miR-29b Mimic Research Articles

Downregulation of microRNA-29b in cancer and fibrosis: molecular insights and clinical implications

MicroRNA-29b (miR-29b) is known for its therapeutic potential as an antifibrotic and anticancer agent. In fibrotic conditions, miR-29b inhibits fibrogenesis by downregulating crucial regulators such as collagens, extracellular matrix proteins and the transforming growth factor-β pathway. Similarly, in cancer, it acts as a tumor suppressor by downregulating various oncogenes and signaling pathways involved in cancer progression, such as Wnt-β-catenin, p38-mitogen-activated protein kinase and nuclear factor-κB. However, the upregulation of these pathways suppresses miR-29b, contributing to fibrosis and cancer development. Preclinical research and clinical trials have shown that delivering exogenous miR-29b mimics can restore its expression, attenuating tumorigenesis and fibrogenesis. This review discusses miR-29b's potential and its possible therapeutic development for cancer and fibrotic disorders.

MiR-29b inhibits COC expansion and oocyte in vitro maturation via induction of ROS by targeting CYCS

Cumulus-oocyte complex (COC) expansion and oocyte maturation are crucial processes for embryo development and fertility across species. Although miR-29b has been detected in porcine ovarian granulosa cells, its specific role in regulating oocyte maturation remains largely unknown. In this study, using the pig as a model, we report that over-expression of miR-29b lead to a decrease of COC expansion area and inhibits oocyte maturation (P<0.05). This suppression correlated with a decrease expression of COC-expansion-associated genes, including SHAS2, ADAMTS1, ADAMTS2, ADAMTS17 and PTX 3 in both mural granulosa cells (mGCs) and cumulus granulosa cells (cGCs). Further investigation revealed that miR-29b over-expression induces reactive oxygen species (ROS) accumulation in both mGCs and cGCs, conversely, knock-down of miR-29b reverses all these effects. Treatment with the antioxidant β-mercaptoethanol alleviates ROS accumulation, rescues COC expansion and restores oocyte polar body formation impaired by miR-29b mimics. Computational analysis predicted CYCS, the gene encoding cytochrome C, as a potential target of miR-29b. Subsequent examination demonstrated that miR-29b downregulates CYCS at both mRNA and protein levels. Dual-luciferase reporter assays further confirmed that miR-29b interacts with the 3’-untranslated region (3’UTR) of CYCS. Over-expression of CYCS decreases ROS accumulation and promotes COC expansion (P<0.05). These results indicate that miR-29b regulates COC expansion and oocyte maturation in vitro by inducing ROS, likely through targeting of CYCS. This study sheds light on the role of miR-29b in oocyte maturation and provides insight into the regulatory function of miRNAs in ovarian physiology.

Ling-gui-zhu-gan promotes adipocytes browning via targeting the miR-27b/PRDM16 pathway in 3T3-L1 cells.

Introduction: Obesity, a global epidemic, is caused by an imbalance between energy intake and expenditure. The induction of white adipose browning to increase heat production has emerged as a potential effective strategy to address obesity. Ling-gui-zhu-gan (LGZG), a traditional Chinese medicine formula, has been proved to achieve promising results to combat obesity and related metabolic diseases, yet the mechanisms remain largely unexplored. This study aimed to elucidate the anti-obesity properties and the mechanisms of LGZG by investigating its browning effect on 3T3-L1 adipocytes. Methods: LGZG-containing serum obtained by oral administration of LGZG to animals was added to 3T3-L1 adipocytes to simulate in vivo conditions. Results: The results showed that 49 compounds were identified in LGZG-containing serum by UHPLC-Q-Orbitrap HRMS, including compounds such as atractylenolides and polyporenic acid C, etc. LGZG-containing serum alleviated the lipid accumulation and decreased both intracellular and extracellular triglyceride contents in a dose-dependent manner. This reduction is accompanied by enhanced mitochondrial respiratory and heat production function. Mechanistically, LGZG-containing serum led to a decrease in miR-27b expression and an increase in the mRNA and protein levels of browning-related markers, including UCP1, PRDM16, PGC-1α, PPARγ, CTBP1, and CTBP2. Further investigation using miR-27b mimic transfection confirmed that miR-27b/PRDM16 pathway might be a potential mechanism by which LGZG-containing serum promotes browning of 3T3-L1 adipocytes. Discussion: These results underscore the therapeutic potential of LGZG in addressing obesity and its associated metabolic disorders through the promotion of adipose browning.

Exogenous miR-29b Reduces DNA Methylation and Apoptosis in Transgenic Cells

Background: MicroRNA-29b (miR-29b) is a 22-base pair long nucleotide, that post-transcriptionally affects the processes like proliferation, differentiation and apoptosis by changes in DNA methyl transferes expressions (DNMTs family gene), which regulate DNA methylation. Methods: This study aimed to investigate the effect of miR-29b mimic and inhibitor on buffalo fibroblast cells containing the human insulin (hINS) gene. Transgenic buffalo cell line containing (hINS) gene was treated with 40 nM miR-29b mimic and inhibitor by lipofectamin-3000. Result: miRNA-29b mimic treated cells showed a significant decrease (P less than 0.05) in expression of DNMT1, DNMT3A, DNMT3B genes but did not find any significant change (P less than 0.05) in HDAC1 expression as compared to control. Whereas, miR-29b inhibitor-treated cells revealed significantly increased (P less than 0.05) the expression of DNMT1, DNMT3A, DNMT3B and HDAC1 genes as compared to control. These miR-29b mimic-treated cells had significantly increased (P less than 0.05) expression of BCL-XL and MCL-1 whereas miR-29b inhibitor-treated cells showed a significantly decreased (P less than 0.05) expression of MCL-1 while BCL-XL showed no significant (P less than 0.05) change in expression. These findings indicate that treatment of miR-29b mimic on transgenic cells reduces the DNA methylation which helps in the reduction of apoptosis levels in cells. miR-29b mimic reduces the methylation status and help in nuclear reprogramming of transgenic cells. Thus in future, these cells can be a better choice as a nuclear donor for efficient animal cloning.

Posttranscriptional Modification to Modulate Progenitor Differentiation on Heterotypic Spheroids.

Cell aggregates are widely used to study heterotypic cellular interactions during the development of vascularization in vitro. In this study, we examined heterotypic cellular spheroids made of adipose-derived stem cells and CD34+/CD31- endothelial progenitor cells induced by the transfection of miR-148b mimic for de novo induction of osteogenic differentiation and miR-210 mimic for de novo induction of endotheliogenesis, respectively. The effect of the microRNA (miRs) mimic treatment group and induction time on codifferentiation was assessed in spheroids formed of transfected cells over the course of a 4-week culture. Based on gene and protein markers of osteogenic and endotheliogenic differentiation, as well as mineralization assays, our results showed that miRs directed cell differentiation and that progenitor maturity influenced the development of heterotypic cellular regions in aggregates. Overall, the success of coculture to create a prevascularized bone model is dependent on a number of factors, particularly the induction time of differentiation before combining the multiple cell types in aggregates. The approach that has been proposed could be valuable in creating vascularized bone tissue by employing spheroids as the building blocks of more complex issues through the use of cutting-edge methods such as 3D bioprinting.

#2641 MiR-200b induces ZEB1 mediated upregulation of Matriptase and affects expression of podocin

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and a predominant reason for renal failure. Evidence from previous research indicates that abnormal expression of microRNAs (miRs) links closely to the occurrence and progression of DKD. Methods Expression of miRs in urine samples from DKD patients and in cultured renal cells stressed with transforming growth factor beta 1 (TGF-β1) was evaluated by TaqMan assay. The emerging miR of interest and its downstream targets were analyzed in human renal sections, in cultured human podocytes and in zebrafish. Results Cell free urine screening showed increased abundance of miR-200b in samples from patients with DKD when compared to healthy controls. In situ hybridization confirmed expression of miR-200b in kidney specimens from DKD patients, but was not detected in healthy renal parenchyma. Transcriptome profiling of cultured human podocytes overexpressing miR-200b identified this miR as a regulator of the ZEB1 mediated matriptase (ST14)/ podocin pathway and found TGF- β1 stress to be upstream activators of miR-200b expression in these cells. Immunofluorescence analysis of kidney sections from DKD patients showed increased expression of ST14 and decreased expression of podocin when compared to healthy controls. Furthermore, injection of a miR-200b mimic in zebrafish larvae caused an upregulation of ST14 and was accompanied by phenotypic changes such as edema, proteinuria and podocyte effacement. Conclusion Our results show that miR-200b is expressed in glomeruli and found at elevated levels in urine of DKD patients. Overexpression of miR-200b in cultured podocytes and in zebrafish induces upregulation of matriptase, a serine protease previously shown to cleave podocin. The observed increase in matriptase and decrease in podocin levels in glomeruli from DKD patients, together with the effect of a miR-200b mimic on proteinuria and ultrastructure of the pronephros in zebrafish positions miR-200b as a key regulator that modulates matriptase overactivation in DKD and warrants further studies.

Effect of miR-181b Treatment on Senescent Human Aortic Smooth Muscle Cells

Large Artery Stiffness (LAS) is independently associated with increased cardiovascular morbidity and mortality and its development is multifactorial and has been associated with the onset of cellular senescence (CS). CS is defined as a stable cell cycle arrest, characterized by an altered cell phenotype with impaired function and detrimental secretory profile. Vascular smooth muscle cells (VSMCs) are present in two main phenotypes: the contractile phenotype seen in young populations, and the synthetic phenotype related to aging and senescent VSMCs. Micro-RNAs (miR) are non-coding RNAs that can alter protein expression. miR-181b has been demonstrated to undergo downregulation with aging with a close to 80% decrease, this correlates with an upregulation of markers of senescence. To test the hypothesis that miR-181b could modulate the senescent phenotype in VSMCs and promote a more contractile phenotype, a cellular model comprising young and old human aortic smooth muscle cells (HASMCs) was employed. These cells were transfected with either a miR-181b mimic or a scrambled miR control (SCR), resulting in an 11-fold increase in the young and a 3.3-fold increase in the old. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology, where it was found to be lower in older HASMCs (1.05±0.001) compared to the young (1.1±0.001, p&lt;0.05). When miR-181b was introduced, there was a significant increase in proliferation for both age groups by 0.03-fold compared to SCR (p&lt;0.05). Old HASMCs demonstrated a decreased average ionomycin-induced contractile response (3.65%±0.46) when compared to the young (7.17%±0.60, p&lt;0.05). Lipofectamine-mediated transfection of HASMCs with miR-181b resulted in an increased contractility response in the old HASMCs (5.46%±0.91) compared to scrambled control (1.41%±0.39, p&lt;0.05), while there was no significant difference for the young cells. The percentage of Senescence-Associated-β-galactosidase (Saβ-gal) positive cells, a marker of senescence, was higher in old (39.74%±3.04) vs young (4.35%±0.72, p&lt;0.05). The percentage of Saβ-gal positive cells tended to decrease in old VSMCs after miR-181b transfection (SCR 79.65%±4.38 vs Old181b 70.39±4.03, p=0.2.02), with no effect on young cells. Immunofluorescence in situ hybridization (IF-FISH) staining for the double-stranded DNA break marker (53BP1) was lower in the young (4.65%) compared to old HASMCs (32.69%). Likewise, the percentage of cells with one or more 53BP1 foci colocalization in telomeres, referred to as Telomere dysfunction-induced foci (TIF), was higher in old (11.54%) compared to young (2.33%) VSMCs (p=0.002). However, miR-181b transfection did not impact these markers regardless of age (p=0.94). Transfection of HASMCs with miR-181b did not impact senescent cell abundance, but improved contractility and cellular proliferation. Combined with our prior data demonstrating a senomorphic effect of miR-181b transfection to improve cellular stiffness and favorably modulate of senescence-associated secretory factors, our results demonstrate an enhancement in the phenotypic characteristics of the old HASMCs group, suggesting that miR-181b participates in the modulation of the senescent VSMCs phenotype. This translates to a potential improvement in HASMC health that could reduce the development of LAS. National Institutes of Health Awards: K08 AG070281 (ET), T32 HL139451 (DM), R01 AG048366 (LAL), R01 AG060395 (AJD), and Western Institute for Veterans Research (ET). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes

BackgroundActivation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies.MethodsThis study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads.ResultsWe show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival.ConclusionsAltogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

Upregulated miR-125b mitigates inflammation, astrocyte activation, and dysfunction of spinal cord injury by inactivating the MAPK pathway.

Since the abnormal expression of miR-125b in spinal cord injury (SCI) and the regulatory effect of miR-125b on the MAPK pathway have been expounded, we attempt to investigate whether miR-125b exerts a regulatory effect on SCI by modulating the MAPK pathway. A SCI rat model was established. The rats were treated with miR-125b antagomir or agomir, and their motor function affected by miR-125b was further detected by Basso-Beattie-Bresnahan (BBB) scoring. The histopathological changes and neuronal loss in the spinal cord were evaluated using hematoxylin-eosin and Nissl staining. Microglia-conditioned medium (MCM) was prepared and further used to treat the astrocytes, the activation of which was evaluated via immunofluorescence staining. The expressions of miR-125b, inflammation-related factors (IL-6, IL-1β, TNF-α, and IL-10), and MAPK pathway-related proteins (p38, ERK1/2, and JNK1/2 as well as their phosphorylated (p) forms) in the spinal cord, serum, and MCM-treated astrocytes of rats were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay, and Western blot. MiR-125b was lowly expressed in SCI-modeled rats. MiR-125b downregulation aggravated the impaired motor function, the disorder within the tissue, astrocyte activation, and neuron loss in the spinal cord tissues of SCI-modeled rats, while miR-125b upregulation did oppositely. MiR-125b downregulation enhanced the levels of IL-6, IL-1β, TNF-α, p38, p-p38, p-ERK1/2, and p-JNK1/2, whilst reducing that of IL-10. Contrarily, miR-125b upregulation exerted the opposite effects in SCI-modeled rats and MCM-treated astrocytes. Up-regulation of miR-125b mitigates inflammation, astrocyte activation, and dysfunction in SCI by inactivating the MAPK pathway.

MICRORNAS 125B, 199A-5P, AND 214 AS MODULATORS OF BONE HOMEOSTASIS

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Enhancing wound healing and overcoming cisplatin resistance in ovarian cancer.

Ovarian cancer (OC) poses significant oncological challenges, notably impaired wound healing in the context of cisplatin (DDP) resistance. This study investigates the role of miR-200b in OC, emphasizing its impact on wound healing processes through DNMT3A/TGF-β1 pathway. The primary aim was to explore how miR-200b regulates autophagy and its consequential effects on wound healing in OC, alongside its influence on cisplatin resistance. Utilizing DDP-sensitive (A2780) and resistant (A2780/DDP) OC cell lines, along with human fibroblast cultures, the study employed an array of in vitro techniques. These included cell transfection with miR-200b mimic or inhibitor, chromatin immunoprecipitation (ChIP), dual-luciferase reporter (DLR) assays, quantitative PCR, Western blotting, MTT and particularly, wound healing assays. The research highlighted the role of miR-200b in wound healing within OC. Inhibition of miR-200b in A2780 cells and its mimic in A2780/DDP cells affected cell viability, indicating the link with DDP resistance. Crucially, miR-200b mimic significantly delayed fibroblast-mediated wound closure in assays, underscoring its impact on wound healing. Bioinformatics analysis and subsequent DLR assays confirmed miR-200b's interaction with DNMT3A, affecting TGF-β1 expression, the key factor in wound repair. Further, ChIP, quantitative PCR and Western blot analyses validated the interaction and expression changes in DNMT3A and TGF-β1. The study demonstrated that miR-200b played a pivotal role in OC by modulating autophagy, which in turn significantly affected wound healing through the DNMT3A/TGF-β1 pathway.

Ultrasound-Induced Microbubble Cavitation for Targeted Delivery of MiR-29b Mimic to Treat Cardiac Fibrosis.

Cardiac fibrosis contributes to adverse ventricular remodeling and is associated with loss of miR-29b. Overexpression of miR-29b via plasmid or intravenous injection of microRNA mimic has blunted fibrosis, but these are inefficient and non-targeted delivery strategies. In this study, we tested the hypothesis that delivery of microRNA-29b (miR-29b) using ultrasound-targeted microbubble cavitation (UTMC) of miR-29b-loaded microbubbles would attenuate cardiac fibrosis and preserve left ventricular (LV) function. Lipid microbubbles were loaded with miR-29b mimic (miR-29b-MB) or negative control (NC) mimic (NC-MB), placed with cardiac fibroblasts (CFs) and treated with pulsed ultrasound. Cells were harvested to measure downstream fibrotic mediators. Mice received angiotensin II (ANG II) infusion causing afterload increase and direct ANG II-induced cardiac fibrosis. UTMC of miRNA-loaded microbubbles was administered to the heart at days 0, 3 and 7. Serial echocardiography was performed, and hearts were harvested on day 10. UTMC treatment of CFs with miR-29b-MB increased miR-29b and decreased fibrotic transcripts compared with NC-MB treatment. In vivo UTMC+NC-MB led to increased LV mass, reduction in cardiac function and increase in fibrotic markers, demonstrating ANGI II-induced adverse cardiac remodeling. Mice treated with UTMC+miR-29b-MB had preservation of cardiac function, downregulation of cardiac fibrillin and trends of lower COL1A1, COL1A2 and COL3 mRNA and decreased cardiac α-smooth muscle protein. UTMC-mediated delivery of miR-29b mimic blunts expression of fibrosis markers and preserves LV function in ANG II-induced cardiac fibrosis.

Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway

Objective: To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance. Methods: Drug-resistant colon cancer cell line (named as HCT-8-7T cells) was established and transplanted into immunodeficient mice. The metastasis in vivo were observed. Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay, respectively. Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays. The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition (EMT) were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR), respectively. Results: In comparison with the mice transplanted with HCT-8 cells, which were survival with limited metastatic tumor cells in organs, aggressive metastases were observed in liver, lung, kidney and ovary of HCT-8-7T transplanted mice (P<0.05). The levels of ATP [(0.10±0.01) mmol/L], glycolysis [(81.77±8.21) mpH/min] and the capacity of glycolysis [(55.50±3.48) mpH/min] in HCT-8-7T cells were higher than those of HCT-8 cells [(0.04±0.01) mmol/L, (27.77±2.55) mpH/min and(14.00±1.19) mpH/min, respectively, P<0.05]. Meanwhile, the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells (P<0.05). However, the level of miRNA-125b (2.21±0.12) in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells (1.00±0.00, P<0.001). In HCT-8-7T cells, forced-expression of p53 reduced the colon number (162.00±24.00) and the migration [(18.53±5.67)%] as compared with those in cells transfected with control vector [274.70±40.50 and (100.00±29.06)%, P<0.05, respectively]. Similarly, miR-125b mimic decreased the glycolysis [(25.28±9.51) mpH/min] in HCT-8-7T cells as compared with that [(54.38±12.70)mpH/min, P=0.003] in HCT-8-7T cells transfected with control. Meanwhile, in comparison with control transfected HCT-8-7T cells, miR-125b mimic also significantly led to an increase in the levels of p53 and β-catenin, in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells (P<0.05). Conclusions: Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance. Targeting miR-125b could be a strategy to overcome Alisertib resistance.

MiR-200b/MYBL2/CDK1 suppresses proliferation and induces senescence through cell cycle arrest in ovine granulosa cells

Normal growth of granulosa cells (GCs) is essential for follicular development. miR-200b plays a vital role in litter size, estrous cycle, ovulation, and follicular development in sheep. However, it is unclear that the specific effect and regulatory mechanism of miR-200b on ovine GCs. miR-200b mimic inhibited GCs proliferation and induced cellular senescence through downregulating mitochondrial membrane potential (MMP), concentration of ATP and mitochondrial respiratory chain complex Ⅰ, and upregulating SA-β-gal positive rate and ROS production. A total of 597 differentially expressed genes were identified by RNA-Seq in GCs transfected with miR-200b mimic and mimic NC, and they were involved in cell cycle and cellular senescence. miR-200b directly targeted and downregulated MYBL2 and CDK1. Overexpression of MYBL2 promoted GCs proliferation and genes expression (CDK1, CDC20, MAD2L1 and FOXM1), which were suppressed by miR-200b mimic. Furthermore, MYBL2 negatively regulated miR-200b-induced GC senescence. In conclusion, miR-200b/MYBL2/CDK1 regulated proliferation and senescence through cell cycle pathway in ovine granulosa cells. Our study provides a novel insight that miR-200b regulates ovine follicular development.

Abstract 4749: Effects of miR-34a-5p and miR-181b-5p silencing and induction on their potential targets in uterine leiomyosarcoma cells

Abstract Background: Leiomyosarcoma (LMS) is a malignant tumor of the uterine body that represents 40-60% of all gynecological sarcomas. Up to now, there is no specific treatment or diagnostic biomarkers for this tumor. The identification and characterization of miRNAs signature could help to its management. In addition, miRNAs represent targets for therapies due to their ability to modulate various signaling pathways simultaneously. Thus, the present study aimed to evaluate the effects of genetic manipulation of miR-34a and miR-181b on their predicted target genes in LMS cells. Methods: SK-UT-1 cells from uterine LMS were transfected with synthetic miRNA mimics and inhibitors (siRNA) of miR-34a and miR-181b. The efficacy of transfection was assessed by miRNA expression (qRT-PCR) assays. In order to identify potential genetic interactions network of miR-34a and 181b, predicted target genes were selected by in silico analysis (miRTargetLink 2.0 and miRDB). After SK-UT-1 cells transfection, the expression of target genes was evaluated by qRT-PCR. Results: SK-UT-1 cells showed greater efficiency of mimics and siRNA transfections after 48h treatment for both miR-34a and 181b. MDM4, TP53, BCL2, KMT2D, NOTCH2, and CCND1 were identified as predicted target genes of miR-34a; and the TIMP3, FGFR1, BCL2, NOTCH2, ATM, and IRS1 target genes for miR-181b. Expression profile of selected target genes was assessed after each treatment. The miR-34a mimic significantly increased the expression of MDM4 after 96h of treatment, while the BCL2 and TP53 showed an increase in expression with siRNA after 48h of treatment. CCND1 showed a greater expression in both treatments at 48h. An increase in KMT2D and NOTCH2 expression was also observed after treatment, even though without statistically significant. Treatment with miR-181b mimic led to increased FGFR1 expression within 72h. Although some expression alterations were observed in TIMP3, BCL2, NOTCH2, ATM, and IRS1 genes after mimic and siRNA treatments, there was no statistical significance. Conclusion: The effect of miR-34a upregulation leads to induction MDM4 and CCND1 expression, the first being a TP53 inhibitor and the second, responsible for cell cycle maintenance. The miR-34a downregulation had an inductor effect on expression of MDM4, BCL2 (apoptosis regulator), TP53 (tumor suppressor), and CCND1. The miR-181b upregulation induced the expression of fibroblast growth factor receptor, FGFR1. In uterine LMS cells, miR-34a seems to exert an important role in regulating the expression of cell cycle and apoptosis-related genes. Citation Format: Bruna C. de Almeida, Laura G. dos Anjos, Katia C. Carvalho. Effects of miR-34a-5p and miR-181b-5p silencing and induction on their potential targets in uterine leiomyosarcoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4749.

MiR-27b attenuates mitochondrial oxidative stress and inflammation in endothelial cells

MiR-27b is highly expressed in endothelial cells (EC) but its function in this context is poorly characterized. This study aims to investigate the effect of miR-27b on inflammatory pathways, cell cycle, apoptosis, and mitochondrial oxidative imbalances in immortalized human aortic endothelial cells (teloHAEC), human umbilical vein endothelial cells (HUVEC), and human coronary artery endothelial cells (HCAEC) exposed to TNF-α. Treatment with TNF-α downregulates the expression of miR-27b in all EC lines, promotes the activation of inflammatory pathways, induces mitochondrial alteration and reactive oxygen species accumulation, fostering the induction of intrinsic apoptosis. Moreover, miR-27b mimic counteracts the TNF-α-related cytotoxicity and inflammation, as well as cell cycle arrest and caspase-3-dependent apoptosis, restoring mitochondria redox state, function, and membrane polarization. Mechanistically, hsa-miR-27b-3p targets the 3′untranslated regions of FOXO1 mRNA to downregulate its expression, blunting the activation of the Akt/FOXO1 pathway. Here, we show that miR-27b is involved in the regulation of a broad range of functionally intertwined phenomena in EC, suggesting its key role in mitigating mithochondrial oxidative stress and inflammation, most likely through targeting of FOXO1. Overall, results reveal for the first time that miR-27b could represent a possible target for future therapies aimed at improving endothelial health.

MicroRNA-125b mediates Interferon-γ-induced downregulation of the vitaminD receptor in systemic lupus erythematosus.

The inflammatory factor interferon (IFN)-γ is related to the occurrence and development of systemic lupus erythematosus (SLE). The vitaminD receptor (VDR) has an anti-inflammatory effect and its downregulation is involved in the onset of SLE. Our previous studies have confirmed that the expression of VDR in SLE peripheral blood mononuclear cells (PBMCs) is downregulated, which is negatively correlated with disease activity and inflammation. However, the mechanism underlying VDR downregulation in SLE is unknown. Based on the results of computer simulation analysis, the expression of VDR and four microRNAs (miR-17-3p, miR-34a, miR-346, and miR-125b) in SLE PBMC cells was analyzed under proinflammatory cytokine IFN‑γ treatment, and miR-125b was identified as the target miRNA. The relationship between IFN‑γ, miR-125b, and VDR was further assessed in THP‑1 cells. We showed that IFN‑γ inhibited the expression of VDR and miR-125b. Further study revealed that VDR mRNA was positively correlated with miR-125b in THP‑1 cells after IFN‑γ intervention. After transfection of miR-125b mimic or inhibitor, the expression of VDR in the miR-125b inhibitor group was lower than in the control group and miR-125b mimic group, while expression in the control group was lower than in miR-125b mimic group. Transfection of miR-125b inhibitor into THP‑1 cells could further promote the ability of IFN‑γ to inhibit VDR. The decrease in VDR expression promotes development of inflammation and SLE. These data suggest that miR-125b may mediate inflammatory factor IFN-γ-induced downregulation of VDR in the pathogenesis of SLE.

Lysosome-related exosome secretion mediated by miR-26b / Rab31 pathway was associated with the proliferation and migration of MCF-7 cells treated with BPA

Bisphenol A (BPA), one of the typical environmental endocrine disruptors (EEDs), can promote the proliferation and migration of cancer cells, but the mechanism of which remains largely unclear. Exosome secretion plays an important role in the stress response of cells to environmental stimuli. This study was designed to explore whether exosome secretion was involved in the toxic effect of BPA on the proliferation and migration of MCF-7 cells, and the related mechanism. Our data shows that the IC50 value of MCF-7 exposure to BPA was about 65.82 µM. The exposure of MCF‐7 to 10 µM BPA resulted in a decreased miR-26b expression and the activation of miR-26b/Rab-31 pathway, consequently, the number and activity of lysosomes decreased, the secretion of exosomes increased, cell proliferation and migration were enhanced obviously. Interestingly, miR-26b mimic up-regulated the number and activity of lysosomes via miR-26b/miR-31 pathway, exosome secretion was down-regulated, cell proliferation and migration decreased. Further, when GW4869 was used to directly inhibit the exosome secretion of MCF-7 treated with BPA, their proliferation and migration were down-regulated. Herein, we concluded that the stimulating effect of BPA on the proliferation and migration of MCF-7 cells was associated with the lysosome - related exosome secretion via miR-26b / Rab31 pathway.

Involvement of microRNA miR-125b in the control of porcine ovarian cell functions

The existing knowledge of the involvement of miR-125b in the control of ovarian functions is insufficient. To evaluate the role of miR-125b in the control of basic porcine ovarian granulosa cell functions, we examined the upregulation (using miR-125b mimics) and downregulation (using miR-125b inhibitor) of this miR-125b. Expression levels of miR-125b, viability, proliferation (expression and accumulation of PCNA and cyclin B1), the proportion of proliferative active cells, apoptosis (expression and accumulation of bax and caspase 3), the proportion of cells containing DNA fragmentation, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analysed by RT-qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT and TUNEL assays, and ELISA. Transfection of cells with miR-125b mimics decreased cell viability, proliferation, and the release of progesterone, testosterone, estradiol, and oxytocin, but stimulated apoptosis and prostaglandin E2 output. Transfection of cells with miR-125b inhibitor had the opposite effect. Moreover, it prevented the effects of miR-125b mimics. Our observations suggest that miR-125b is a potent physiological inhibitor of granulosa ovarian cell functions – cell cycle, apoptosis, and secretory activity.

MiRNA29B Induces Fetal Hemoglobin through Targeting DNMT3 and MYB in Vitro and In Vivo in Preclinical Townes Sickle Cell Mice

Introduction: Therapies aimed at inducing fetal hemoglobin (HbF) expression is an effective approach for ameliorating the clinical severity of sickle cell disease (SCD) in adults and children, however drug treatment options are limited. DNA methyltransferase (DNMT) inhibitors tested in clinical trials induce HbF expression by producing γ-globin gene promoter DNA hypomethylation. Alternative approaches to increase HbF involve development of small non-coding microRNAs (miR) to silence major repressors of γ-globin gene transcription. Studies from our group demonstrated that miR29b inhibits DNA methylation through binding the 3' UTR of DNMT3 genes and is associated with high HbF levels in SCD patients. Studies performed in KU812 cells showed concomitant DNMT3 silencing along with ү-globin gene activation and HbF induction. Moreover, miR29b decreased expression of the γ-globin repressor protein MYB in normal adult CD34+ stem cells. Therefore, we tested the hypothesis that miR29b activates γ-globin and HbF expression via DNA hypomethylation and MYB gene silencing in normal erythroid progenitors and in Townes SCD mice. Methods: Human KU812 were cultured and normal CD34+ cells (n=3) were differentiated using a two-phase erythropoiesis culturing system. KU812 cells were co-transfected with the MYB luciferase reporter plasmid and the effects of miR29b (50 nM and 100 nM) overexpression or negative Scrambled (Scr) mimic on luciferase activity was quantified after 48 hours. The effect of MYB knockdown on ү-globin activation and HbF induction in KU812 cells using a retroviral vector encoding a MYB shRNA was also assessed. Subsequent studies were conducted in CD34 cells electroporated with miR29b mimic (50nM and 100nM) or negative Scrambled (Scr) control on day 8 and collected after 24 hours. Erythroid differentiation was assessed using Giemsa staining, and flow cytometry was used to measure the percentage of HbF positive cells (F-cells). Real-time PCR quantified expression of miR29b, γ-globin, and β-globin gene expression, and Western blot measured DNMT, HbF and HbA protein. In vivo studies were performed in Townes SCD mice (6 per group), treated by 28-day continuous infusion using subcutaneous mini-osmotic pumps. The treatment groups consisted of miR29b (2, 3, and 4 mg/kg/day) and corresponding doses of Scr control. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes percent by flow cytometry. Gene expression was determined by real-time PCR. Results: Co-transfection of miR29b and a luciferase reporter plasmid containing the MYB 3'-UTR into KU812 cells resulted in a >90% decrease in luciferase activity at both miR29b concentrations. MYB knockdown by shRNA significantly increased the ү/ү+β ratio and miR29b expression in KU812 cells and normal CD34 cells. We observed efficient erythroid differentiation at day 7, day 12, and day 14 of cell culture assessed by Giemsa stain. Treatment with miR29b increased the ү/ү+β mRNA ratio by 2.5-fold (p<0.01) and F-cell levels increased from ~6.0% (Scr) to 15% with miR29b (100 nM); we observed lower DNMT3 mRNA and protein levels in cells transfected with 100 nM miR29b. Analysis of HbF induction by miR29b revealed >85% increase in F-cells by flow cytometry in normal erythroid progenitors as compared to Scr cells. Subsequent in vivo studies were conducted with Townes SCD mice treated with miR29b for 28 days; treatment was well tolerated with good weight gain and no deaths. There was minimal effects on hematopoiesis with a decrease in total white blood count and increase in neutrophils. Hemoglobin, reticulocyte and platelet counts remained stable. By week 4, we observed a dose-dependent 2.6-fold, 2.8-fold, and 3.0-fold (p<0.05) increase in F-cells at the respective miR29b doses compared to Scr control. We also observed a significant decrease in the percent of sickle cells on peripheral smear (p<0.05) mediated by miR29b with 20% inhibition (p<0.05) at week 4. MiR29b significantly decreased DNMT3 mRNA expression at week 2 and MYB mRNA at week 4 with a concomitant increase in the ү/ү+β mRNA ratio at weeks 2 and 4. Conclusions: Our findings support the ability of miR-29b to induce HbF in sickle erythroid progenitors without significant toxicity in vitro and SCD mice in vivo. This research highlights a novel miRNA-based epigenetic approach to induce HbF supporting discovery of new drugs to expand treatment options for SCD.