miR-146a Levels Research Articles

Potential of Circulating miRNA Biomarkers and Exosomes for Early Pregnancy Diagnoses in Cattle.

Circulating microRNAs (miRNAs) were investigated as biomarkers for the diagnosis of early pregnancy in cattle. The levels of prospective miRNA biomarkers and the features of extracellular vesicles (EVs) in the blood were evaluated. In Study 1, plasma samples from cows 21 days after artificial insemination (AI) were examined using RT-qPCR to determine the levels of seven circulating miRNAs. Only the levels of miR-126-3p were significantly lower in the pregnant group than in the non-pregnant group. In Study 2, among individuals not pregnant at the first AI, the miRNA levels were compared between the individuals pregnant at the second AI and those who remained non-pregnant. The miR-25 levels were significantly higher in the pregnant group at the second AI than in the pregnant group at the first AI; miR-19b, miR-27b, and miR-29a levels were also high. In the non-pregnant group, changes were absent in the miRNA levels in the same individual between the first and second AIs. In Study 3, Western blotting and RT-qPCR showed the presence of miRNAs in EVs and their levels were lower than in plasma. Thus, circulating miR-126-3p may serve as a biomarker for the diagnosis of early pregnancy in cattle. In addition, the expression of some miRNAs tended to be higher during pregnancy than during non-pregnancy in the same individual, suggesting their potential as an index to determine pregnancy and non-pregnancy rates using a comparative method.

Ameliorative effect of Metformin / alpha-lipoic acid combination on diabetic nephropathy via modulation of YAP/ miR-29a/PTEN/p-AKT axis

Diabetic nephropathy (DN) is the most frequent and serious complication of type 2 diabetes (T2DM). Lack of a precise remedy and socio-economic burden of DN patients implements searching about alternative therapies. This study aims to evaluate the possible beneficial effect of alpha-lipoic acid (α-LA) alone or in combination with metformin (Met) in ameliorating STZ/High fat diet (HFD)-induced DN. T2DM was induced via HFD administration for 15 weeks and single ip injection of STZ (35 mg/kg) at week 7. Male Sprague–Dawley rats were randomly grouped as follows: control group, STZ/HFD-induced DN, Met/T; daily treated with 150 mg/kg Met, α-LA/T group; daily treated with 100 mg/kg α-LA, and Met/T + α-LA/T group; daily treated with Met and α-LA at same doses. Administration of Met and α-LA succeeded in attenuating STZ/HFD-induced DN as manifested by significant decrease in kidney weight as well as renal and cardiac hypertrophy index. Moreover, Met and α-LA improved glycemic control, kidney functions and lipid profile as well as restored redox balance. Additionally, Met and α-LA administration significantly upregulated PTEN level accompanied by significant downregulation in renal p-AKT and miR-29a levels. Histopathologically, Met and α-LA administration mitigated STZ/HFD-induced histopathological alterations in kidney and heart. Moreover, immunohistochemical examination revealed a significant decrease in renal YAP, collagen I and Ki-67. Taken together, these observations revealed that Met and α-LA administration could protect against STZ/HFD-induced DN.

The relationship of peripheral blood lncRNA-PVT1 and miR-146a levels with Th17/Treg cytokines in patients with Hashimoto's thyroiditis and their clinical significance.

Hashimoto's thyroiditis (HT) is a prevalent autoimmune disease. We investigated the relationship of peripheral blood long noncoding RNA-plasmacytoma variant translocation 1 (lncRNA-PVT1) and microRNA (miR)-146a levels with Th17/Treg-related cytokines in HT patients and their clinical significance. Correlations of PVT1 and miR-146a with Th17/Treg-related cytokines were analyzed, and its clinical value in diagnosing HT was assessed. Results showed reduced lncRNA-PVT1 and interleukin (IL)-10 levels and increased miR-146a and IL-17 levels in HT patients. lncRNA-PVT1 negatively interrelated with miR-146a, IL-17, IL-23 and IL-6, and positively interrelated with IL-10; miR-146a positively correlated with IL-17, IL-23 and IL-6, but negatively correlated with IL-10 in HT patients. The area under the curve (AUC) of lncRNA-PVT1and miR-146a levels for diagnosing HT were 0.822 and 0.844, respectively (sensitivity 88.73% and 86.62%, specificity 67.02% and 69.15%, cut-off values 0.76 and 2.73), with their combined detections yielding a higher AUC. Patients with poorly expressed lncRNA-PVT1 and highly expressed miR-146a had elevated HT incidence. lncRNA-PVT1 and miR-146a levels were also found to be an independent influencing factor for HT occurrence. Our findings suggest that HT patients have low peripheral blood lncRNA-PVT1expression and high miR-146a expression. lncRNA-PVT1and miR-146a level changes were correlated with Th17/Treg cytokine imbalance and could be a potential diagnostic tool and independent influencing factor for HT.

Niaodukang mixture inhibits micro-inflammation in CKD rats by enhancing MiR-146a levels in enterogenous exosomes

Ethnopharmacological relevanceThe Niaodukang mixture (NDK) is a preparation known for its ability to lower serum creatine levels in individuals with chronic kidney disease (CKD) and is commonly administered at medical facilities like the Zhongshan Hospital of Traditional Chinese Medicine. The initial use of NDK was mainly to treat CKD. Our hospital frequently utilizes NDK, which consists of Rheum officinaleBaill., Salvia miltiorrhiza Bunge., Astragalus aaronii (Eig) Zohary., Carthamus tinctorius L., and Sanguisorba officinalis L., for treating patients with CKD-MBD. It has the effects of eliminating dampness and turbidity and dredging kidney collaterals. However, The impact and process of NDK in chronic kidney disease remain unknown. Aim of the studyTo determine whether microRNA-146a (miR-146a) is associated with CKD micro-inflammationand whether NDK protects against CKD micro-inflammation by modulating the miR-146a/nuclear factor kappa-B (NF-κB) signaling pathway. Materials and methods(1) An adenine-induced rat model of chronic kidney disease was created through the use of materials and methods. The levels of miR-146a in exosomes from plasma and ileum were determined by RT-PCR. (2) Human cloned colon adenocarcinoma (Caco-2)cellswere stimulated with lipopolysaccharide (LPS)and transfected with miR-146a mimic and inhibitor. Following that, the Western blot and RT-PCR techniques were used to measure the protein and mRNA quantities of Toll-like receptor 4 (TLR4), NF-κB, and TNF receptor-associated factor 6 (TRAF6). (3) Enzyme-linked immunosorbent assay (ELISA) was used to identify serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). (4) Plasma exosomes were extracted, and the exosomes in intestinal tissues were extracted via ultrahigh-speed centrifugation.Negative staining electron microscopy was used to analyze the morphology of exosomes and the ultrastructure of intestinal tissue and exosomes. The particle size of the exosomes was measured using nanoparticle tracking analysis. ResultsThe pathological characteristics of CKD rats included those associated with systemic micro-inflammation, which may be associated with the release of exosomes in intestinal tissue. NDK suppressed the inflammatory response in Caco-2 cells and decreased the levels of IL-1β, IL-6, and TNF-α in rats with CKD. The expression of miR-146a, which regulates inflammation, differed between plasma-derived and enterogenous exosomes in CKD rats, which may be due to stimulation of ileal exosome release into the blood. NDK effectively reduced the levels of TRAF6, NF-κB, and TLR4 in the ileum tissue of CKD rats. ConclusionNDK can effectively improve micro-inflammation in CKD ratsby enhancing the release of enterogenous exosomes, thereby enhancing the release of exosome-associated miR-146a and inhibiting micro-inflammation.

Downregulation of miR-29a as a possible diagnostic biomarker for schizophrenia

BackgroundMicroRNAs (miRNAs) are epigenetic factors regulating many genes involved in brain development. Dysregulation of miRNA could result in dysregulation of genes which may contribute to diseases affecting the brain and behavior (e.g., schizophrenia). miR-29 family is a miRNA family contributing to brain maturation. miR-29 knockout in animal studies is reported to correlate with psychiatric disorders very similar to those seen in schizophrenia. In this study, we aimed to evaluate the miR-29a level in patients with schizophrenia and its potential value in the diagnosis of schizophrenia.Materials and methodsThe serum sample of 42 patients with schizophrenia and 40 healthy subjects were obtained from the Azeri Recent onset/Acute phase psychosis Survey (ARAS) Cohort study. After preparations, the expression level of miR-29a was investigated by real-time PCR. The SPSS and GraphPad prism software were used to analyze the relation between miR-29a level and clinical parameters and its potential as a biomarker for the diagnosis of schizophrenia.ResultsOur study showed a significantly lower miR-29a level in patients compared to healthy controls (p = 0.0012). Furthermore, miR-29a level was significantly lower in some types of schizophrenia (p = 0.024). miR-29a level was not related to sex, age, or heredity (p > 0.05). miR-29a also showed 80% specificity and 71.43% sensitivity in the diagnosis of schizophrenia.ConclusionDownregulation of miR-29a in schizophrenia is significantly related to the development of this illness. It might have the potential as a biomarker for schizophrenia.

Abstract PO3-25-03: Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？

Abstract Background: MicroRNA-125a (miR-125a) has been shown to function as tumor suppressive miRNA by pre-clinical in vitro studies. However, to the best of our knowledge, there has been no study that investigated the clinical relevance of miR-125a in breast cancer patients. We hypothesized that miR-125a expression in breast cancer associate with its aggressiveness and can be a prognostic biomarker. Material and Methods: Non-tumorigenic epithelial cell line, MCF-10A and breast cancer cell lines, MCF-7, T47D, and MDA-MB231, were used to analyze the expression levels of miR-125a as well as cell viability. The data of 2042 patients were extracted from publicly available databases, The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC), The Cancer Genome Atlas (TCGA), and GSE57897. These databases included clinicopathological and survival data as well as transcriptome data. The survival analysis, gene set enrichment analysis (GSEA) were conducted comparing miR-125a high expressing and low expressing tumors, divided by the median cutoff. The association between the miR-125a expression and tumor immune microenvironment was assessed by xCell. Results: The expression levels of miR-125a were suppressed in breast cancer cell lines, MCF-7, T47D, and MDA-MB231compared with MCF-10A. This was concordant with human samples. The expression levels of miR-125a were lower in tumors compared with normal breast tissues in both TCGA and GSE57897 cohorts. These results were in agreement with the notion that it is a tumor suppressive miRNA (p< 0.001 and p< 0.001, respectively). ER-positive/HER2-negative (ER+/HER2-) showed the highest miR-125a expression among the subtypes in TCGA (p< 0.001). MiR-125a expression was not associated with cancer staging in any of the subtypes in neither TCGA nor METABRIC cohorts. To our surprise, high miR-125a tumors demonstrated worse disease free survival (DFS), disease specific survival (DSS), and overall survival (OS) compared with low expressing in ER+/HER2- breast cancer patients (p=0.008, p=0.005, and p=0.037) which was not the case for the other subtypes in METABRIC cohort. Interestingly, miR-125a expression was associated with Nottingham histological grade only in ER+/HER2- among the subtypes (p< 0.001). Also, high miR-125a tumors significantly demonstrated higher expression of MKI67, one of the most commonly used parameters for cell proliferation, correlated with miR-125a expression in ER+/HER2- (p=0.02), but not in other subtypes. Furthermore, high miR-125a tumors enriched four out of five cell proliferation-related gene sets in Hallmark collection, such as E2F Targets, G2M Checkpoint, Mitotic Spindle, and MYC Targets V2 in ER+/HER2- subtype, but not in TNBC. This was also the case in immune-related gene sets; interferon-alpha response and interferon-gamma response that enriched to high miR-125a tumors in ER+/HER2-, but not in TNBC. Anti-tumor immune cells, CD8 cells, T-helper type 1 cells, and M1 macrophages, were all elevated in high miR-125a tumors in ER+/HER2- subtype (all p< 0.03), but none in TNBC. Also, pro-cancerous immune cell such as T-helper type 2 cells were similarly highly infiltrated with high miR-125a tumors only in ER+/HER2- subtype (p=0.026). Given its association with the survival outcome, we cannot help but speculate that miR-125a expression is parallel with highly proliferative ER+/HER2- breast cancer, but its tumor suppressive effect is not enough to improve survival outcome. Conclusion: High miR-125a levels in tumors were associated with aggressive biology and worse survival, thus it can be a candidate for a prognostic biomarker in ER+/HER2- patients. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Mai Okawa, Saki Ando, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-03.

Downregulation of miR-4284 can Inhibit the Apoptosis of Human Arterial Smooth Muscle Cells (HASMCs) in Arteriosclerosis Obliterans (ASO).

The disease arteriosclerosis obliterans (ASO) affects the lower extremities. ASO's mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). The miR-4284 is involved in several biological processes of the cardiovascular system, including VSMC proliferation, migration, and death. However, it is unknown if the miR-4284 gene is involved in the control of ASO. Furthermore, the molecular processes behind the contribution of human arterial smooth muscle cells (HASMCs), one of the most significant components of the arterial wall, to arteriosclerosis obliterans (ASO) pathogenesis remain unknown. Previously, we explored the alterations of miRNAs in the blood of ASO patients, and now we wanted to test further whether these changes also take place in the HASMCs that are responsible for the pathogenesis of ASO. The expression levels of miR-29a in arterial walls were analyzed via a real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR- 4284 on HASMCs. The Transwell system and CCK-8 detection were used to assess the migration and proliferation of HASMCs. The proportion of apoptotic cells as well as the concentrations of apoptotic signal protein production were assessed using flow cytometry. A Western blot technique was used to identify B cell lymphoma-2 (Bcl2), Bcl2-associated X protein (BAX), as well as Xlinked inhibitors of apoptosis protein (XIAP). The results showed that PCR confirmed that the qualified production or expression of miR-4284 was significantly reduced in HASMCs after they were cultured without FBS and in an atmosphere of 1% O2 + 5% CO2 + 94% N2 and that glucose had no effect on its expression. MiR- 4284 has no effect on migration and proliferation, but downregulation of miR-4284 can decrease the apoptotic rate of HASMCs, as revealed by flow cytometry. Furthermore, western blot experiments showed that the expression of BAX was low, while the expression of the other two proteins, viz., Bcl2 and XIAP, was over-expressed. We found that miR-4284 downregulation enhanced Bcl2, as well as XIAP, and decreased Bax. This shows that downregulated miR-4284 regulates apoptosis-related protein expression in HASMCs. The mechanism is not clear, and we need further study to confirm it.

Abstract 3000: Mir-146a Deficiency Differentially Regulates Sexual Dimorphism Of Abdominal Aortic Aneurysms In Mice

Background and Objective: Abdominal aortic aneurysms (AAAs) are permanent aortic dilations with 80% mortality after rupture. AAA exhibits sexual dimorphism, with males at higher risk in both mice and humans. In spite of lower incidence, women acquire aggressive aneurysm growth and rupture at smaller sizes, compared to men. Despite this oddity, mechanisms driving the sexual dimorphism of AAA have not been defined. Previously, we demonstrated that Lysyl Oxidase (LOX) inhibition abolishes sexual dimorphism of AAA in mice. Recent clinical studies showed elevated miR-146a levels, an anti-inflammatory miRNA, in plasma and AAA tissues from patients. In our preliminary studies, we found that aortic miR-146a is significantly lower in females compared to male mice. However, the contribution of miR-146a in the sexual dimorphism of AAAs is unknown. In this study, we examined the role of miR-146a in AAA sexual dimorphism in mice by administering β-aminopropionitrile (BAPN), a LOX inhibitor and Angiotensin II (AngII) concurrently. Methods and Results: 8 -10 week old male and female C57BL/6J mice that were either miR146a wild type (WT) or deficient (KO; n=18-24/per group) were infused with saline or Ang II (1,000 ng/kg/min) via subcutaneous osmotic mini-pump for 28 days. In addition, mice were co-administered with BAPN (1 mg/ml; 0.15g/kg/d in drinking water) from day 0-14. miR-146a deficiency had no effect on body weight and blood pressure in both male and female mice. AngII+BAPN-administration increased AAA in both male (82%;19/23) and female (72%;18/25) mice compared to saline+BAPN controls. Interestingly, miR-146a deficiency profoundly but significantly increased AAA in males (WT=1.5 ± 0.1 versus KO=2.3 ± 0.1 mm, P<0.05), but significantly decreased AAA in females (WT=1.5 ± 0.1 versus KO=1.2 ± 0.1 mm, P<0.05). By RNA sequencing and qPCR, we identified several differentially expressed genes in the absence of miR-146a, with RFLNA upregulated and ELAVL2 downregulated in male KO mice, and SLC38A5, CCND2, DUSP10 genes upregulated in female KO mice. Ongoing studies aim to delineate the role of these genes in miR-146a-mediated sexual dimorphism of AAA. Conclusion: These findings demonstrate that miR-146a plays a critical role in mediating sexual dimorphism of AAA in mice.

Effects of Multi-species Synbiotic Supplementation on Circulating miR-27a, miR-33a Levels, and Lipid Parameters in Adult Men with Dyslipidemia; A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

MicroRNAs (miRNAs) have emerged as important regulators of lipid metabolism. Recent studies have suggested synbiotics may modulate miRNA expression and lipid metabolism. This study aimed to investigate the effects of synbiotic supplementation on circulating miR-27a, miR-33a, and lipid parameters in patients with dyslipidemia. Fifty-six eligible participants were randomly allocated to receive either synbiotic or placebo sachets twice a day for 12 weeks. Each synbiotic sachet contained 3×1010 CFU six species of probiotic microorganisms and 5 grams of inulin and fructooligosaccharide (FOS) as prebiotics. Serum miR-27a and miR-33a expression levels, serum lipids, and apolipoproteins, the fecal concentration of short-chain fatty acids (SCFAs), and Firmicutes and Bacteroidetes phyla were assessed before and after the study. Real-time PCR was used to determine the relative expression levels of miRNAs. The results showed synbiotic supplementation significantly downregulated the expression levels of miR-27a and miR-33a compared to the placebo group (p = 0.008 and p = 0.001, respectively). Furthermore, the intervention group exhibited significant improvements in serum high-density lipoprotein (HDL-C), small dense low-density lipoprotein (sdLDL-C), apoA-I, and apoB-100 (p = 0.008, p = 0.006, p = 0.003, p = 0.001, respectively). The results showed a significant negative correlation between miR-33a expression levels with HDL-C, butyrate, propionate, and a significant positive correlation with total cholesterol (TC), low-density lipoprotein (LDL-C), and sdLDL-C in the intervention group. Fecal bacteria and SCFAs were significantly increased in the intervention group. This study provides evidence that synbiotic supplementation can modulate miR-27a and miR-33a expression and improve lipid metabolism in patients with dyslipidemia.

Dysregulation of miR-146a is associated with exacerbated inflammation, oxidative and endoplasmic reticulum stress in the progression of diabetic foot ulcer.

Recent evidence has implicated the role of microRNA-146a (miR-146a) in regulating inflammatory responses. In the present study, we investigated the role of miRNA-146a in the progression of diabetic foot ulcer (DFU) in type 2 diabetes mellitus patients (T2DM) and studied its correlation with stress mediators such as Endoplasmic Reticulum (ER) and oxidative stress. Ninety subjects were enrolled and evenly distributed among three groups: Controls (n = 30), T2DM without complications (n = 30) and T2DM with foot ulcers (n = 30). Subsequently, each group was further subdivided based on the University of Texas classification. Peripheral blood was collected from all the study subjects, while tissue biopsies were taken only from DFU patients. Total RNA from both PBMCs and wound tissues were isolated using miRNA isolation kit and qPCR was performed to check the expression of miR-146a, ER stress and oxidative stress markers. Our findings revealed a significant decrease in miR-146a expression among T2DM patients with Grade 2 and Grade 3 DFUs compared with those with Grade 0 and Grade 1 DFUs. Notably, inflammatory genes regulated by miR-146a, including TRAF6, IRAK-1 and ADAM, were all upregulated in T2DM patients with Grade 2 and Grade 3 DFUs. Moreover, reduced miR-146a levels were correlated with increased markers of ER stress and oxidative stress in Grade 2 and Grade 3 DFU patients. Furthermore, our in vitro experiment using mouse 3T3 fibroblasts demonstrated a downregulation of miR-146a following induction of hyperglycaemia, ER stress and oxidative stress in these cells. These findings suggest a potential link between diminished miR-146a expression and heightened oxidative and ER stress in T2DM patients with more severe grades of DFUs. Our results imply that targeting miR-146a may hold therapeutic promise for managing disease progression in DFU patients, as it could help alleviate oxidative and ER stress associated with diabetic complications.

Serum miR-124a and miR-34a as Potential Biomarkers for Rheumatoid Arthritis

Abstract Background: MicroRNAs (miRs) are defined as noncoding small RNAs that are involved in the regulation of various immune functions, indicating they could be possible biomarkers for immune-mediated disorders. We aimed to evaluate miR-124a and miR-34a levels in serum as diagnostic biomarkers for rheumatoid arthritis (RA) and to investigate their correlation with the disease activity in RA patients. Methods: Our study consisted of 40 patients with RA and 40 controls. The disease activity for the RA patients was evaluated using the Disease Activity Score in 28 joints (DAS28). Relative quantification of miR-124a and miR-34a expressions in serum was conducted by reverse transcriptase quantitative real-time polymerase chain reaction. Results: Expression levels of miR-124a and miR-34a in serum were significantly lower in RA patients (median: 0.64 and 0.30, respectively) compared to controls (median: 3.12 and 1.88, respectively), P < 0.001. Their serum levels were negatively associated with disease activity and inversely correlated with DAS28, C-reactive protein, and erythrocyte sedimentation rate (P < 0.001). Conclusion: Serum miR-124a and miR-34a could serve as potential noninvasive biomarkers for RA diagnosis and reflect the disease activity in RA patients.

Identification of miR-125a and miR-106b signature as a potential diagnostic biomarker in breast cancer tissues

MicroRNAs (miRNAs) have essential roles in the etiology of breast cancer and are regarded as possible markers in this malignancy. In order to find new markers for breast cancer, the current study has measured expression level of four miRNAs, namely miR-125a, miR-106b, miR-96 and miR-92a-3p in the paired breast samples. Expression levels of miR-125a and miR-106b were higher in tumoral tissues compared with control tissues (Expression ratios (95% CI) = 4.01 (1.96–8.19) and 3.9 (1.95–7.81); P values = 0.0005 and 0.0003, respectively). miR-106b and miR-125a differentiated between malignant and non-malignant tissues with AUC values of 0.7 and 0.67, respectively. We detected association between expression of miR-106b and clinical stage (P = 0.03), in a way that its expression was the lowest in the advanced stages. Finally, significant relationships were found between miR-96 and miR-125a in both tumoral and non-tumoral specimens (ρ = 0.76 and 0.69, respectively). This nonparametric measure of rank correlation also showed relationship between miR-106b and miR-96 in both sets of samples (ρ = 0.63 and 0.61, respectively). Cumulatively, the assessed miRNAs, particularly miR-125a and miR-106b are putative targets for further expression and functional assays in breast cancer.

Identification of miR-29a as a novel biomarker for lumpy skin disease virus exposure in cattle

ABSTRACT Micro RNAs (miRNAs) have been implicated in the regulation of maturation, proliferation, differentiation, and activation of immune cells. In this study, we demonstrated that miR-29a antagonizes IFN-γ production at early times post-LSDV infection in cattle. miR-29a was predicted to target upstream IFN-γ regulators, and its inhibition resulted in enhanced IFN-γ production in sensitized peripheral blood mononuclear cells (PBMCs). Further, stimulation of PBMCs with LSDV antigen exhibited lower levels of miR-29a, concomitant with a potent cell-mediated immune response (CMI), characterized by an increase in LSDV-specific CD8+ T cell counts and enhanced levels of IFN-γ, which eventually facilitated virus clearance. In addition, a few immunocompromised cattle (developed secondary LSDV infection at ~ 6 months) that failed to mount a potent cell-mediated immune response, were shown to maintain higher miR-29a levels. Furthermore, as compared to the sensitized crossbred cattle, PBMCs from sensitized Rathi (a native Indian breed) animals exhibited lower levels of miR-29a along with an increase in CD8+ T cell counts and enhanced levels of IFN-γ. Finally, we analysed that a ≥ 60% decrease in miR-29a expression levels in the PBMCs of sensitized cattle correlated with a potent CMI response. In conclusion, miR-29a expression is involved in antagonizing the IFN-γ response in LSDV-infected cattle and may serve as a novel biomarker for the acute phase of LSDV infection, as well as predicting the functionality of T cells in sensitized cattle. In addition, Rathi cattle mount a more potent CMI response against LSDV than crossbred cattle.

Abstract 2013: Extracellular vesicles transfer miR-181a to confer PARP inhibitor resistance and induce STING degradation in BRCA-mutated triple-negative breast and ovarian cancers cells

Abstract Background: Patients with BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC), and ovarian cancer (OvCa) are treated with poly (ADP-ribose) polymerase inhibitors (PARPi). BC and OvCa tumors develop similar mechanisms of resistance to platinum-based agents and PARPi. This study aimed to identify microRNAs (miRs) inducing PARPi resistance in cancer cells that can be transferred by extracellular vesicles (EV). Methods: Olaparib-resistant (OlaR) cell lines were analyzed for 2,083 miRs using HTG miRNA Whole Transcriptomic Analysis (WTA) and NGS. Functional assays were performed in BRCA1-mutated TNBC and OvCa cell lines. In-silico analysis were performed using TCGA, GTEx, CCLE, GDSC, and GEO databases. Results: The miR-181 family (including miR-181a, p=0.001) was significantly upregulated in OlaR TNBC cell lines. High miR-181a levels occurred in tumor tissues of TNBC patients (p=0.001). miR-181a binds to the stimulator of interferon genes (STING) mRNA to promote STING mRNA degradation in OvCa. We hypothesized that miR-181a downregulates STING and downstream proinflammatory cytokines, to mediate PARPi resistance. miR-181a-overexpression induced olaparib resistance and STING downregulation in BRCA1-mutated TNBC cell lines. EV isolated from OlaR TNBC cell lines horizontally transferred miR-181a to non-resistant parental cell lines, which conferred PARPi-resistance and decreased STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p=0.01). Low-IFNG response scores in HER2-negative BC patients showed a worse response to neoadjuvant treatment (p=0.001). Consistently, OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to cisplatin showed resistance to olaparib; but miR-181a knockout significantly improved olaparib sensitivity (p=0.001). Conclusions: High levels of miR-181a decrease STING pathway, correlate with IFNG scores, drive PARPi and platinum-based drug resistance. Horizontal miR-181a EVs transfer can facilitate drug resistance in cells. The miR-181a-STING axis can be used for predicting PARPi responses in TNBC and OvCa tumors. Citation Format: Matias A. Bustos, Takamichi Yokoe, Yoshiaki Shoji, Yuta Kobayashi, Shodai Mizuno, Tomohiro Murakami, Sreeja C. Sekhar, Matthew Knarr, Steven A. Vasilev, Analisa DiFeo, Ronny Drapkin, Dave S. Hoon. Extracellular vesicles transfer miR-181a to confer PARP inhibitor resistance and induce STING degradation in BRCA-mutated triple-negative breast and ovarian cancers cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2013.

The critical role and functional mechanism of microRNA-146a in doxorubicin-induced apoptosis in breast cancer cells

Background Breast cancer among women is the most frequently diagnosed cancer and the leading cause of death worldwide. There many advances in diagnosing and treating this disease, early diagnosis and treatment are still a significant challenge in the early stages. In recent years, microRNAs have attracted much attention in cancer diagnosis and treatment. However, the role of miR-146a in breast cancer is still controversial. We aimed to investigate the roles of miR-146a in apoptosis in breast cancer cells. Methods A microarray dataset from the GEO database was selected, and using the GEO2R tool, the gene expression profile of this dataset was extracted. Then, the target scan database was used to explore the miR-146a target genes. The link between the signaling pathways was collected. We used miR-146a mimic, which was transfected to the MCF-7 cells to investigate the miR-146a roles in the apoptosis. The expression levels of miR-146a and BAX, BCL-2, and p-21(most essential genes in the apoptosis) were quantified by qPCR and western blot analysis. Results Our findings indicated that doxorubicin induces miR-146a expression. In addition, overexpression of miR-146a affected MCF-7 cell viability, induced apoptosis, and led to reduced expression levels of BCL-2 and P-21, as well as increased BAX expression levels. Conclusion Considering the role of doxorubicin in inducing apoptosis and increasing the expression of miR-146a, it can be suggested that this miR is involved in inducing apoptosis in BC cells. In addition, miR-146a can be considered a therapeutic candidate.

CircABCB10 Promotes the Apoptosis and Inflammatory Response of 16HBE Cells by Cigarette Smoke Extract by Targeting miR-130a/PTEN Axis.

Chronic obstructive pulmonary disease (COPD) has become a global public health problem due to its high mortality. So there is an urgent need to find an effective treatment. The targeting relationship among circABCB10, miR-130a and PTEN was predicted by the targetscan database (TargetScanHuman 8.0, https://www.targetscan.org/vert_80/). A total of 60 patients which were from the second affiliated hospital of Qiqihar Medical University from 2022 to 2023 were enrolled. The lung condition was detected by CT (Computed Tomography). The expression levels of circABCB10, miR-130a and PTEN in lung tissues were determined by qRT-PCR. The COPD model was established by stimulating normal and silenced 16HBE cells in circABCB10 genes with cigarette smoke extract (CSE) at different concentrations. qRT-PCR was conducted for the expression levels of circABCB10, miR-130a and PTEN, WB for the expression levels of apoptotic proteins, ELISA for the content of inflammatory factors, and CCK8 for the effect of CSE on the proliferation of cells. CircABCB10 expression increased in lung tissues from patients with COPD and in 16HBE cells treated with CSE. The stimulation on cells with CSE increased the expression of inflammatory factors, while knocking down circABCB10 could reverse this response. The inflammatory response to the knockdown of circABCB10 was reversed by miR-130a inhibitor, which increased the expression of c-caspase 3. The targetscan database predicted the target factor downstream miR-130a was PTEN. Transfecting OE-PTEN reversed the inflammation of knocking down circABCB10, and increased the apoptosis and inflammation. CircABCB10 can cause the inflammatory response by targeting miR-130a/PTEN axis, which is a mechanism that may lead to the occurrence and development of COPD.

Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery.

JOURNAL/nrgr/04.03/01300535-202501000-00030/figure1/v/2024-05-14T021156Z/r/image-tiff Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery. Our previous in vitro study demonstrated that exosomes/small extracellular vesicles (sEVs) isolated from cerebral endothelial cells (CEC-sEVs) of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a (miR-27a) is an elevated miRNA in ischemic CEC-sEVs. In the present study, we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a (27a-sEVs) further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs. 27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector. Small EVs isolated from CECs transfected with a scramble vector (Scra-sEVs) were used as a control. Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs. An array of behavior assays was used to measure neurological function. Compared with treatment of ischemic stroke with Scra-sEVs, treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side, and significantly improved neurological outcomes. In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth. Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone, while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a, and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone. Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs. Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes. Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

MiR-29a-KLF4 signaling inhibits breast tumor initiation by regulating cancer stem cells

Cancer stem cells (CSCs) are known for their potent ability to drive tumor initiation and recurrence, yet the molecular mechanisms regulating CSCs are still unclear. Our study found a positive correlation between increased levels of miR-29a and better survival rates in early-stage breast cancer patients, but a negative correlation in late-stage patients, suggesting a dual function of miR-29a in regulating breast cancer. Furthermore, miR-29a showed significant downregulation in the ALDH+ breast cancer stem cell population compared to non-stem cancer cells. Overexpression of miR-29a in human breast cancer cells reduced the proportion of CSCs, suppressed their ability to form mammospheres, and inhibited the expression of stemness genes SOX2, KLF4, and hTERT in vitro. Conversely, knockdown of miR-29a in breast cancer cells showed opposite effects. Tumor xenograft experiments revealed that miR-29a overexpression significantly inhibited tumorigenesis initiated by MDA-MB-231 cell transplantation in nude mice. We further demonstrated that Krüppel-like factor 4 (KLF4), a key gene that regulates cell stemness, was a direct target of miR-29a in breast cancer cells. miR-29a suppressed the expression of KLF4 at both mRNA and protein levels. Reintroduction of KLF4 into breast cancer cells rescued the miR-29a-induced CSC suppression phenotype. In summary, our study is the first to demonstrate that miR-29a-KLF4 signaling inhibits breast tumor initiation by regulating CSCs, which provides novel therapeutic targets for preventing breast tumor initiation.

Oncogenic miR-146a-PBX2 axis, a novel leukemic target in xenograft acute lymphoblastic leukemia model

Acute lymphoblastic leukemia (ALL) is one of the major hematological malignancies both in adults and children. Bone marrow biopsy is the main requirement for its accurate diagnostic and prognostic purpose till now that is a very painful process especially for children. The reluctance to go for bone marrow biopsy leads to delayed diagnosis treatment of ALL. MicroRNAs are emerging as diagnostic and therapeutic targets for cancer including ALL using less painful methods. The current study was conducted to evaluate the potential of miR-146a-PBX2 axis as a leukemic target of ALL using ALL-induced rabbit model. A total of 8 rabbits were divided into two groups: ALL-induced (diseased) and normal (control). The whole blood was collected from both groups and plasma isolation was performed. The levels of miR-146a and PBX2 gene in plasma of diseased and control samples were quantified by the real-time PCR (RT-qPCR). The normalized fold expression was calculated using miR-16 and the reference gene GAPDH. The results showed that there was a significant increase in miR-146a expression in diseased (ALL-induced) samples as compared to the control. Moreover, a significant upregulation of PBX2 gene was found in the diseased model as compared to that in the control. The results implied that miR-146a-PBX2 axis may be utilized as a potential target for ALL.

The Role of miR-29a and miR-143 on the Anti-apoptotic MCL-1/cIAP-2 Genes Expression in EGFR Mutated Non-small Cell Lung Carcinoma Patients.

The survival rate of lung cancer is low due to the high frequency of drug resistance in patients with mutations in the driver genes. Overexpression of anti-apoptotic genes is one of the most prominent features of tumor drug resistance. EGFR signaling induces the expression of anti-apoptotic genes. Also, microRNAs (miRNAs) have a critical role in regulating biological functions such as apoptosis; a process mostly eluded in cancer progression. The mutation screening was performed on one thousand non-small cell lung carcinoma patients to enroll clinical samples in this study. Bioinformatics analysis predicted that miRNAs (miR-29a, miR-143) might regulate MCL-1 and cIAP-2 expression. We investigated the expression of MCL-1, cIAP-2, miR-29a, and miR-143 encoding genes in adenocarcinoma patients with or without EGFR mutations before treatment. The potential role of miR-29a and miR-143 on gene expression was evaluated by overexpression and luciferase assays in HEK-293T cells. EGFR mutations were found in 262 patients (26.2%) with a greater incidence in females (36.23% vs. 20.37%, P = 0.001). The expression levels of MCL-1 and cIAP-2 genes in patients with mutated EGFR were higher than those of wild-type EGFR. In contrast, compared to those of patients with wild-type EGFR, the expression levels of miR-29a and miR-143 were lower in the patients carrying EGFR mutations. In cell culture, overexpression of miR-29a and miR-143 significantly downregulated the expression of MCL-1 and cIAP-2. Dual-luciferase reporter experiments confirmed that miR-29a and miR-143 target MCL-1 and cIAP-2 mRNAs, respectively. Our results suggest that upregulation of EGFR signaling in lung cancer cells may increase anti-apoptotic MCL-1 and cIAP-2 gene expression, possibly through downregulation of miR-29a-3p and miR-143-3p.