MicroRNA-29b-3p degenerates terminally differentiated dopaminergic SH-SY5Y cells by perturbation of mitochondrial functions.

Mitochondrial dysfunction is the main cause of gradual deterioration of structure and function of neuronal cells, eventually resulting in neurodegeneration. Studies have revealed a complex interrelationship between neurotoxicant exposure, mitochondrial dysfunction, and neurodegenerative diseases. Alteration in the expression of microRNAs (miRNAs) has also been linked with disruption in mitochondrial homeostasis and bioenergetics. In our recent research (Cellular and Molecular Neurobiology (2023) https://doi.org/10.1007/s10571-023-01362-4), we have identified miR-29b-3p as one of the most significantly up-regulated miRNAs in the blood of Parkinson's patients. The findings of the present study revealed that neurotoxicants of two different natures, that is, arsenic or rotenone, dramatically increased miR-29b-3p expression (18.63-fold and 12.85-fold, respectively) in differentiated dopaminergic SH-SY5Y cells. This dysregulation of miR-29b-3p intricately modulated mitochondrial morphology, induced oxidative stress, and perturbed mitochondrial membrane potential, collectively contributing to the degeneration of dopaminergic cells. Additionally, using assays for mitochondrial bioenergetics in live and differentiated SH-SY5Y cells, a reduction in oxygen consumption rate (OCR), maximal respiration, basal respiration, and non-mitochondrial respiration was observed in cells transfected with mimics of miR-29b-3p. Inhibition of miR-29b-3p by transfecting inhibitor of miR-29b-3p prior to exposure to neurotoxicants significantly restored OCR and other respiration parameters. Furthermore, we observed that induction of miR-29b-3p activates neuronal apoptosis via sirtuin-1(SIRT-1)/YinYang-1(YY-1)/peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)-regulated Bcl-2 interacting protein 3-like-dependent mechanism. Collectively, our studies have shown the role of miR-29b-3p in dysregulation of mitochondrial bioenergetics during degeneration of dopaminergic neurons via regulating SIRT-1/YY-1/PGC-1α axis.

Laminar shear stress alleviates monocyte adhesion and atherosclerosis development via miR-29b-3p/CX3CL1 axis regulation.

Laminar shear stress (Lss) is an important anti-atherosclerosis (anti-AS) factor, but its mechanism network is not clear. Therefore, this study aimed to identify how Lss acts against AS formation from a new perspective. In this study, we analyzed high-throughput sequencing data from static and Lss-treated human aortic and human umbilical vein endothelial cells (HAECs and HUVECs, respectively) and found that the expression of CX3CL1, which is a target gene closely related to AS development, was lower in the Lss group. Lss alleviated the inflammatory response in TNF-α (also known as TNF)-activated HAECs by regulating the miR-29b-3p/CX3CL1 axis, and this was achieved by blocking nuclear factor (NF)-κB signaling. In complementary in vivo experiments, a high-fat diet (HFD) induced inflammatory infiltration and plaque formation in the aorta, both of which were significantly reduced after injection of agomir-miRNA-29b-3p via the tail vein into HFD-fed ApoE-/- mice. In conclusion, this study reveals that the Lss-sensitive miR-29b-3p/CX3CL1 axis is an important regulatory target that affects vascular endothelial inflammation and AS development. Our study provides new insights into the prevention and treatment of AS.

Circ-CARD6 inhibits oxidative stress-induced apoptosis and autophagy in ARPE-19 cells via the miR-29b-3p/PRDX6/PI3K/Akt axis.

BACKGROUND: Oxidative stress-induced damage and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD) and hereditary retinopathy diseases (HRDs). This study aimed to elucidate the roles and mechanisms of circ-CARD6 and miR-29b-3p in oxidative stress-induced RPE and provide new ideas for the diagnosis and treatment of retinopathy disease (RD). METHODS: A model of oxidative stress-induced RPE (ARPE-19) was established, and the level of malondialdehyde (MDA) and concentration of reactive oxygen species (ROS) were detected by a DCFH-DA fluorescent probe and MDA kit. The cell viability was measured by a CCK-8 assay. The expression of PRDX6/PI3K/Akt axis genes and proteins related to apoptosis and autophagy were determined by RT‒qPCR and Western blot analyses. The dual-luciferase reporter system confirmed the targeting relationship between miR-29b-3p and circ-CARD6 and between miR-29b-3p and PRDX6. RESULTS: In H2O2-treated ARPE-19 cells, the expression of circ-CARD6 and PRDX6 was decreased, while the expression of miR-29b-3p was increased. The overexpression of circ-CARD6 inhibits oxidative stress-induced increases in ROS, apoptosis and autophagy in ARPE-19 cells. circ-CARD6 targets miR-29b-3p, miR-29b-3p targets PRDX6, and circ-CARD6 regulates PRDX6 via miR-29b-3p. Further studies showed that circ-CARD6 acts as a competitive endogenous RNA of miR-29b-3p to affect the expression of PRDX6, thereby inhibiting autophagy and apoptosis in ARPE-19 cells. CONCLUSION: circ-CARD6 can inhibit oxidative stress and apoptosis by regulating the miR-29b-3p/PRDX6/PI3K/Akt axis.

miR-29b-3p regulates cardiomyocytes pyroptosis in CVB3-induced myocarditis through targeting DNMT3A.

BACKGROUND: Viral myocarditis (VMC) is a disease resulting from viral infection, which manifests as inflammation of myocardial cells. Until now, the treatment of VMC is still a great challenge for clinicians. Increasing studies indicate the participation of miR-29b-3p in various diseases. According to the transcriptome sequencing analysis, miR-29b-3p was markedly upregulated in the viral myocarditis model. The purpose of this study was to investigate the role of miR-29b-3p in the progression of VMC. METHODS: We used CVB3 to induce primary cardiomyocytes and mice to establish a model of viral myocarditis. The purity of primary cardiomyocytes was identified by immunofluorescence. The cardiac function of mice was detected by Vevo770 imaging system. The area of inflammatory infiltration in heart tissue was shown by hematoxylin and eosin (H&E) staining. The expression of miR-29b-3p and DNMT3A was detected by quantitative real time polymerase chain reaction (qRT-PCR). The expression of a series of pyroptosis-related proteins was detected by western blot. The role of miR-29b-3p/DNMT3A in CVB3-induced pyroptosis of cardiomyocytes was studied in this research. RESULTS: Our data showed that the expression of miR-29b-3p was upregulated in CVB3-induced cardiomyocytes and heart tissues in mice. To explore the function of miR-29b-3p in CVB3-induced VMC, we conducted in vivo experiments by knocking down the expression of miR-29b-3p using antagomir. We then assessed the effects on mice body weight, histopathology changes, myocardial function, and cell pyroptosis in heart tissues. Additionally, we performed gain/loss-of-function experiments in vitro to measure the levels of pyroptosis in primary cardiomyocytes. Through bioinformatic analysis, we identified DNA methyltransferases 3A (DNMT3A) as a potential target gene of miR-29b-3p. Furthermore, we found that the expression of DNMT3A can be modulated by miR-29b-3p during CVB3 infection. CONCLUSIONS: Our results demonstrate a correlation between the expression of DNMT3A and CVB3-induced pyroptosis in cardiomyocytes. These findings unveil a previously unidentified mechanism by which CVB3 induces cardiac injury through the regulation of miR-29b-3p/DNMT3A-mediated pyroptosis.

miR-29b-3p Affects the Hypertrophy of Ligamentum Flavum in Lumbar Spinal Stenosis and its Mechanism.

To explore the effect of miR-29b-3p on fibrosis and hypertrophy of ligamentum flavum (LF) in lumbar spinal stenosis (LSS) and its underlying mechanism. Patients with LSS and lumbar disc herniation (LDH) (control) undergoing posterior lumbar laminectomy were included in this study. Human LF samples were obtained for LF cell isolation, RNA, and protein extraction. Histomorphological analysis of LF was performed using hematoxylin-eosin (HE) staining. After isolation, culture, and transfection of primary LF cells, different transfection groups were constructed: NC-mimic, miR-29b-3p-mimic, NC-inhibitor, and miR-29b-3p-inhibitor. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29b-3p in LF and LF cells. Western blot analysis detected the protein expressions of P16 and CyclinD1. ELISA detected the protein expressions of TGF-ß1, Smad2, Smad3, TLR4, Type I collagen, and Type III collagen. Finally, LF cell viability was detected using the Cell Counting Kit-8 (CCK8) assay. The thickness of LF was significantly thicker in the LSS group compared to the LDH group (p < 0.05), accompanied by a higher calcification degree, more fibroblasts, and a larger area of collagen fiber proliferation. miR-29b-3p expression was significantly lower in LSS-derived LF tissues and cells than in LDH-derived tissues and cells (both p < 0.05). Compared to the NC-mimic group, the miR-29b-3p-mimic group exhibited significantly higher miR-29b-3p expression, decreased protein expressions of Type I collagen, Type III collagen, TGF-ß1, Smad2, Smad3, TLR4, P16, and CyclinD1, and inhibited LF cell proliferation (all p < 0.05). As expected, the miR-29b-3p-inhibitor group displayed contrasting expression patterns (all p < 0.05). Compared to the phosphate buffer saline (PBS) group, the Trimethylamine-N-Oxide (TMAO) group showed significantly increased expressions of TGF-ß1, Smad2, Smad3, TLR4, Type I collagen, Type III collagen, P16, and CyclinD1, as well as enhanced LF cell proliferation (all p < 0.05). However, there was no significant difference between the TMAO group and the Ang II group (all p > 0.05). Upregulation of miR-29b-3p expression may play a role in improving LF fibrosis and hypertrophy in LSS by inhibiting P16 expression and suppressing the activation of the TGF-ß/Smad signaling pathway. This finding offers new insights into future gene modification therapy for this patient population.

miR-29b-3p targetedly regulates VEGF to inhibit tumor progression and cisplatin resistance through Nrf2/HO-1 signaling pathway in non-small cell lung cancer.

BACKGROUNDS: Non-small cell lung carcinoma (NSCLC) is a common type of lung cancer. Prior investigations have elucidated the pivotal role of miR-29b-3p in restraining tumor growth and metastasis. Nonetheless, it remains to be determined whether miR-29b-3p can effectively suppress NSCLC progression and enhance the sensitivity of NSCLC cells to cisplatin. This investigation sought to determine the mechanism by which miR-29b-3p inhibited the advancement of NSCLC and mitigated resistance to cisplatin. METHODS: We initially assessed miR-29b-3p and VEGF levels in NSCLC tissues and cell lines. Next, miR-29b-3p expression was elevated in NSCLC cell lines H1975 and A549 by overexpression plasmid transfection. Following this, a sequence of molecular biology experiments was conducted to evaluate the impact of miR-29b-3p on the biological behaviors of NSCLC cells and their resistance to cisplatin. Additionally, we predicted VEGF was a target gene of miR-29b-3p by bioinformatics analysis. We next employed western blot to evaluate the protein expression of Nrf2 and HO-1 in NSCLC cells. Finally, we elucidated the effects of VEGF and Nrf2/HO-1pathway on NSCLC progression and cisplatin resistance by in vitro assays. RESULTS: In comparison to paracancerous tissues and human normal lung epithelial cells, the expression of miR-29b-3p was notably reduced and VEGF expression was clearly elevated in NSCLC tissues and cells. Moreover, miR-29b-3p upregulated obviously suppressed the biological activities of NSCLC cells and increased their sensitivity to cisplatin. Furthermore, in NSCLC cells, miR-29b-3p bound to VEGF and negatively regulate its transcription. Additionally, miR-29b-3p overexpression also inhibited the Nrf2/HO-1 signaling pathway. Finally, the overexpression of VEGF and the activation of the Nrf2/HO-1 pathway reversed miR-29b-3p-mediated inhibitory effect on biological behaviors of NSCLC cells and increased the cisplatin resistance. CONCLUSION: Our findings indicate that miR-29b-3p impedes NSCLC cells' biological behaviors and augments their sensitivity to cisplatin by targeting VEGF to modulate the Nfr2/HO-1 signaling pathway.

Upregulation of miR-29b-3p alleviates coronary microembolization-induced myocardial injury via regulating BMF and GSK-3ß.

Coronary microembolization (CME) is an intractable complication results from acute coronary syndrome. CME-induced myocardial apoptosis was associated with progressive cardiac contractile dysfunction. miR-29b-3p has been reported implicated in variety cardiovascular diseases, but its function in CME-induced myocardial injury is yet unknown. Herein, a rat model of CME was established by injecting microspheres into the left ventricle and found that the expression level of miR-29b-3p was markedly decreased in the CME rat heart tissues. By using echocardiography, CD31 immunohistochemistry staining, hematoxylin basic fuchsin picric acid (HBFP) staining, TUNEL staining, and western blotting analysis after CME, it was found that upregulating miR-29b-3p improved cardiac dysfunction, promoted angiogenesis, decreased myocardial microinfarct area, and inhibited myocardial apoptosis. Additionally, miR-29b-3p inhibition can reverse the protective benefits of miR-29b-3p overexpression. Mechanistically, the target genes of miR-29b-3p were identified as glycogen synthase kinase 3 (GSK-3ß) and Bcl-2 modifying factor (BMF) by bioinformatics analysis and luciferase reporter experiment. Overall, our findings imply that induction of miR-29b-3p, which negatively regulates GSK-3ß and BMF expression, attenuates CME-induced myocardial injury, suggesting a novel potential therapeutic target for cardioprotective after CME.

MAD2B Blunts Chronic Unpredictable Stress and Corticosterone Stimulation-Induced Depression-Like Behaviors in Mice.

BACKGROUND: Depression is a prevalent and recurrent psychiatric disorder. Aberrant neural structure and activity play fundamental roles in the occurrence of depression. Mitotic arrest deficient protein (MAD2B) is highly expressed in neurons and may be implicated in synaptic plasticity in the central nervous system. However, the effect of MAD2B in depression, as well as the related molecular mechanism, is uncertain. METHODS: Here, we employed mouse models of depression induced by chronic unpredictable stress exposure or corticosterone (CORT) stimulation. Depression-like behaviors in mice were evaluated by sucrose preference, forced swimming, and tail suspension tests. Hippocampal MAD2B overexpression was mediated by adeno-associated virus 8 containing enhanced green fluorescent protein. In vitro primary neuronal cells were obtained from the hippocampus of rat embryos and were treated with CORT, and MAD2B overexpression was performed using lentivirus. MAD2B and glutamate metabotropic receptor 4 (GRM4) levels were evaluated by western blots and quantitative PCR. Primary neuronal miR-29b-3p expression was detected by quantitative PCR. RESULTS: MAD2B expression was reduced in the hippocampus in mice exhibiting depressive-like behaviors. However, hippocampal MAD2B overexpression protected mice from developing either chronic unpredictable stress- or CORT-induced depression-like behaviors, an effect associated with reduced expression of GRM4, a presynaptic receptor involved in depression. Moreover, MAD2B overexpression in primary neuronal cells also decreased GRM4 expression while enhancing the level of miR-29b-3p; this phenomenon was also observed under CORT stimulation. CONCLUSIONS: Our results suggest an important role of neuronal MAD2B in the pathogenesis of depression via the miR-29b-3p/GRM4 signaling pathway. MAD2B could be a potential therapeutic target for depressive disorders.

Blood exosome sensing via neuronal insulin-like growth factor-1 regulates autism-related phenotypes.

The development and progression of autism spectrum disorder (ASD) is characterized by multiple complex molecular events, highlighting the importance of the prefrontal brain regions in this process. Exosomes are nanovesicles that play a critical role in intercellular communication. Peripheral systems influence brain function under both physiological and pathological conditions. We investigated whether this influence was mediated by the direct sensing of peripheral blood exosomes by brain cells. Administration of serum exosomes from rats with valproic acid-induced ASD resulted in ASD-related phenotypes in mice, whereas exosomes from normal rats did not exhibit such effects. RNA sequencing and bioinformatics analysis suggested that negative regulation of medial prefrontal cortex (mPFC) insulin-like growth factor 1 (IGF-1) by exosome-derived miR-29b-3p may contribute to these ASD-associated effects. Further evidence showed that miR-29b-3p-enriched exosomes crossed the blood-brain barrier to reach the mPFC, subsequently inducing the suppression of IGF-1 expression in neurons. Optogenetic activation of excitatory neurons in the mPFC improved behavioral abnormalities in exosome-treated mice. The addition of exogenous IGF-1 or inhibition of miR-29b-3p expression in the mPFC also rescued the ASD-related phenotypes in mice. Importantly, administration of miR-29b-3p-enriched serum exosomes from human donors with ASD into the mouse medial prefrontal cortex was sufficient to induce hallmark ASD behaviors. Together, our findings indicate that blood-brain cross-talk is crucial for ASD pathophysiology and that the brain may sense peripheral system changes through exosomes, which could serve as the basis for future neurological therapies.

Bone marrow mesenchymal stem cell-derived exosome miR-29b-3p alleviates UV irradiation-induced photoaging in skin fibroblast.

BACKGROUND: Mesenchymal stem cells-derived exosome (MSCs-exo) was identified to reduce photoaging. The purpose of this study was to investigate the potential role of microRNA (miR)-29b-3p derived from bone marrow MSCs-exo (BMSCs-exo) in photoaging. METHODS: Exosomes were isolated from BMSCs and verified by Western blot. A photoaging cell model was constructed by UVB irradiation of human dermal fibroblasts (HDFs). Quantitative real-time PCR (RT-qPCR) was performed to detect the mRNA levels of miR-29b-3p, collagen type I and matrix metalloproteinases (MMPs). CCK-8, Transwell and flow cytometry were applicated to examine cell viability, migration and apoptosis. Commercial kits are used to measure levels of oxidative stress indicators. Finally, a dual-luciferase reporter assay was applied to validate the target of miR-29b-3p. RESULTS: Extracted exosomes were positive for HSP70 and CD9. Survival of HDFs increased in an exosome concentration-dependent manner. UVB irradiation inhibited miR-29b-3p levels compared with controls, but BMSCs-exo treatment restored miR-29b-3p levels (p < .05). Additionally, BMSCs-exo-miR-29b-3p reversed the inhibition of HDFs migration and oxidative stress by UVB irradiation, as well as the promotion of apoptosis. However, this reversal was attenuated by the suppression of miR-29b-3p (p < .05). Furthermore, BMSCs-exo-miR-29b-3p also inhibited the degradation of collagen type I and the production of MMPs in photoaging, and they were also eliminated by the reduced miR-29b-3p. Finally, MMP-2 was the target gene of miR-29b-3p. CONCLUSION: Our study presented a novel role for BMSCs-exo-miR-29b-3p in improving skin photoaging function, and these findings may provide new insights into the targeted treatment of skin photoaging.

Guizhi Fuling Wan inhibits autophagy of granulosa cells in polycystic ovary syndrome mice via H19/miR-29b-3p.

OBJECTIVE: To investigate the potential molecular mechanism of traditional Chinese medicine Guizhi Fuling Wan (GZFLW) inhibiting granulosa cells (GCs) autophagy in polycystic ovary syndrome (PCOS). METHODS: Control GCs and model GCs were cultured and treated with blank serum or GZFLW-containing serum. The levels of H19 and miR-29b-3p in GCs were detected using qRT-PCR, target genes of miR-29b-3p were identified using luciferase assay. The protein expressions of Phosphatase and tensin homolog (PTEN), Matrix Metalloproteinase (MMP)-2, and Bax were measured using western blot. The level of autophagy was detected via MDC staining, the degree of autophagosomes and autophagic polymers was observed using dual fluorescence-tagged mRFP-eGFP-LC3. RESULTS: GZFLW intervention reduced the expression of autophagy-related proteins PTEN, MMP-2 and Bax, by upregulating the expression of miR-29b-3p and downregulated the expression of H19 (p < .05 or p < .01). The number of autophagosomes and autophagy polymers was significantly decreased by GZFLW treatment. However, the inhibition of miR-29b-3p and overexpression of H19 induced a significant increase in the number of autophagosomes and autophagic polymers, which attenuated the inhibitory effect of GZFLW on autophagy (p < .05 or p < .01). In addition, inhibition of miR-29b-3p or overexpression of H19 can attenuate the effect of GZFLW on the expression of PTEN, MMP-2 and Bax proteins (p < .05 or p < .01). CONCLUSION: Our study found that GZFLW inhibits autophagy in PCOS GCs via H19/miR-29b-3p pathway.

Discovering the Biological Significance and Therapeutic Potential of miR-29b-3p in Triple-Negative Breast Cancer.

The lack of estrogen or progesterone receptors and absence of HER2 amplification/overexpression in triple-negative breast cancer (TNBC) restricts therapeutic options used in clinical management. MicroRNAs (miRNAs) are small, non-coding transcripts which affect important cellular mechanisms by regulating gene expression at the post-transcriptional level. Among this class, attention was focused on miR-29b-3p with a high profile in TNBC and correlated with the overall survival rates, as TCGA data revealed. This study aims to investigate the implication of the miR-29b-3p inhibitor in TNBC cell lines by identifying a potential therapeutic transcript, improving the clinical outcomes of this disease. The experiments were performed on two TNBC cell lines (MDA-MB-231 and BT549) as in vitro models. An established dose of 50 nM was used for all functional assays performed on the miR-29b-3p inhibitor. A decreased level of miR-29b-3p determined a significant reduction in cell proliferation and colony-forming capacity. At the same time, the changes occurring at the molecular and cellular levels were highlighted. We observed that, when inhibiting the expression level of miR-29b-3p, processes such as apoptosis and autophagy were activated. Further, microarray data revealed that the miRNA expression pattern was altered after miR-29b-3p inhibition, pointing out 8 overexpressed and 11 downregulated miRNAs specific for BT549 cells and 33 upregulated and 10 downregulated miRNAs that were specific for MDA-MB-231 cells. As a common signature for both cell lines, three transcripts were observed, two downregulated, miR-29b-3p and miR-29a, and one upregulated, miR-1229-5p. According to DIANA miRPath, the main predicted targets are related to ECM (extracellular matrix) receptor interaction and TP53 signaling. An additional validation step through qRT-PCR was performed, which showed an upregulation of MCL1 and TGFB1. By inhibiting the expression level of miR-29b-3p, it was shown that complex regulatory pathways targeted this transcript in TNBC cells.

lncRNA MSTRG4710 Promotes the Proliferation and Differentiation of Preadipocytes through miR-29b-3p/IGF1 Axis.

Obesity, a major global health issue, is increasingly associated with the integral role of long non-coding RNA (lncRNA) in adipogenesis. Recently, we found that lncRNA-MSTRG4710 was highly expressed in the liver of rabbits fed a high-fat diet, but whether it is involved in lipid metabolism remains unclear. A series of experiments involving CCK-8, EDU, qPCR, and Oil Red O staining demonstrated that the overexpression of MSTRG4710 stimulated the proliferation and differentiation of preadipocytes while its knockdown inhibited these processes. Bioinformatics analysis showed that miR-29b-3p was a potential target gene of MSTRG4710, and IGF1 was a downstream target gene of miR-29b-3p. Luciferase reporter gene analysis and qPCR analysis confirmed that miR-29b-3p was a potential target gene of MSTRG4710, and miR-29b-3p directly targeted the 3'UTR of IGF1. The overexpression of miR-29b-3p was observed to regulate IGF1 protein and mRNA levels negatively. Additionally, a total of 414 known differentially expressed genes between the miR-29b-3p mimic, miR-29b-3p negative control (NC), siMSTRG4710, and siMSTRG4710-NC group were screened via transcriptome sequencing technology. The GO- and KEGG-enriched pathways were found to be related to lipid metabolism. The study also established that miR-29b-3p targets IGF1 to inhibit preadipocyte proliferation and differentiation. Notably, IGF1 knockdown significantly reduced preadipocyte proliferation and differentiation. Furthermore, co-transfection of pcDNA3.1(+)-MSTRG4710 and mimics into rabbit preadipocytes revealed that the mimics reversed the promotional effect of pcDNA3.1(+)-MSTRG4710. In conclusion, these results uncover that MSTRG4710 positively regulated cell proliferation and adipogenesis by the miR-29b-3p/IGF1 axis. Our findings might provide a new target for studying adipogenesis in rabbit preadipocytes and obesity.

Downregulation of MicroRNA-29-3p Following Percutaneous Coronary Intervention: An Implication of YY1/IRAK1 Pathway in the Post-Vascular Injury Inflammation.

This study explored the expression of microRNA (miR)-29b-3p following percutaneous coronary intervention (PCI) and the implication of its downstream Yin Yang 1 (YY1)/interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) pathway in post-vascular injury inflammation. Blood samples were collected for analysis of plasma miR-29b-3p from patients with acute coronary syndrome before surgery, 1 day after PCI, and 30 days after PCI. Lipopolysaccharide (LPS)-treated human coronary artery endothelial cells (HCAECs) were transfected with miR-29b-3p mimic/inhibitor or YY1 shRNA and underwent viability tests. Enzyme-linked immunosorbent assay was performed to detect the levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), IL-1ß, IL-6, and tumor necrosis factor (TNF)-α in serum and cell culture supernatant. Dual-luciferase reporter and RNA/chromatin immunoprecipitation were used to confirm the targeting relationships among miR-29b-3p, YY1, and IRAK1. A rat model of intraluminal injury of the common femoral artery was established to address the role of miR-29b-3p and relevant mechanisms. miR-29b-3p was lowly expressed, and sVCAM-1, IL-1ß, IL-6, and TNF-α were upregulated 1 day after PCI and 24 h after LPS treatment. miR-29b-3p overexpression or YY1 knockdown alleviated LPS-induced inflammatory responses and improved the viability of HCAECs. miR-29b-3p inhibition aggravated LPS-induced inflammatory injury in HCAECs. miR-29b-3p bound to YY1 mRNA and inhibited the expression of YY1 protein. YY1 bound to the IRAK1 promoter and activated the transcription of IRAK1. Upregulation of miR-29b-3p suppressed the inflammatory response after intraluminal injury of the common femoral artery in rats. In conclusion, dysregulation of the YY1/IRAK1 pathway via miR-29b-3p downregulation may be implicated in post-vascular injury inflammation.

miR-29b-3p suppresses the malignant biological behaviors of AML cells via inhibiting NF-κB and JAK/STAT signaling pathways by targeting HuR.

BACKGROUND: HuR/ELAVL1 (embryonic lethal abnormal vision 1) was a downstream target of miR-29b in some cancer cells. HuR protein exerts important prognostic effects of involving in the pathogenesis and development of acute myeloid leukemia (AML). This study aims to investigate the role of miR-29b-3p in biological behaviors of AML cells by targeting HuR and the involvement of the NF-κB and JAK/STAT signaling pathways. METHODS: The expressions of HuR and miR-29b-3p in AML cells were determined using RT-qPCR and Western blot, and the association between them was analyzed using the Spearman method. Next, the target relationship between HuR and miR-29b-3p was predicted by biological information databases and verified by the dual-luciferase reporter gene assay. MTS, methyl cellulose, flow cytometry and transwell assay were employed to detect the cell proliferation, clone formation, cell cycle and apoptosis, invasion and migration respectively, the effect of miR-29b-3p targeted HuR on the biological behaviors of AML cells was explored after over- /down-expression of miR-29b-3p and rescue experiment. Then, immunofluorescence assay and western blot were employed to detect location expression and phosphorylation levels of NF-κB and JAK/STAT signaling pathways related molecules respectively. RESULTS: HuR was negatively correlated with miR-29b-3p, and was the downstream target of miR-29b-3p in AML cells. When miR-29b-3p was overexpressed in AML cells, HuR was down-regulated, accompanied by cell viability decreased, cell cycle arrest, apoptosis increased, invasion and migration weakened. Moreover, the opposite result appeared after miR-29b-3p was down-regulated. The rescue experiment showed that miR-29b-3p inhibitor could reverse the biological effect of HuR down-regulation in AML cells. Molecular pathway results showed that miR-29b-3p could inhibit p65 expression in nucleus and phosphorylation levels of p65, IκBα, STAT1, STAT3 and STAT5. CONCLUSION: miR-29b-3p can inhibit malignant biological behaviors of AML cells via the inactivation of the NF-κB and JAK/STAT signaling pathways by targeting HuR. miR-29b-3p and its target HuR can be used as a new potential molecular for AML treatment.

Extracellular vesicles from human umbilical cord mesenchymal stem cells reduce lipopolysaccharide-induced spinal cord injury neuronal apoptosis by mediating miR-29b-3p/PTEN.

OBJECTIVE: This study investigated the molecular mechanism of whether hUC-MSCs-EVs repressed PTEN expression and activated the PI3K/AKT pathway through miR-29b-3p, thus inhibiting LPS-induced neuronal injury. METHODS: hUC-MSCs were cultured and then identified. Cell morphology was observed. Alizarin red, oil red O, and alcian blue staining were used for inducing osteogenesis, adipogenesis, and chondrogenesis. EVs were extracted from hUC-MSCs and identified by transmission electron microscope observation and Western blot. SCI neuron model was established by 24h lipopolysaccharide (LPS) induction. After the cells were cultured with EVs without any treatment, uptake of EVs by SCI neurons, miR-29b-3p expression, cell viability, apoptosis, caspase-3, cleaved caspase-3, caspase 9, Bcl-2, PTEN, PI3K, AKT, and p-Akt protein levels, caspase 3 and caspase 9 activities, and inflammatory factors IL-6 and IL-1ß levels were detected by immunofluorescence labeling, RT-qPCR, MTT, flow cytometry, Western blot, caspase 3 and caspase 9 activity detection kits, and ELISA. The binding sites between PTEN and miR-29b-3p were predicted by the database and verified by dual-luciferase assay. RESULTS: LPS-induced SCI cell model was successfully established, and hUC-MSCs-EVs inhibited LPS-induced apoptosis of injured spinal cord neurons. EVs transferred miR-29b-3p into LPS-induced injured neurons. miR-29b-3p silencing reversed EV effects on reducing LPS-induced neuronal apoptosis. miR-29b-3p reduced LPS-induced neuronal apoptosis by targeting PTEN. After EVs-miR-inhi and si-PTEN treatment, inhibition of the PI3K/AKT pathway reversed hUC-MSCs-EVs effects on reducing LPS-induced neuronal apoptosis. CONCLUSION: hUC-MSCs-EVs activated the PI3K/AKT pathway by carrying miR-29b-3p into SCI neurons and silencing PTEN, thus reducing neuronal apoptosis.

The Effect of Interferon-Beta Therapy on T-Helper 17/miR-326 and T-Helper 1/miR-29b-3p Axis in Relapsing-Remitting Multiple Sclerosis Patients.

BACKGROUND: We aimed to evaluate the therapeutic effects of interferon-beta (IFN-ß) on hsa-miR29b-3p and hsa-miR326 in isolated T-helper (Th)1 and Th17 cells expressed by relapsing-remitting multiple sclerosis (RRMS) patients before and after 1 year of treatment with IFN-ß. METHODS: The study was done on 19 RRMS patients pre- and posttreatment with IFN-ß to evaluate the frequency of Th1 and Th17 cells by flow cytometry. The expression level of hsa-miR-29b-3p and hsa-miR-326 in isolated Th1 and Th17 cells was assessed by quantitative polymerase chain reaction. Enzyme-linked immunosorbent assay was also used to measure the plasma levels of I interferon -gamma and interleukin (IL)-17A. RESULTS: Th17 cells and plasma levels of IL-17A decreased in RRMS patients after IFN-ß therapy but hsa-miR-29b-3p and hsa-miR-326 expression had no significant change in treated RRMS patients versus baseline. MxA gene expression was significantly induced upon IFN-ß therapy in patients with RRMS. CONCLUSION: IFN-ß therapy is more effective on Th17 than Th1, but it does not reform altered expression of hsa-miR-326 and hsa-miR-29b-3p in Th17 and Th1, respectively.

MiR-29b-3p Inhibits the Inflammation Injury in Human Umbilical Vein Endothelial Cells by Regulating SEC23A.

This study aims to investigate the effects of miR-29b-3p on the inflammation injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) and explore the underlying mechanisms. The effects of different concentrations of LPS (0, 1, 5 and 10 µg/mL) on inflammation injury in HUVECs are detected by ELISA, CCK-8, EdU, flow cytometry and western blot analyses to determine the optimal stimulus concentration. After stimulating HUVECs with 10 µg/mL LPS, the expression levels of miR-29b-3p are detected, and the effects of miR-29b-3p on inflammation injury are detected by ELISA, CCK-8, EdU, flow cytometry and western blot analyses. Bioinformatic analysis, luciferase reporter assay and confirmatory experiments are applied to identify the target gene bound with miR-29b-3p. Rescue experiments have verified the roles of miR-29b-3p and the target gene in inflammation injury. We found that pro-inflammatory factor was increased, apoptosis was promoted, and cell proliferation was inhibited after the treatment of LPS in HUVECs. Overexpression of miR-29b-3p inhibited LPS-induced inflammatory response and apoptosis while promoting proliferation in HUVECs. Besides, bioinformatics analysis indicated that SEC23A was the target gene of miR-29b-3p and the confirmatory experiments showed that SEC23A was negatively correlated with miR-29b-3p and positively correlated with LPS concentration. Rescue experiments revealed that overexpression of SEC23A partially enhanced the inflammation injury effects in LPS-induced HUVECs with overexpression of miR-29b-3p. Hence, miR-29b-3p repressed inflammatory response, cell apoptosis and promoted cell proliferation in LPS-induced HUVECs by targeting SEC23A, providing a potential target for treating sepsis.

Cellular and Molecular Profiling of Tumor Microenvironment and Early-Stage Lung Cancer.

Lung cancers are broadly divided into two categories: non-small-cell lung carcinoma (NSCLC), which accounts for 80-85% of all cancer cases, and small-cell lung carcinoma (SCLC), which covers the remaining 10-15%. Recent advances in cancer biology and genomics research have allowed an in-depth characterization of lung cancers that have revealed new therapy targets (EGFR, ALK, ROS, and KRAS mutations) and have the potential of revealing even more biomarkers for diagnostic, prognostic, and targeted therapies. A new source of biomarkers is represented by non-coding RNAs, especially microRNAs (miRNAs). MiRNAs are short non-coding RNA sequences that have essential regulatory roles in multiple cancers. Therefore, we aim to investigate the tumor microenvironment (TME) and miRNA tumor profile in a subset of 51 early-stage lung cancer samples (T1 and T2) to better understand early tumor and TME organization and molecular dysregulation. We analyzed the immunohistochemistry expression of CD4 and CD8 as markers of the main TME immune populations, E-cadherin to evaluate early-stage epithelial-to-mesenchymal transition (EMT), and p53, the main altered tumor suppressor gene in lung cancer. Starting from these 4 markers, we identified and validated 4 miRNAs that target TP53 and regulate EMT that can be further investigated as potential early-stage lung cancer biomarkers.

Epigallocatechin-3-O-gallate promotes extracellular matrix and inhibits inflammation in IL-1ß stimulated chondrocytes by the PTEN/miRNA-29b pathway.

CONTEXT: Epigallocatechin-3-O-gallate (EGCG) exhibits anti-arthritic activity. MiR-29b-3p provokes chondrocyte apoptosis and promotes the initiation and development of osteoarthritis (OA). OBJECTIVE: To explore the roles of EGCG and miR-29b-3p in interleukin-1ß (IL-1ß)-stimulated chondrocytes. MATERIALS AND METHODS: HE and Safranin O staining were used to detect the pathological changes of cartilage tissue in OA patients and healthy people. OA-like chondrocyte injury was mimicked by 5 ng/mL IL-1ß stimulation for 24 h in vitro, and after transfection with miR-29b-3p mimics and pcDNA-PTEN, IL-1ß-stimulated chondrocytes were pre-treated with EGCG (20 and 50 µM) for 2 h. Cell viability, colony numbers, apoptosis rate, the levels of IL-6 and matrix metalloproteinase-13 (MMP-13), miR-19b-3p, PTEN and apoptosis-associated proteins in chondrocytes were evaluated. RESULTS: MiR-29b-3p level was upregulated in cartilage tissues of OA patients (3.5-fold change, p < 0.001) and IL-1ß stimulated chondrocytes (two fold change, p < 0.001). The matrix staining was weakened and unevenly distributed, and the chondrocytes were arranged disorderly in the tissues of patients with OA. EGCG (20 and 50 µM) increases viability and decreases the levels of miR-29b-3p and MMP-13 and IL-6 in IL-1ß stimulated chondrocytes (p < 0.05). MiR-29b-3p mimics reversed the effects above 50 µM EGCG (p < 0.05). Furthermore, PTEN overexpression abrogated the effects of miR-29b-3p mimics on viability, colony numbers, apoptosis rate and the levels of Bcl-2, MMP-13, IL-6, Bax and cleaved caspase 3 in IL-1ß-stimulated chondrocytes (p < 0.01). DISCUSSION AND CONCLUSIONS: EGCG is a potential candidate for the treatment of OA, which also can be explored in a novel therapeutic method for other degenerative or inflammatory disorders.