeostasis. In this study, we integrated gene and miRNA expression profiles from the same cells to propose miRNA biomarkers of adverse exposure to metal-based nanoparticles. We employed RNA sequencing together with a quantitative strategy that also enables analysis of the overlooked repertoire of length and sequence miRNA variants called isomiRs. Whilst only modest changes in expression were observed within the first 6 h of exposure, the miRNA/isomiR (miR) profiles of each nanoparticle were unique. Via canonical correlation and pathway enrichment analyses, we identified a co-regulated miR-mRNA cluster, predicted to be highly relevant for cellular response to metal ion homeostasis. These miRs were annotated to be canonical or variant isoforms of hsa-miR-142-5p, -342-3p, -5100, -6087, -6894-3p, and -7704. Hsa-miR-5100 was differentially expressed in response to each nanoparticle in both the 6 h and 24 h exposures. Taken together, this co-regulated miR-mRNA cluster could represent potential biomarkers of sub-toxic metal-based nanoparticle exposure.

Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS

Astroglia play active and diverse roles in modulating neuronal/synaptic functions in the CNS. How these astroglial functions are regulated, especially by neuronal signals, remains largely unknown. Exosomes, a major type of extracellular vesicles (EVs) that originate from endosomal intraluminal vesicles (ILVs), have emerged as a new intercellular communication process. By generating cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) mice and immuno-EM/confocal image analysis, we found that neuronal CD63-GFP+ ILVs are primarily localized in soma and dendrites, but not in axonal terminals in vitro and in vivo. Secreted neuronal exosomes contain a subset of microRNAs (miRs) that is distinct from the miR profile of neurons. These miRs, especially the neuron-specific miR-124-3p, are potentially internalized into astrocytes. MiR-124-3p further up-regulates the predominant glutamate transporter GLT1 by suppressing GLT1-inhibiting miRs. Our findings suggest a previously undescribed neuronal exosomal miR-mediated genetic regulation of astrocyte functions, potentially opening a new frontier in understanding CNS intercellular communication.

Diabetic Nephropathy Alters the Distribution of Circulating Angiogenic MicroRNAs Among Extracellular Vesicles, HDL, and Ago-2.

Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m2), and patients with DN (eGFR <30 mL/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132).

The long noncoding RNA PTENP1 regulates human endometrial epithelial adhesive capacity in vitro: implications in infertility.

There is general consensus that the synchronous development of the embryo and endometrium is absolutely essential for successful implantation. Recent studies have strongly suggested that embryo-secreted factors are able to deliver into the endometrial cavity/endometrium and alter its protein profile in preparation for implantation. However, there is limited research focusing on long noncoding RNA (lncRNA) changes in the endometrium that brought about by the embryonic derived factors. It has been suggested that lncRNA has intricate interplay with microRNA (miR), small (~19-22 nucleotides), non-protein-coding RNA, to regulate protein production in the endometrium, thus controlling adhesive capacity. Here through microarray assays, we compare the lncRNA profile of the primary human endometrial epithelial cells (HEECs) that have been precultured with blastocyst-conditioned media (BCM) from embryos that implanted versus nonimplanted. Our data indicate a substantial change of lncRNA expression in HEECs, including 9 up-regulated and 12 down-regulated lncRNAs after incubation with implanted BCM. Selective knockdown of PTENP1, the most increased lncRNA after implanted BCM treatment in the HEECs, compromised the spheroid adhesion (P<0.001). Characterization of PTENP1 confirmed its expression in the luminal epithelium with staining appeared most intense in the midsecretory phase. Furthermore, we have recorded a substantial change of miR profile upon PTENP1 knockdown in HEECs. Overexpression of miR-590-3p, a novel predicted target of PTENP1, impaired spheroid adhesion (P<0.001). Collectively, these data have supported a novel regulation system that lncRNAs were able to participate in the regulation of implantation through association with miRs.

Serum Exosomal MicroRNAs Predict Acute Respiratory Distress Syndrome Events in Patients with Severe Community-Acquired Pneumonia.

Background Severe community-acquired pneumonia (SCAP) requiring intensive care unit (ICU) treatment commonly causes acute respiratory distress syndrome (ARDS) with high mortality. This study was aimed at evaluating whether microRNAs (miRNAs) in circulating exosomes have the predictive values for patients at risk of developing ARDS due to SCAP. Methods ARDS/ALI-relevant miRNAs were obtained by literature search. Exosomes in serum were isolated by ultracentrifugation method and identified by Transmission Electron Microscopy. Then the miR profiling in the exosomes using real-time PCR was analyzed in SCAP patients with or without ARDS. Moreover, multivariate Cox proportional regression analysis was performed to estimate the odds ratio of miRNA for the occurrence of ARDS and prognosis. The receiver operating characteristics (ROC) curves were calculated to discriminate ARDS cases. Finally, the Kaplan-Meier curve using log-rank method was performed to test the equality for survival distributions with different miRNA classifiers. Results A total of 53 SCAP patients were finally recruited. Ten miRNAs were picked out. Further, a subset of exosomal miRNAs, including the miR-146a, miR-27a, miR-126, and miR-155 in ARDS group exhibited significantly elevated levels than those in non-ARDS group. The combined expression of miR-126, miR-27a, miR-146a, and miR-155 predicted ARDS with an area under the curve of 0.909 (95 % CI 0.815 –1). Only miR-126 was selected to have potential to predict the 28-day mortality (OR=1.002, P=0.024) with its median value classifier. Conclusions The altered levels of circulating exosomal microRNAs may be useful biologic confirmation of ARDS in patients with SCAP.

Effect of low sperm quality on progeny: a study on zebrafish as model species

Nowadays a decrease tendency in human sperm quality has been reported mainly in developed countries. Reproductive technologies have been very valuable in achieving successful pregnancies with low quality sperm samples. However, considering that spermatozoa molecular contribution is increasingly important in recent studies, it is crucial to study whether fertilization with low sperm quality could leave a molecular mark on progeny. This study explores the consequences that fertilization with low sperm quality may have on progeny, using zebrafish as a model. Good and bad breeders were established attending to sperm quality analyses and were individually tracked. Significant differences in fertilization and malformation rates were obtained in progenies between high and low quality sperm samples. Moreover an altered miR profile was found in the progenies of bad zebrafish breeders (upregulation of miR-141 and miR -122 in 24 hpf embryos) and as a consequence, some of their targets involved in male sex development such as dmrt1, suffered downregulation. Our results indicate that fertilizing with high sperm quality samples becomes relevant from a new perspective: to avoid molecular alterations in the progeny that could remain masked and therefore produce unexpected consequences in it.

Abstract LB-305: Circulating miR-652-3p as a biomarker of resistance to regorafenib in metastatic colorectal cancer patients

Abstract MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis and carcinogenesis and control multiple oncogenic pathways. Numerous miRs are aberrantly expressed in colorectal cancer (CRC) and their deregulation is associated with clinical outcome and cancer progression. Chemo-refractory metastatic CRC (mCRC) patients are often treated with regorafenib, a multi-tyrosine kinase inhibitor with anti-angiogenic effect. Given the limited clinical benefits of regorafenib in unselected patient populations, there is an unmet need for better patient selection and identification of mechanisms of resistance. miRs are highly stable and have shown encouraging value as potential biomarkers for CRC detection and prognosis. Here we aimed to identify circulatory miRs that might be exploited for the upfront selection of patients’ candidate to regorafenib treatment. We ran a translational phase II trial of regorafenib in chemo-refractory mCRC patients. Serum, plasma and tissue biopsies were obtained at baseline (BL), every four weeks and at disease progression (PD). Patient Derived Organoids (PDOs) and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. MiR profiling was performed in baseline serum of all patients by NanoString nCounter platform of 800 genes and validated with digital droplet (dd)PCR in serum, plasma, PDOs and by In Situ Hybridization (ISH) in matching tissue biopsies. Progression Free Survival (PFS) was measured from date of registration to date of first progression/relapse or death from cancer progression. Overall Survival (OS) was measured from date of randomisation to death from cancer. Further validation was performed by ddPCR in 97 patients from an independent patient’s cohort. MiR expression was tested in 43 BL sera and dysregulation in 28 miRs was associated with PFS and OS. Up-regulation of miR-652-3p was associated with worse PFS and OS. These results were validated by ddPCR on the same serum samples, and matching plasmas. ISH confirmed upregulation of this miR in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance to regorafenib. Validation in an independent patient’s cohort confirmed direct association of miR-652-3p dysregulation with PFS and OS. Functional experiments to define miR652-3p mediated resistance have shown that there is a decrease of tumour growth and migration in miR-652-3p inhibition in PDOs. We provide initial evidence suggesting that circulating miR-652-3p might work as a negative predictive biomarker for the upfront selection of patients’ candidate to regorafenib treatment. Citation Format: Somaieh Hedayat, Khurum Khan, David Cunningham, George Vlachogiannis, Andrea Lampis, Silvia Marchetti, Matteo Fassan, Ruwaida Begum, Marta Schirripa, Fotios Fotios Loupakis, Nicola Valeri. Circulating miR-652-3p as a biomarker of resistance to regorafenib in metastatic colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-305.

Identification of microRNA-target modules from rice variety Pusa Basmati-1 under high temperature and salt stress.

High temperature and salinity stress are major factors limiting the growth and productivity of rice crop on a global scale. It is therefore an essential prerequisite to understand the molecular genetic regulation of plant responses to dual stresses. MicroRNAs (miRs) are recognized as key controllers of gene expression which act mainly at the post-transcriptional level to regulate various aspects of plant development. The present study attempts to investigate the miR circuits that are modulated in response to high temperature and salinity stress in rice. To gain insights into the pathway, preliminary miR profiles were generated using the next-generation sequencing (NGS) datasets. The identified molecules were filtered on the basis of fold differential regulation under high temperature, and time kinetics of their expression under the two individual stresses was followed to capture the regulatory windows. The analysis revealed the involvement of common miR regulatory nodes in response to two different abiotic stresses, thereby broadening our perspective about the stress-mediated regulatory mechanisms operative in rice.

Mesenchymal stem cells of Systemic Sclerosis patients, derived from different sources, show a profibrotic microRNA profiling

Systemic Sclerosis (SSc) is a disease with limited therapeutic possibilities. Mesenchymal stem cells (MSCs)-therapy could be a promising therapeutic option, however the ideal MSCs source has not yet been found. To address this problem, we perform comparison between bone marrow (BM)-MSCs and adipose (A)-MSCs, by the miRs expression profile, to identify the gene modulation in these two MSCs source. MicroRNAs (miRs) are RNAs sequences, regulating gene expression and MSCs, derived from different tissues, may differently respond to the SSc microenvironment. The miRs array was used for the miRs profiling and by DIANA-mirPath tool we identified the biological functions of the dysregulated miRs. In SSc-BM-MSCs, 6 miRs were significantly down-regulated and 4 miRs up-regulated. In SSc-A-MSCs, 11 miRs were significantly down-regulated and 3 miRs up-regulated. Interestingly, in both the sources, the involved pathways included the senescence mechanisms and the pro-fibrotic behaviour. Furthermore, both the MSCs sources showed potential compensatory ability. A deeper knowledge of this miRs signature might give more information about some pathogenic steps of the disease and in the same time clarify the possible therapeutic role of autologous MSCs in the regenerative therapy in SSc.

Senescence cell-associated extracellular vesicles serve as osteoarthritis disease and therapeutic markers.

Senescent cells (SnCs) are increasingly recognized as central effector cells in age-related pathologies. Extracellular vesicles (EVs) are potential cellular communication tools through which SnCs exert central effector functions in the local tissue environment. To test this hypothesis in a medical indication that could be validated clinically, we evaluated EV production from SnCs enriched from chondrocytes isolated from human arthritic cartilage. EV production increased in a dose-responsive manner as the concentration of SnCs increased. The EVs were capable of transferring senescence to nonsenescent chondrocytes and inhibited cartilage formation by non-SnCs. microRNA (miR) profiles of EVs isolated from human arthritic synovial fluid did not fully overlap with the senescent chondrocyte EV profiles. The effect of SnC clearance was tested in a murine model of posttraumatic osteoarthritis. miR and protein profiles changed after senolytic treatment but varied depending on age. In young animals, senolytic treatment altered expression of miR-34a, -30c, -125a, -24, -92a, -150, and -186, and this expression correlated with cartilage production. The primary changes in EV contents in aged mice after senolytic treatment, which only reduced pain and degeneration, were immune related. In sum, EV contents found in synovial fluid may serve as a diagnostic for arthritic disease and indicator for therapeutic efficacy of senolytic treatment.

Diffusion-weighted magnetic resonance imaging and micro-RNA in the diagnosis of hepatic fibrosis in chronic hepatitis C virus

BACKGROUNDDiffusion-weighted magnetic resonance imaging has shown promise in the detection and quantification of hepatic fibrosis. In addition, the liver has numerous endogenous micro-RNAs (miRs) that play important roles in the regulation of biological processes such as cell proliferation and hepatic fibrosis.AIMTo assess diffusion-weighted magnetic resonance imaging and miRs in diagnosing and staging hepatic fibrosis in patients with chronic hepatitis C.METHODSThis prospective study included 208 patients and 82 age- and sex-matched controls who underwent diffusion-weighted magnetic resonance imaging of the abdomen, miR profiling, and liver biopsy. Pathological scoring was classified according to the METAVIR scoring system. The apparent diffusion coefficient (ADC) and miR were calculated and correlated with pathological scoring.RESULTSThe ADC value decreased significantly with the progression of fibrosis, from controls (F0) to patients with early fibrosis (F1 and F2) to those with late fibrosis (F3 and F4) (median 1.92, 1.53, and 1.25 × 10-3 mm2/s, respectively) (P = 0.001). The cut-off ADC value used to differentiate patients from controls was 1.83 × 10-3 mm2/s with an area under the curve (AUC) of 0.992. Combining ADC and miR-200b revealed the highest AUC (0.995) for differentiating patients from controls with an accuracy of 96.9%. The cut-off ADC used to differentiate early fibrosis from late fibrosis was 1.54 × 10-3 mm2/s with an AUC of 0.866. The combination of ADC and miR-200b revealed the best AUC (0.925) for differentiating early fibrosis from late fibrosis with an accuracy of 80.2%. The ADC correlated with miR-200b (r = - 0.61, P = 0.001), miR-21 (r = - 0.62, P = 0.001), and miR-29 (r = 0.52, P = 0.001).CONCLUSIONCombining ADC and miRs offers an alternative surrogate non-invasive diagnostic tool for diagnosing and staging hepatic fibrosis in patients with chronic hepatitis C.