miR-26a Expression Research Articles

MiR-15a Participated in the Pathogenesis of Pterygium via Targeting BCL-2: An Experimental Research

ABSTRACT Purpose To compare the expression levels of miR-15a between pterygium and normal conjunctiva, and further investigate the potential role of miR-15a in the progression of pterygium. Methods 21 cases of primary pterygium were enrolled in our study. The length of the pterygium invaded into the cornea and the total thickness of the pterygium were measured with anterior segment optical coherence tomography (AS-OCT). The pterygial and adjacent normal conjunctival samples of the 21 patients were collected. Expressions of miR-15a, BCL-2, Bax in both pterygium and normal conjunctiva were measured, and correlations between miR-15a and BCL-2, miR-15a and Bax, miR-15a and clinical parameters were made. Pterygium epithelial cells (PECs) were isolated, cultured and transfected with miR-15a mimic or miR-15a inhibitor to interfere the miR-15a expression levels. The regulation of BCL-2 expression by miR-15a was examined with Real-Time PCR (RT-PCR), Western blot and immunofluorescence. The regulation of Bax expression by miR-15a was also examined with Real-Time PCR (RT-PCR) and Western blot. The cell viability of the transfected PECs was measured with the CCK-8 assay and the apoptosis in these cells was detected using the TUNEL assay. Results The expression of miR-15a, Bax were significantly decreased while the BCL-2 was significantly increased in pterygium (p < .05). There was a negative correlation in expression between miR-15a and BCL-2 in pterygium tissues (r = −0.516, p < .05). We also found that relative miR-15a level was positively correlated with the length of pterygium invaded into the cornea (r = −0.570, p < .05). In cultured PECs, miR-15a could downregulate the expression of BCL-2 and upregulate the expression of Bax. Promotion of miR-15a could suppress cell proliferation and promote cell apoptosis in cultured PECs. Conclusions Our study demonstrated that decreased expression of miR-15a in pterygium might be associated with the apoptosis and proliferation of abnormal cell via regulating BCL-2, which could subsequently contribute to the development of pterygium. Downregulation of miR-15a might also contribute to the pathogenesis of pterygium by other mechanisms including abnormal proliferation and neovascularization, which remain to be investigated.

Circulating Extracellular Vesicle Proteins and MicroRNA Profiles in Subcortical and Cortical-Subcortical Ischaemic Stroke.

In order to investigate the role of circulating extracellular vesicles (EVs), proteins, and microRNAs as damage and repair markers in ischaemic stroke depending on its topography, subcortical (SC), and cortical-subcortical (CSC) involvement, we quantified the total amount of EVs using an enzyme-linked immunosorbent assay technique and analysed their global protein content using proteomics. We also employed a polymerase chain reaction to evaluate the circulating microRNA profile. The study included 81 patients with ischaemic stroke (26 SC and 55 CSC) and 22 healthy controls (HCs). No differences were found in circulating EV levels between the SC, CSC, and HC groups. We detected the specific expression of C1QA and Casp14 in the EVs of patients with CSC ischaemic stroke and the specific expression of ANXA2 in the EVs of patients with SC involvement. Patients with CSC ischaemic stroke showed a lower expression of miR-15a, miR-424, miR-100, and miR-339 compared with those with SC ischaemic stroke, and the levels of miR-339, miR-100, miR-199a, miR-369a, miR-424, and miR-15a were lower than those of the HCs. Circulating EV proteins and microRNAs from patients with CSC ischaemic stroke could be considered markers of neurite outgrowth, neurogenesis, inflammation process, and atherosclerosis. On the other hand, EV proteins and microRNAs from patients with SC ischaemic stroke might be markers of an anti-inflammatory process and blood–brain barrier disruption reduction.

Retraction Note: MicroRNA-23a enhances migration and invasion through PTEN in osteosarcoma.

To investigate the biological significance of abundant microRNA-23a (miR-23a) expression in osteosarcoma and its correlation with PTEN in the pathogenesis of osteosarcoma migration and invasion. The human osteosarcoma cell lines MG63, HOS58 and SaoS-2, and the human normal osteoblasts (hFOB1.19) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. Gene and protein levels of miR-23a and PTEN were examined to determine the molecular relationship between them in the pathogenesis of osteosarcoma. Inhibition of miR-23a effectively reduced migration and invasion of osteosarcoma cell lines. Bioinformatics and luciferase-reporter assay revealed that miR-23a specifically targeted the 3'-untranslational region of PTEN and regulated its expression. Downregulation of PTEN enhanced migration and invasion of osteosarcoma cell lines. Furthermore, in tumor tissues obtained from osteosarcoma patients, the expression of miR-23a was negatively correlated with PTEN and the high expression of miR-23a combined with low expression of PTEN might serve as a risk factor for cancer patients. Besides, miR-23a-mediated suppression of PTEN led to activation of AKT/ERK pathways and epithelial-mesenchymal transition (EMT) in osteosarcoma cells, and finally enhanced the activity of osteosarcoma cell proliferation and movement and promoted osteosarcoma xenograft tumor growth in mouse models. Our study showed that miR-23a, by downregulation of PTEN, enhanced migration and invasion in osteosarcoma cells.

P-232 Prognostic inflammatory and microRNA biomarkers in stage IV colorectal cancer patients

Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. Stage IV CRC patients have poor prognosis with a five-year survival rate of 14%. Systemic inflammation has a key determinant role in clinicopathological outcomes among CRC patients. Furthermore, microRNA (miRNA), small RNA non-coding molecules, have been detected to be dysregulated in tissues, as well as in biological fluids taken from colorectal cancer patients. Consequently, we are interested in investigating the prognostic potential of inflammatory markers and miRNA in Stage IV colorectal cancer patients. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum albumin, fibrinogen, ferritin, haptoglobin, lymphocyte/monocyte ratio, neutrophil/lymphocyte ratio and platelets/lymphocytes ratio, and Glasgow prognostic score were prospectively evaluated pre-chemotherapy in 67 Stage IV CRC patients presenting at the American University of Beirut Medical Center between May 2018 till November 2020. miRNA (miR-21, miR-19a, miR-23a, miR-29a, miR-155, miR-210, miR-145, miR-203, miR-31 and miR-345) expression was determined using reverse transcription real-time PCR in plasma samples collected prechemotherapy. Cutoff values of the biomarkers were determined using ROC curve. Statistical analysis was performed with SPSS version 25. The median overall survival for the 67 patients was not reached and the median progression-free survival was 13 months. Patients with low pre-chemotherapy WBC count (< 9600 /cu.mm), CRP (< 3.1 mg/L) or ESR (< 20.5 mm/hr) experienced significant improvements in progression-free survival (P=0.012, P=0.016, P < 0.05 respectively) compared with patients with high pre-chemotherapy CRP, ESR or WBC count. Patients with low pre-chemotherapy haptoglobin (< 3.1 g/L) had significantly better overall survival (OS) (P < 0.05) compared with patients with high pre-chemotherapy haptoglobin. Low levels of miR-23a and miR-19a were significantly associated with better PFS (P=0.021 and P=0.031 respectively). Elevated levels of miR-345 and miR-210 were correlated with better OS (P=0.029 and P=0.035 respectively). Subsequent Cox regression multivariate analysis showed that high pre-chemotherapy of CRP, WBC count, miR-19a, and miR-23a were independent favorable prognostic factors for PFS with a HR=3.8; 95%CI: 1.149-12.648; HR=2.426; 95%CI: 1.159-5.075, HR= 2.37; 95%CI: 1.092-5.15 and, HR=2.31; 95%CI: 1.029-4.414), respectively. CRP level, WBC count, miR-19a, and miR-23a are potential prognostic biomarkers for progression-free survival in Stage IV colorectal cancer that need further validation in larger cohorts.

Abstract 2842: 6-gingerol attenuates metastasis in aged mice model of colorectal cancer through modulation of epithelial-mesenchymal transition mediators genes and e-cadherin upregulation

Abstract Colorectal cancer (CRC) is the third most common cancer in America, and the third cause of cancer related death. Metastasis is the major cause of death in patient with CRC. Many CRC are likely to spread from primary site to the other parts of the body before and after detection of malignant tumor. 6-gingerol, the bioactive component of fresh ginger have been reported to have myriad of biological activities including anti-inflammatory and antioxidant properties. We have previously reported the anti-CRC effect of 6-gingerol. However there is dearth of information on the anti-metastatic effect 6-gingerol in CRC. Thus, the effect of 6-gingerol on epithelial-mesenchymal transition mediators' genes and E-Cadherin repression in aged mice model of CRC was assessed in this study. Following institutional ethical approval. Aged male BALB/c mice (10 months) were divided into four groups of 10 mice each. Mice were fed with AIN-93 diet and allowed access to water ad libitum. Group 1 mice serve as control, group 2 mice received 6-gingerol 10mg/kg/day by oral gavage for 10 weeks. Group 3 mice received a single dose of azoxymethane (AOM) 10mg/kg (iP) and on the seventh day they received three cycles of dextran sulfate sodium (DSS) 2% (W/V) in drinking water ( each cycle is define as one week of DSS followed by two weeks of plain drinking water) Group 4 mice received 6-gingerol 10mg/kg/day by oral gavage in combination with single dose of azoxymethane 10mg/kg (iP) and on the seventh day they received three cycles of dextran sulfate sodium salt 2% (W/V) in drinking water. Mice were humanely sacrificed under anesthesia and colon tissues were processed for RT-PCR and multiplex immunohistochemistry analysis. Mice that received both 6-gingerol and AOM and DSS showed a significant decreased expression of Lymphocyte function-associated antigen-1, N-cadherin, SNAIL 1, ZEB 1, AP4 and HMGA1 with concomitant Upregulation miR-34, miR-15a, miR-218 and miR-302 expression as compared with the AOM and DSS only group. The result obtained from this study showed 6-gingerol inhibited metastasis through modulation of epithelial-mesenchymal transition mediators' genes and E-Cadherin upregulation, and as such 6-gingerol could be a promising anti-metastatic agent in CRC treatment. Citation Format: Babajide Oluwaseun Ajayi, Ayomiposi Adenigba, Anuoluwapo Adeshina. 6-gingerol attenuates metastasis in aged mice model of colorectal cancer through modulation of epithelial-mesenchymal transition mediators genes and e-cadherin upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2842.

BCR Unresponsiveness and 13q14 Duplication Underpin Spontaneous Disease Regression in Chronic Lymphocytic Leukemia

Spontaneous regression of chronic lymphocytic leukemia occurs in less than 2% of cases and the mechanisms that underlie this rare event remain largely unknown. We reviewed 1425 patients with CLL across 4 centers and identified 20 who underwent spontaneous disease regression without treatment over a median period of 8.6 years. This included 12 patients with complete and 8 with partial (> 50%) resolution of lymphocytosis and clinical features. Residual circulating CLL cells remained detectable in all cases and phenotypic, functional and genomic analyses were performed on the residual CLL clone, as well as stored cells where available, to investigate potential mechanisms underlying regression.IGH sequence analysis of sequential samples confirmed that the CLL clone was identical between the time of diagnosis and regression (n=6). Moreover, all cases showed somatic mutation of the IGHV gene and VH3 or VH4 usage was seen in 17 cases. Flow cytometric analysis of spontaneous regression cases (n=17) showed that Ki-67 and CD49d expression was markedly decreased compared to stable indolent disease (n=54) and progressive disease (n=40) whereas CXCR4 and FasR expression was increased. No difference was observed with respect to the expression of CD38, ZAP-70, Bcl-2 and Mcl-1 between spontaneous regression and indolent cases.Sequential analysis revealed that these changes in Ki-67 and CXCR4 expression developed during the period of regression. Furthermore, telomere length analysis demonstrated that tumor cells generally had short telomeres (< 5 kb) after regression and this profile, together with the phenotypic progression, indicates that tumors have undergone an initial period of intense proliferation prior to the development of regression. Importantly, telomerase expression remained high in regression tumors and no evidence of cellular senescence was observed. Analysis of the immune compartment revealed a reduction in T cell and NK cell numbers during spontaneous CLL regression and a modest improvement in T cell function as revealed by increased T cell proliferation and decreased expression of PD-1.The response of tumor cells to stimulation through the B cell receptor (BCR) was also investigated. Surface expression of IgM and IgD was low in regression cases and basal Erk phosphorylation was increased. In addition, these tumors were uniformly unresponsive to stimulation separately with anti-IgM F(ab‘)2 antibodies and anti-IgD F(ab‘)2 antibodies, and combined stimulation with both antibodies. These findings indicate that spontaneously regressed CLL tumors are in a state of profound unresponsiveness to BCR stimulation. Consistent with reduced BCR responsiveness, RNA-seq analysis of sorted CLL cells revealed reduced expression of both MYC and Myc target genes in regression cases as well as downregulation of pathways involved in RNA processing, cellular metabolism and proliferation.Finally, the genomic landscape of tumor cells after regression was analyzed by whole exome sequencing and SNP array analysis on paired germline and CLL samples. CLL genomic complexity was generally low although mutations in TP53, MYD88 or HIST1H1B were observed in 5 tumors. Strikingly, 13q14 duplication was present in 13 of 19 tumors and qPCR analysis showed that this was associated with increased expression of miR-15a/16-1.This is the largest and most comprehensive analysis of spontaneous CLL regression to date and reveals a range of novel features. Tumors that have undergone spontaneous regression express somatically mutated immunoglobulin genes, demonstrate short telomeres with a phenotype of BCR unresponsiveness and have a very high prevalence of 13q14 duplication. This profile suggests a model in which CLL clones undergo substantial proliferation initially which subsequently subside into a state of BCR unresponsiveness and low proliferation. The identification of 13q14 duplication as a recurrent genetic abnormality indicates a potential molecular mechanism for this pattern but it is currently unclear if this lesion is established during tumorigenesis or is acquired during the initial proliferative phase. These findings identify CLL regression as a unique subset of B cell lymphoproliferative disease and suggest that therapies which upregulate the expression of miR-15a/16-1 may represent a mechanism to suppress B-CLL proliferation. [Display omitted] DisclosuresRawstron:Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; BD biosciences: Patents & Royalties; Roche: Consultancy, Honoraria; Gilead: Research Funding. Munir:Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; Gilled: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Hillmen:AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; Celgene: Research Funding; GSK: Consultancy, Honoraria, Research Funding.

The regulation of miR-320a/XBP1 axis through LINC00963 for endoplasmic reticulum stress and autophagy in diffuse large B-cell lymphoma

BackgroundThis study incorporates fundamental research referring to considerable amounts of gene-sequencing data and bioinformatics tools to analyze the pathological mechanisms of diffuse large B-cell lymphoma (DLBCL).MethodsA lncRNA-miRNA-mRNA ceRNA network of DLBCL was constructed through database analysis combining GTEx and TCGA. qPCR was used to detect the expression of LINC00963 and miR-320a in DLBCL cell lines. After LINC00963 or miR-320a overexpression in vitro, western blot was performed to assess the protein levels of UPR sensors (GRP78, p-IRE1, IRE1, active ATF6, ATF4 and XBP1), along with apoptosis markers (Bcl-2, Bax, caspase 3) and autophagy indicators (Beclin1, LC3II, LC3I and p62). Additionally, the expression of LC3 was analyzed through immunofluorescence (IF) assay. ResultsFollowing LINC00963 overexpression in vitro, SUDHL4 cell line showed a marked increase in the level of UPR-related GRP78, p-IRE1 and spliced XBP-1/XBP-1(s), apoptosis-related Bax and cleaved caspase 3, as well as autophagy-related Beclin1 and LC3II, whereas miR-320a mimic greatly diminished the effects of LINC00963 overexpression. Moreover, LINC00963 targeted miR-320a while miR-320a bound to the 3’UTR of XBP1. It was also found that LINC00963 overexpression resulted in significantly delayed tumor growth in a xenograft model of DLBCL. ConclusionMechanistically, LINC00963/miR-320a regulated XBP1-apoptosis pathwayand autophagy, implying the therapeutic potential ofthis pathway for selective targeting. The data presented here illustratedthe mechanism of LINC00963/miR-320a/XBP1 in DLBCL forthe first time.

Knockdown circular RNA circGFRA1 inhibits glioma cell proliferation and migration by upregulating microRNA-99a.

Glioma is the most widespread and malignant brain tumor in the central nervous system of adult, causing multiple cancer-associated deaths worldwide. Here, we identified the impact of circGFRA1 on glioma, and aimed to uncover the underlying molecular mechanism. The expression of circGFRA1 of glioma specimens was evaluated by using quantitative reverse transcription PCR. Cell viability, proliferation, colony formation, apoptosis and migration were estimated utilizing CCK-8, EdU staining, colony formation assay, TUNEL staining and Transwell assay, respectively. Bioinformatics analysis, luciferase assay and RNA co-immunoprecipitation was utilized for verification of direct binding between circGFRA1 and miR-99a. Western blot was applied to investigate protein expression in U251 cells. The results showed that circGFRA1 expression was overexpressed in glioma specimens. Knockdown circGFRA1 declined viability, colony formation, proliferation and migrative potential, but enhanced U251 cell apoptosis. Moreover, circGFRA1 acts as a microRNA sponge for miR-99a. Furthermore, miR-99a was involved in the circGFRA1-regulated glioma cell behaviors. Silencing circGFRA1 reduced p/t-AKT, p/t-FOXO1 and p/t-mTOR expression levels via upregulating miR-99a expression. In conclusion, our study demonstrated that knockdown circGFRA1 inhibits glioma cell proliferation and migration by upregulating microRNA-99a.

ZEB1 induces non-small cell lung cancer development by targeting microRNA-320a to increase the expression of RAD51AP1

Lung cancer is the most prevalent cancer worldwide, with its mortality rate reported to be in millions annually; one of the two subtypes is non-small cell lung cancer (NSCLC). In this study, we investigated the interactions and expressions of zinc finger E-box binding homeobox 1 (ZEB1), microRNA-320a (miR-320a) and RAD51-associated protein 1 (RAD51AP1) in NSCLC tissues to determine the roles of ZEB1 in regulation of miR-320a and RAD51AP1 in the development and metastasis of NSCLC. First, the expression levels of miR-320a and ZEB1 were quantified in NSCLC tissues and cells. Transfection assay was conducted to identify the effects of miR-320a on the progression of NSCLC cells. The interaction of miR-320a with ZEB1 and RAD51AP1 was predicted and verified using dual-luciferase reporter gene assay and chromatin immunoprecipitation assay. Finally, subcutaneous xenograft tumors of 6-week mice and metastatic model tumors of 8-week mice were established to further explore the in vivo effect of miR-320a/ZEB1/RAD51AP1 on NSCLC. The findings revealed a lower expression of miR-320a in NSCLC tissues and cells, while this result was reversed regarding ZEB1 expression. ZEB1 suppressed miR-320a expression and upregulation of miR-320a resulted in the reduction of proliferation, invasion and metastasis rate of NSCLC cells, and promoted NSCLC cell apoptosis. ZEB1 promoted the expression of RAD51AP1 via inhibition of miR-320a, promoting tumor growth in vivo. ZEB1 transcriptionally inhibited the expression of miR-320a and upregulated the expression of RAD51AP1, thereby promoting metastasis in NSCLC.

Exercise Improves Endothelial Function via the lncRNA MALAT1/miR-320a Axis in Obese Children and Adolescents.

Background Endothelial dysfunction commonly occurs in obese children and adolescents, leading to an increased risk of cardiovascular diseases. Exercise has significant protective effects against endothelial dysfunction through regulating some noncoding RNAs. This study aimed to investigate the relationship of long noncoding RNA MALAT1 and microRNA-320a (miR-320a) with the exercise-induced improvement of endothelial dysfunction in obese children and adolescents. Methods Sixty obese children and adolescents were included in this study, including 40 cases that received 12-week exercise training and 20 cases that received only diet control. The anthropometric and blood indices before and after exercise were recorded and compared, and the endothelial dysfunction was evaluated by examining the levels of markers, including VCAM-1, ICAM-1, and E-selectin, using an ELISA assay. The expression levels of noncoding RNAs were assessed using real-time quantitative PCR, and their correlation with patients' recorded indices and endothelial dysfunction markers was analyzed. Results The 12-week exercise training significantly decreased the levels of VCAM-1, ICAM-1, and E-selectin and could inhibit MALAT1 but promote miR-320a expression in obese children and adolescents. The expression of MALAT1 and miR-320a was correlated with the changes in the anthropometric and blood indices of obese children and adolescents, and their correlations with endothelial dysfunction markers were obtained. Conclusion All the data revealed that exercise has significantly protective effects against endothelial dysfunction and can regulate the expression of the MALAT1/miR-320a axis. MALAT1 and miR-320a were correlated with endothelial dysfunction markers, indicating that the MALAT1/miR-320a axis may be related with the alleviating effects of exercise on endothelial function in obese children and adolescents.

Relationship of Circulating Mirnas With Insulin Resistance and Metabolic Dysfunction in Adolescents

Abstract Objectives To evaluate the relationship of plasma expression of miR-122, -375, -139, 15-a and -let7c with insulin resistance and factors related to metabolic syndrome (MetS) in adolescents. Methods Data from 188 adolescents aged 12–19 years participating in a cross-sectional population-based study were analyzed, all non-smokers, without acute inflammatory diseases and cancer, and without the use of medication that would interfere in the outcome. The quantification of circulating miRNA was performed by RT-PCR, using the Fluidigm chip technique. The miRNA expression was compared among adolescents grouped by the absence (n = 94) or presence of insulin resistance (n = 94)—categorized according to with the HOMA-IR index -, and the absence (n = 92) or presence of 1 (n = 59), 2 (n = 23), 3 (n = 10) or 4 (n = 4) factors related to MetS. The comparison of means was performed using the Mann-Whitney test for two samples or Kruskal-Wallis followed by Dunn's post-test to compare three or more samples. Results Insulin resistance was identified in 50% of the individuals (HOMA-IR &amp;gt; 2.7). In adolescents with insulin resistance, the plasma expression of miR-122 was higher in comparison to normoglycemic individuals and was positively associated with fasting insulinemia (r = 0.157; P = 0.031), HOMA-IR index (r = 0.160; P = 0.028) and plasma hepcidin (r = 0.209; P = 0.005) levels. When evaluating the expression according to the number of factors related to MetS, the expression of miR-let7c, -139 and -15a increased according to the increase of the factors related to MetS. The opposite effect was observed in relation to miR-375. Individuals with 3 or more factors differed from groups whith 1 or 2 factores for plasma miR-375, -139, 15-a and -let7c expression. MiR-375 showed a negative association with body weight (r = −0.169; P = 0.019), while miR-139 was associated with systolic (r = 0.153; P = 0.055) and diastolic (r = 0.193; P = 0.016) blood pressure. Plasma miR-15a expression had a positive correlation with diastolic blood pressure (r = 0.198; P = 0.0064). MiR-let7c showed a positive correlation plasma TG (r = 0.156; P = 0.029). Conclusions Plasma miR 122 expression was related to insulin resistance, while plasma miR 375, 139, 15 a and let7c expressions were associated with MetS in adolescents. Funding Sources From Sao Paulo Research Foundation and National Council for Scientific and Technological Development

MiR-20a suppresses Treg differentiation by targeting Map3k9 in experimental autoimmune encephalomyelitis

BackgroundExperimental autoimmune encephalomyelitis (EAE) is a model for inflammatory demyelinating diseases of the central nervous system (CNS), a group of autoimmune diseases characterized by inflammatory infiltration, demyelination, and axonal damage. miR-20a is dysregulated in patients with CNS inflammatory demyelinating diseases; however, the function of miR-20a remains unclear. In this study, we intended to explore the role of miR-20a in EAE.MethodsThe expression of miR-20a was detected by quantitative real-time PCR (qRT-PCR) in EAE mice and patients with MOG antibody-associated demyelinating diseases. CD4+ T cells of EAE mice were sorted, stimulated, and polarized with miR-20a knockdown. Activation and differentiation of CD4+ T cells were analyzed by flow cytometry. The expression of target gene Map3k9 was detected by qRT-PCR and western blot experiments. The binding of miR-20a to the 3’ UTR of Map3k9 was tested by luciferase assays. The feasibility of miR-20a as a therapeutic target to alleviate the severity of EAE was explored by intravenous administration of miR-20a antagomirs to EAE mice.ResultsmiR-20a was upregulated in splenocytes and lymph node cells, CD4+ T cells, and spinal cords of EAE mice. Moreover, miR-20a knockdown did not influence the activation of antigen-specific CD4+ T cells but promoted their differentiation into Treg cells. Map3k9 was predicted to be a target gene of miR-20a. The expressions of Map3k9 and miR-20a were negatively correlated, and miR-20a knockdown increased the expression of Map3k9. In addition, miR-20a binded to the 3’ UTR of Map3k9, and simultaneous knockdown of miR-20a and Map3k9 counteracted the enhanced differentiation of Tregs observed when miR-20a was knocked down alone. Furthermore, injection of miR-20a antagomirs to EAE mice reduced the severity of the disease and increased the proportion of Treg cells in peripheral immune organs.ConclusionsmiR-20a suppresses the differentiation of antigen-specific CD4+ T cells into Tregs in EAE by decreasing the expression of Map3k9. miR-20a antagomirs alleviate EAE, suggesting a new therapy for EAE and CNS inflammatory demyelinating diseases.

Combination of Metformin, Sodium Oxamate and Doxorubicin Induces Apoptosis and Autophagy in Colorectal Cancer Cells via Downregulation HIF-1α.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide in both sexes. Current therapies include surgery, chemotherapy, and targeted therapy; however, prolonged exposure to chemical agents induces toxicity in patients and drug resistance. So, we implemented a therapeutic strategy based on the combination of doxorubicin, metformin, and sodium oxamate called triple therapy (Tt). We found that Tt significantly reduced proliferation by inhibiting the mTOR/AKT pathway and promoted apoptosis and autophagy in CRC derived cells compared with doxorubicin. Several autophagy genes were assessed by western blot; ULK1, ATG4, and LC3 II were overexpressed by Tt. Interestingly, ULK1 was the only one autophagy-related protein gradually overexpressed during Tt administration. Thus, we assumed that there was a post-transcriptional mechanism mediating by microRNAs that regulate UKL1 expression during autophagy activation. Through bioinformatics approaches, we ascertained that ULK1 could be targeted by mir-26a, which is overexpressed in advanced stages of CRC. In vitro experiments revealed that overexpression of mir-26a decreased significantly ULK1, mRNA, and protein expression. Contrariwise, the Tt recovered ULK1 expression by mir-26a decrease. Due to triple therapy repressed mir-26a expression, we hypothesized this drug combination could be involved in mir-26a transcription regulation. Consequently, we analyzed the mir-26a promoter sequence and found two HIF-1α transcription factor recognition sites. We developed two different HIF-1α stabilization models. Both showed mir-26a overexpression and ULK1 reduction in hypoxic conditions. Immunoprecipitation experiments were performed and HIF-1α enrichment was observed in mir-26a promoter. Surprisingly, Tt diminished HIF-1α detection and restored ULK1 mRNA expression. These results reveal an important regulation mechanism controlled by the signaling that activates HIF-1α and that in turn regulates mir-26a transcription.

WITHDRAWN: LncRNA MALAT1/miR-320a axis is associated with exercise-induced improvement of endothelial dysfunction in obese children

Endothelial dysfunction commonly occurs in obese children, leading to increased risk of cardiovascular diseases. Exercise has protective effects against endothelial dysfunction through regulating some noncoding RNAs. This study aimed to investigate the relationship of long noncoding RNA MALAT1 and microRNA-320a (miR-320a) with the exercise-induced improvement of endothelial dysfunction in obese children. Sixty obese children were included in this study, and 40 cases received a 12-week exercise training. The morphological and blood indices before and after exercise were recorded and compared, and the endothelial dysfunction was evaluated by examining the levels of VCAM-1, ICAM-1, E-selectin, IL-6 and TNF-α using ELISA kits. The expression of noncoding RNAs was assessed using Real-Time quantitative PCR. Endothelial cells were used to explore the effects of MALAT1 and miR-320a on endothelial function. The 12-well exercise training decreased the levels of VCAM-1, ICAM-1 and E-selectin, and inhibited MALAT1 but promoted miR-320a expression in obese children. The expression of MALAT1 and miR-320a was correlated with the changes of morphological and blood indices in obese children, and their correlations with endothelial dysfunction markers were obtained. Additionally, MALAT1 overexpression or miR-320a reduction led to inhibited proliferation and increased inflammation in HUVECs. All the data revealed that exercise has significantly protective effects against endothelial dysfunction, and can regulate the expression of the MALAT1/miR-320a axis. MALAT1 and miR-320a were correlated with endothelial dysfunction, indicating that the MALAT1/miR-320a axis may be related with the alleviating effects of exercise on endothelial function in obese children.

MIR99AHG is a noncoding tumor suppressor gene in lung adenocarcinoma

Little is known about noncoding tumor suppressor genes. An effective way to identify these genes is by analyzing somatic copy number variation (CNV)-related noncoding genes. By integrated bioinformatics analyses of differentially expressed long noncoding RNAs (lncRNAs) and arm-level CNVs in lung adenocarcinoma (LUAD), we identified a potential antitumor gene, MIR99AHG, encoding lncRNA MIR99AHG as well as a miR-99a/let-7c/miR-125b2 cluster on chromosome 21q. All four of these transcripts were downregulated in LUAD tissues partly due to the copy number deletion of the MIR99AHG gene. Both MIR99AHG and miR-99a expression was positively correlated with the survival of LUAD patients. MIR99AHG suppressed proliferation and metastasis and promoted autophagy both in vitro and in vivo. Mechanistically, the interaction between MIR99AHG and ANXA2 could accelerate the ANXA2-induced ATG16L+ vesicle biogenesis, thus promoting phagophore assembly. Additionally, miR-99a targeted a well-known autophagy suppressor, mammalian target of rapamycin (mTOR), thereby synergistically promoting autophagy and postponing LUAD progression with MIR99AHG. In summary, MIR99AHG emerges as a noncoding tumor suppressor gene in LUAD, providing a new strategy for antitumor therapy.

Modulation of Immune Response to Chlamydia muridarum by Host miR-135a.

Previously, our laboratory established the role of small, noncoding RNA species, i.e., microRNA (miRNA) including miR-135a in anti-chlamydial immunity in infected hosts. We report here chlamydial infection results in decreased miR-135a expression in mouse genital tissue and a fibroblast cell line. Several chemokine and chemokine receptor genes (including CXCL10, CCR5) associated with chlamydial pathogenesis were identified in silico to contain putative miR-135a binding sequence(s) in the 3’ untranslated region. The role of miR-135a in the host immune response was investigated using exogenous miR-135a mimic to restore the immune phenotype associated with decreased miR-135a following Chlamydia muridarum (Cm) infection. We observed miR-135a regulation of Cm-primed bone marrow derived dendritic cells (BMDC) via activation of Cm-immune CD4+ T cells for clonal expansion and CCR5 expression. Using a transwell cell migration assay, we explore the role of miR-135a in regulation of genital tract CXCL10 expression and recruitment of CXCR3+ CD4+ T cells via the CXCL10/CXCR3 axis. Collectively, data reported here support miR-135a affecting multiple cellular processes in response to chlamydial infection.

MicroRNA-mediated extracellular matrix remodeling in squamous cell carcinoma of the oral cavity.

We evaluated microRNAs and extracellular matrix component profiles in squamous cell carcinoma of the oral cavity (OSCC) in comparison to healthy mucosa. Retrospective study investigating 64 microRNAs related to oncogenic process and to constituents of the extracellular matrix. We also performed immunohistochemical assays for molecules involved in the same biological processes. High expression of miR-21-5p (p < 0.001) and miR-106-5p (p < 0.001) and low expression of miR-320a (p = 0.001) and miR-222-3p (p = 0.001) were predictors of malignancy. Individually, miR-21-5p exhibited the best statistical performance (area under the curve = 0.972; 95% confidence interval: 0.911-1.000) in the differentiation between tumor tissue and healthy mucosa. Moreover, tumor sample showed increased expression of MMP-2, MMP-9, α-laminin, and β-laminin in tumor-related fibroblasts and lower continuity of type IV collagen in the basement membrane. The present study demonstrates the biological effects of microRNAs on the carcinogenesis of OSCC as well as the intense modification of the tumor microenvironment.

Binding of microRNA-135a (miR-135a) to homeobox protein A10 (HOXA10) mRNA in a high-progesterone environment modulates the embryonic implantation factors beta3-integrin (ITGβ3) and empty spiracles homeobox-2 (EMX2).

BackgroundPatients with elevated circulating progesterone concentrations on the day of the human chorionic gonadotropin (hCG) trigger had relatively low implantation rates during assisted reproductive treatments. In this study, we assess the hypothesis that different concentrations of progesterone regulate the expression of homeobox protein A10 (HOXA10) and its downstream genes through miRNA-135a.MethodsMicroRNA-135a (miR-135a), HOXA10, beta3-integrin (ITGβ3), and empty spiracles homeobox-2 (EMX2) expression levels in endometrial tissues from patients with elevated progesterone were measured. To determine the threshold of progesterone level which can impair implantation, Ishikawa cells were used to determine the expression of the aforementioned 4 genes after exposure to 5 graded concentrations of progesterone. The dual-luciferase reporter assay was used to verify whether miR-135a regulated the expression of HOXA10. Furthermore, the effects of HOXA10 on the expression of key endometrial receptivity genes ITGβ3 and EMX2 were confirmed.ResultsHigh progesterone levels promoted miR-135a expression in vivo, and miR-135a bound to the 3'-untranslated region (3'-UTR) of HOXA10 mRNA to inhibit HOXA10 expression. Reduction of HOXA10 promoted EMX2 expression and inhibited ITG-3 production. Progesterone promoted the expression of HOXA10 in vitro at low concentrations. However, when the concentration was greater than 10−7 ng/mL, progesterone inhibited HOXA10 by promoting miR-135a expression, thereby altering the expression of related genes and affecting endometrial receptivity.ConclusionsIn vitro, the trend in miR-135a expression (which first decreased and then increased) was in direct contrast to that of HOXA10 expression (which first increased and then decreased) as progesterone levels increased. The key factors regulating endometrial receptivity included ITGβ3 and EMX2, which were confirmed to be regulated by HOXA10. High progesterone levels affected miR-135a expression, and miR-135a inhibited HOXA10 expression, thereby affecting endometrial receptivity.

Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance.

Simple SummaryMelanoma is a highly metastatic and therapy-resistant cancer and is therefore associated with low survival rates of patients. In melanoma, the inactivation of the wild-type form of the p53 tumour suppressor protein is a frequent event, mainly through interactions with MDM2 and MDMX. In this work, our recently disclosed p53-activating agent, SLMP53-2, displayed promising in vitro and in vivo antitumour activity, with particular impacts on melanoma migration and invasion. Moreover, SLMP53-2 (re)sensitized melanoma cells to clinically used chemotherapeutic agents, potentially overcoming the therapeutic resistance issue. As a whole, the p53 activator SLMP53-2 may represent a new therapeutic opportunity for melanoma, particularly in combination with MAPK pathway-targeting drugs.Melanoma is the deadliest form of skin cancer, primarily due to its high metastatic propensity and therapeutic resistance in advanced stages. The frequent inactivation of the p53 tumour suppressor protein in melanomagenesis may predict promising outcomes for p53 activators in melanoma therapy. Herein, we aimed to investigate the antitumor potential of the p53-activating agent SLMP53-2 against melanoma. Two- and three-dimensional cell cultures and xenograft mouse models were used to unveil the antitumor activity and the underlying molecular mechanism of SLMP53-2 in melanoma. SLMP53-2 inhibited the growth of human melanoma cells in a p53-dependent manner through induction of cell cycle arrest and apoptosis. Notably, SLMP53-2 induced p53 stabilization by disrupting the p53–MDM2 interaction, enhancing p53 transcriptional activity. It also promoted the expression of p53-regulated microRNAs (miRNAs), including miR-145 and miR-23a. Moreover, it displayed anti-invasive and antimigratory properties in melanoma cells by inhibiting the epithelial-to-mesenchymal transition (EMT), angiogenesis and extracellular lactate production. Importantly, SLMP53-2 did not induce resistance in melanoma cells. Additionally, it synergized with vemurafenib, dacarbazine and cisplatin, and resensitized vemurafenib-resistant cells. SLMP53-2 also exhibited antitumor activity in human melanoma xenograft mouse models by repressing cell proliferation and EMT while stimulating apoptosis. This work discloses the p53-activating agent SLMP53-2 which has promising therapeutic potential in advanced melanoma, either as a single agent or in combination therapy. By targeting p53, SLMP53-2 may counteract major features of melanoma aggressiveness.

EXOSOMAL MICRORNA-26A RESPONSE TO TGF-B1 STRESS IN HYPERTENSION

Objective: In a previous work we established an exosomal miRNA signature associated to urinary albumin excretion (UAE) in hypertension, which included microRNA (miRNA) miR-26a, and obtaining cell signaling (MAPK, calcium, p53), TGF-ß and VEGF pathways as the most regulated biological processes. The aim of this work was to validate the exosomal miR-26a changes and its potential association with TGF-ß in human podocytes. Design and method: This prospective study analysed the miR-26a levels in 52 hypertensive patients, 24 with UAE (162,8 ± 168.2 mg/g creatinine; mean age 52.7 ± 8.4 years) and 28 normoalbuminuric (mean age 54.5 ± 5.6 years). Exosomes were isolated by differential ultracentrifugation from urine and plasma and miRNA levels were calculated by qRT-PCR. Urinary levels of TGF-ß1 were quantified by ELISA. Finally, human podocytes cultures were stimulated with recombinant human TGF-ß1 at several concentrations (5 ng/mL, 10 ng/mL and 15 ng/mL) for up 24 h to assess miR-26a changes in exosomal and cellular fractions. Results: We found a decrease in the levels of miR-26-5p in plasma and urinary exosomes of patients con increased UAE (p &lt; 0.05 and p &lt; 0.01, respectively). In addition, miR-26a-5p correlated inversely with UAE levels, both in urinary and plasma exosomes (r = -0.49, p &lt; 0.01; r = -0.53, p &lt; 0.01). Our patients also showed a significant increase in urinary levels of TGF-ß1 (2-fold increase, p &lt; 0.05) in hypertensive patients with UAE. Finally, we analysed the effect in vitro of TGF-ß1 stress. Significant changes in scaffolding of podocytes, such as an increase of vimentin levels, has been also observed. Finally, significant down-regulated miR-26a expression in exosomes from podocytes after TGF-ß1 stimulation (5 – 15 ng / mL): 2.23, 1.96 and 1.7-fold decrease, with p &lt; 0.001 and p &lt; 0.05, respectively. No significant changes were obtained at intracellular level. Conclusions: Our results show that miR-26a-5p is decreased in urinary and plasma exosomes of microalbuminuric patients. In addition, exosomal miR-26a podocyte release is associated with TGF-ß1 pathway, key molecule in the development and maintenance structure of the glomerular filtration barrier and fibrosis.