Sperm-borne miR-34c-5p and miR-191-3p as markers for sperm motility and embryo developmental competence.

BACKGROUND: Sperm-borne microRNAs play a pivotal role in influencing essential cellular processes during fertilization, impacting the quality of embryo development. Dysregulated microRNA profiles have been associated with compromised embryonic development and increased incidences of pregnancy loss. OBJECTIVE: This study aimed to investigate the potential associations between the abundance of miR-34c-5p and miR-191-3p in human spermatozoa with sperm quality, as well as with embryo quality and metabolic performance during in vitro development. MATERIALS AND METHODS: Thirteen couples who underwent a total of 13 cycles participated in this study. The sperm quality was assessed using conventional methods following World Health Organization guidelines. Quantitative polymerase chain reaction was employed to measure microRNA abundance in spermatozoa. Embryos were categorized as good, lagging, or bad based on morphokinetic evaluation. Evaluation of embryo metabolic performance involved tracking changes in specific metabolites within the cultured media using nuclear magnetic resonance spectroscopy. Statistical analysis was conducted to explore the correlation between microRNA abundance in human spermatozoa and all other collected data. RESULTS: Our findings revealed a negative correlation between the abundance of miR-34c-5p (but not miR-191-3p) and total sperm motility, potentially mediated by the modulation of key signaling pathways. Additionally, higher levels of miR-34c-5p in spermatozoa were strongly associated with the consumption or release of key metabolites by developing embryos, particularly those linked with lipid and glucose metabolism, suggesting enhanced metabolic performance, while miR-191-3p was mostly associated with glucose consumption. Concurrently, only miR-34c-5p content in spermatozoa correlated with higher embryo quality. DISCUSSION AND CONCLUSION: This study provides evidence suggesting that the abundance of miR-34c-5p in spermatozoa is correlated not only with total sperm motility but also with markers of embryo developmental competence, highlighting the potential significance of this sperm microRNA content as a biomarker in assisted reproduction.

Circ_0007611 modulates the miR-34c-5p/LPAR2 cascade to suppress proliferation and enhance apoptosis of HTR-8/SVneo cells.

INTRODUCTION: The aberrant biological behaviors of trophoblast cells actively take part in the pathogenesis of preeclampsia (PE). Herein, we defined the action of the circular RNA (circRNA) circ_0007611 on trophoblast cell apoptosis and growth to understand its role in PE. METHODS: Expression of circ_0007611, miR-34c-5p and lysophosphatidic acid receptor 2 (LPAR2) mRNA was analyzed by qPCR. LPAR2 protein was determined by western blotting. Cell proliferation was analyzed by EdU assay. We assessed apoptosis through flow cytometry and analysis of caspase3 activity and apoptosis-related marker proteins. The binding of miR-34c-5p and circ_0007611 or LPAR2 was verified by dual-luciferase and pull-down assays. RESULTS: Circ_0007611 and LPAR2 levels were augmented, while miR-34c-5p was diminished in blood samples of PE. Circ_0007611 deficiency repressed cell apoptosis and enhanced the growth of HTR-8/SVneo cells. Circ_0007611 interacted with miR-34c-5p, and miR-34c-5p depletion reversed circ_0007611 deficiency-induced HTR-8/SVneo cell apoptotic inhibition and growth enhancement. MiR-34c-5p targeted LPAR2, and circ_0007611 affected LPAR2 expression via miR-34c-5p competition. Circ_0007611 deficiency-induced HHTR-8/SVneo cell apoptotic inhibition and growth enhancement were also counteracted by LPAR2 overexpression. DISCUSSION: Circ_0007611 modulates the miR-34c-5p/LPAR2 cascade to enhance apoptosis and inhibit proliferation in HTR-8/SVneo cells, thereby contributing to the progression of PE.

Scutellarin Protects against Myocardial Ischemia-reperfusion Injury by Enhancing Aerobic Glycolysis through miR-34c-5p/ALDOA Axis.

Background: Aerobic glycolysis has recently demonstrated promising potential in mitigating the effects of ischemia-reperfusion (IR) injury. Scutellarin (Scu) possesses various cardioprotective properties that warrant investigation. To mimic IR injury in vitro, this study employed hypoxia/reoxygenation (H/R) injury. Methods and Results: First, we conducted an assessment of the protective properties of Scu against HR in H9c2 cells, encompassing inflammation damage, apoptosis injury, and oxidative stress. Then, we verified the effects of Scu on the Warburg effect in H9c2 cells during HR injury. The findings indicated that Scu augmented aerobic glycolysis by upregulating p-PKM2/PKM2 levels. Following, we built a panel of six long noncoding RNAs and seventeen microRNAs that were reported to mediate the Warburg effect. Based on the results, miR-34c-5p was selected for further experiments. Then, we observed Scu could mitigate the HR-induced elevation of miR-34c-5p. Upregulation of miR-34c-5p could weaken the beneficial impacts of Scu in cellular viability, inflammatory damage, oxidative stress, and the facilitation of the Warburg effect. Subsequently, our investigation revealed a decrease in both ALDOA mRNA and protein levels following HR injury, which could be restored by Scu administration. Downregulation of ALDOA or Mimic of miR-34c-5p could reduce these effects induced by Scu. Conclusions: Scu provides cardioprotective effects against IR injury by upregulating the Warburg effect via miR-34c-5p/ALDOA.

MicroRNAs dysregulated in multiple sclerosis affect the differentiation of CG-4 cells, an oligodendrocyte progenitor cell line.

Introduction: Demyelination is one of the hallmarks of multiple sclerosis (MS). While remyelination occurs during the disease, it is incomplete from the start and strongly decreases with its progression, mainly due to the harm to oligodendrocyte progenitor cells (OPCs), causing irreversible neurological deficits and contributing to neurodegeneration. Therapeutic strategies promoting remyelination are still very preliminary and lacking within the current treatment panel for MS. Methods: In a previous study, we identified 21 microRNAs dysregulated mostly in the CSF of relapsing and/or remitting MS patients. In this study we transfected the mimics/inhibitors of several of these microRNAs separately in an OPC cell line, called CG-4. We aimed (1) to phenotypically characterize their effect on OPC differentiation and (2) to identify corroborating potential mRNA targets via immunocytochemistry, RT-qPCR analysis, RNA sequencing, and Gene Ontology enrichment analysis. Results: We observed that the majority of 13 transfected microRNA mimics decreased the differentiation of CG-4 cells. We demonstrate, by RNA sequencing and independent RT-qPCR analyses, that miR-33-3p, miR-34c-5p, and miR-124-5p arrest OPC differentiation at a late progenitor stage and miR-145-5p at a premyelinating stage as evidenced by the downregulation of premyelinating oligodendrocyte (OL) [Tcf7l2, Cnp (except for miR-145-5p)] and mature OL (Plp1, Mbp, and Mobp) markers, whereas only miR-214-3p promotes OPC differentiation. We further propose a comprehensive exploration of their change in cell fate through Gene Ontology enrichment analysis. We finally confirm by RT-qPCR analyses the downregulation of several predicted mRNA targets for each microRNA that possibly support their effect on OPC differentiation by very distinctive mechanisms, of which some are still unexplored in OPC/OL physiology. Conclusion: miR-33-3p, miR-34c-5p, and miR-124-5p arrest OPC differentiation at a late progenitor stage and miR-145-5p at a premyelinating stage, whereas miR-214-3p promotes the differentiation of CG-4 cells. We propose several potential mRNA targets and hypothetical mechanisms by which each microRNA exerts its effect. We hereby open new perspectives in the research on OPC differentiation and the pathophysiology of demyelination/remyelination, and possibly even in the search for new remyelinating therapeutic strategies in the scope of MS.

miR-34c-5p inhibited fibroblast proliferation, differentiation and epithelial-mesenchymal transition in benign airway stenosis via MDMX/p53 pathway.

Benign airway stenosis (BAS) means airway stenosis or obstruction that results from a variety of non-malignant factors, including tuberculosis, trauma, benign tumors, etc. In consideration of the currently limited research on microRNAs in BAS, this study aimed to explore the role and mechanism of miR-34c-5p in BAS. The expression of miR-34c-5p in BAS granulation tissues showed a significant down-regulation compared with the normal control group. Moreover, miR-34c-5p mimics suppressed the proliferation and differentiation of human bronchial fibroblasts (HBFs) and the epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells (HBE). Conversely, miR-34c-5p inhibitors aggravated those effects. A dual-luciferase reporter assay confirmed that miR-34c-5p can target MDMX rather than Notch1. The over-expression of MDMX can reverse the inhibiting effect of miR-34c-5p on HBFs proliferation, differentiation and EMT. Furthermore, the expressions of tumor protein (p53) and PTEN were down-regulated following the over-expression of MDMX. In addition, the expressions of PI3K and AKT showed an up-regulation. In conclusion, miR-34c-5p was down-regulated in BAS and may inhibit fibroblast proliferation differentiation and EMT in BAS via the MDMX/p53 signaling axis. These findings expand the understanding of the role of miR-34c-5p and will help develop new treatment strategies for BAS.

Early life stress enhances the susceptibility to depression and interferes with neuroplasticity in the hippocampus of adolescent mice via regulating miR-34c-5p/SYT1 axis.

Early life events are major risk factors for the onset of depression and have long-term effects on the neurobiological changes and behavioral development of rodents. However, little is known about the specific mechanisms of early life adversity in the susceptibility to subsequent stress exposure in adolescence. This study characterized the effect of maternal separation (MS), an animal model of early life adversity, on the behavioral responses to restraint stress in mice during adolescence and investigated the molecular mechanism underlying behavioral vulnerability to chronic stress induced by MS. Our results showed that MS exposure could further reinforce the depressive vulnerability to restraint stress in adolescent mice. In addition, miR-34c-5p expression was obviously up-regulated in the hippocampi of MS mice at postnatal day (P) 14 and P42. Further, synaptotagmin-1 (SYT1) was deemed as a target gene candidate of miR-34c-5p on the basis of dual luciferase assay. It was found that the downregulation of miR-34c-5p expression in the hippocampi of MS mice could ameliorate dysfunction of synaptic plasticity by targeting molecule SYT1, effects which were accompanied by alleviation of depressive and anxious behaviors in these mice. The results demonstrated that the miR-34c-5p/SYT1 pathway was involved in the susceptibility to depression induced by MS via regulating neuroplasticity in the hippocampi of mice.

The Expression of miR-34c-5p Induces G0/G1 Cell Cycle Arrest and Apoptosis in SW480 Colon Cancer Cell.

Background: Expression of the miR-34 family, including miR-34a/b/c, has been reported to inhibit the progression of several cancer types by inhibiting cell proliferation and inducing apoptosis. Objectives: We attempted to investigate the effect of SW480 cell transfection with miR-34c-5p mimics on cell proliferation. Methods: To do this, SW480 colon cancer cell line was transfected with miR-34c-5p mimics, scramble sequence, and the vehicle in PBS mock, and then cell proliferation was assessed by MTT assay. The population of cells in cell cycle phases, ROS generation, and apoptosis rate were evaluated by flow cytometry. Additionally, we determined the relative expression of apoptotic genes through real-time PCR technique. Results: We observed a reduced proliferation rate in cells transfected with miR-34c-5p compared to the control group (P <0.05). We also found that miR-34c-5p caused a significant increase in apoptosis rate (P < 0.001) and cell cycle arrest in the G0 and G1 phases (P < 0.05). Moreover, a significant increase was reported in the expression of pro-apoptotic genes, including BAK (P < 0.001), BAX and BAD (P < 0.0001), and Caspase 7/9 (P < 0.0001). Conclusions: However, no remarkable difference was seen in the expression of MCL1, BCL2, and CASPASE 3 genes. Our conclusion is that overexpression of miR-34c-5p could be considered a promising approach for colorectal cancer treatment.

CircRNA0007766 accelerates cancer progression via miR-34c-5p/cyclin D1 axis in adenocarcinoma of the esophagogastric junction (AEG).

Growing empirical evidence shows that circular RNAs (circRNAs) are implicated in tumor pathogenesis. However, little is known about the mechanism by which circRNAs contribute to the progression of adenocarcinoma of the esophagogastric junction (AEG). We conducted RNA high-throughput sequencing and bioinformatic analyses on 22 AEG tissues and their matching healthy gastric mucosal tissues and found that circRNA0007766 may act as a tumor promoter in AEG pathogenesis. BaseScope® in situ hybridization revealed that circRNA0007766 was strongly upregulated in AEG. We then constructed co-expression and ceRNA networks to elucidate the relationships among specific circRNAs, microRNAs (miRNAs), and mRNAs. We also demonstrated that circRNA0007766 acted as the sponge of miR-34c-5p, thereby positively regulating cyclin D1. In vivo and in vitro experiments demonstrated the roles of circRNA0007766 in promoting AEG progression and invasion. AEG tissues are characterized by circRNA0007766 upregulation which is correlated with lymph node metastasis and poor survival. To the best of our knowledge, the present study is one of the first to show that the circRNA0007766/miR-34c-5p/cyclin D1 axis is important in AEG progression. Furthermore, the results of this work imply that circRNA0007766 is potentially a novel AEG biomarker.

LncRNA CYP4A22-AS1 promotes the progression of lung adenocarcinoma through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

OBJECTIVES: Lung adenocarcinoma (LUAD) exhibits a higher fatality rate among all cancer types worldwide, yet the precise mechanisms underlying its initiation and progression remain unknown. Mounting evidence suggests that long non-coding RNAs (lncRNAs) exert significant regulatory roles in cancer development and progression. Nevertheless, the precise involvement of lncRNA CYP4A22-AS1 in LUAD remains incompletely comprehended. METHODS: Bioinformatics analyses evaluated the expression level of CYP4A22-AS1 in lung adenocarcinoma and paracancer. The LUAD cell line with a high expression of CYP4A22-AS1 was constructed to evaluate the role of CYP4A22-AS1 in the proliferation and metastasis of LUAD by CCK8, scratch healing, transwell assays, and animal experiments. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. Luciferase reporter gene analyses, west-blotting, and qRT-PCR were carried out to reveal the interaction between CYP4A22-AS1, miR-205-5p/EREG, and miR-34c-5p/BCL-2 axes. RESULTS: CYP4A22-AS1 expression was significantly higher in LUAD tissues than in the adjacent tissues. Furthermore, we constructed a LUAD cell line with a high expression of CYP4A22-AS1 and noted that the high expression of CYP4A22-AS1 significantly enhanced the proliferation and metastasis of LUAD. We applied transcriptome and microRNA sequencing to examine the mechanism of CYP4A22-AS1 enhancing the proliferation and metastasis of LUAD. CYP4A22-AS1 increased the expression of EREG and BCL-2 by reducing the expression of miR-205-5p and miR-34-5p and activating the downstream signaling pathway of EGFR and the anti-apoptotic signaling pathway of BCL-2, thereby triggering the proliferation and metastasis of LUAD. The transfection of miR-205-5p and miR-34-5p mimics inhibited the role of CYP4A22-AS1 in enhancing tumor progression. CONCLUSION: This study elucidates the molecular mechanism whereby CYP4A22-AS1 overexpression promotes LUAD progression through the miR-205-5p/EREG and miR-34c-5p/BCL-2 axes.

Long-term iTBS Improves Neural Functional Recovery by Reducing the Inflammatory Response and Inhibiting Neuronal Apoptosis Via miR-34c-5p/p53/Bax Signaling Pathway in Cerebral Ischemic Rats.

To investigate intermittent theta-burst stimulation (iTBS) effect on ischemic stroke and the underlying mechanism of neurorehabilitation, we developed an ischemia/reperfusion (I/R) injury model in Sprague-Dawley (SD) rats using the middle cerebral artery occlusion/reperfusion (MCAO/r) method. Next, using different behavioral studies, we compared the improvement of the whole organism with and without iTBS administration for 28 days. We further explored the morphological and molecular biological alterations associated with neuronal apoptosis and neuroinflammation by TTC staining, HE staining, Nissl staining, immunofluorescence staining, ELISA, small RNA sequencing, RT-PCR, and western blot assays. The results showed that iTBS significantly protected against neurological deficits and neurological damage induced by cerebral I/R injury. iTBS also significantly decreased brain infarct volume and increased the number of surviving neurons after 28 days. Additionally, it was observed that iTBS decreased synaptic loss, suppressed activation of astrocytes and M1-polarized microglia, and simultaneously promoted M2-polarized microglial activation. Furthermore, iTBS intervention inhibited neuronal apoptosis and exerted a positive impact on the neuronal microenvironment by reducing neuroinflammation in cerebral I/R injured rats. To further investigate the iTBS mechanism, this study was conducted using small RNA transcriptome sequencing of various groups of peri-infarcted tissues. Bioinformatics analysis and RT-PCR discovered the possible involvement of miR-34c-5p in the mechanism of action. The target genes prediction and detection of dual-luciferase reporter genes confirmed that miR-34c-5p could inhibit neuronal apoptosis in cerebral I/R injured rats by regulating the p53/Bax signaling pathway. We also confirmed by RT-PCR and western blotting that miR-34c-5p inhibited Bax expression. In conclusion, our study supports that iTBS is vital in inhibiting neuronal apoptosis in cerebral I/R injured rats by mediating the miR-34c-5p involvement in regulating the p53/Bax signaling pathway.

Bone Marrow Stromal Cell-Secreted Extracellular Vesicles Containing miR-34c-5p Alleviate Lung Injury and Inflammation in Bronchopulmonary Dysplasia Through Promotion of PTEN Degradation by Targeting OTUD3.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is the predominant chronic disorder in preterm neonates. This study explored impacts of miR-34c-5p carried by bone marrow stromal cells-secreted extracellular vesicles (BMSC-EVs) on BPD progression. METHODS: A BPD mouse model was established, followed by measurement of miR-34c-5p, OTUD3, and PTEN expression. EVs were isolated from BMSCs transfected with miR-34c-5p mimic or mimic NC and intratracheally injected into mice. CD31 and Ki67 expression was detected and the pathological changes of lung tissues and lung function indexes were observed for mice. A neonatal human pulmonary microvascular endothelial cell (HPMEC) model was developed with hyperoxia, followed by co-culture with extracted EVs and ectopic experiments for measurement of cell viability, migration, and angiogenesis. IL-4, IL-13, IL-1ß, and IL-6 levels were measured in cell supernatants and lung tissues. Dual-luciferase reporter, ubiquitination, Co-IP, and RIP assays were adopted to determine the relationship among miR-34c-5p, OTUD3, and PTEN. RESULTS: Lung tissues of BPD mice had downregulated miR-34c-5p expression and upregulated OTUD3 and PTEN expression. BMSC-EVs and BMSC-EVs-miR-34c-5p treatment improved lung injury and alveolar structure, decreased lung resistance and IL-4, IL-13, IL-1ß, and IL-6 levels, and elevated dynamic lung compliance in BPD mice, as well as enhanced proliferation, angiogenesis, and migration and restrained inflammation in HPMECs. Mechanistically, miR-34c-5p negatively targeted OTUD3 which restrained ubiquitination to promote PTEN protein stabilization. Upregulation of OTUD3 or PTEN negated the changes in the proliferation, angiogenesis, migration, and inflammation of hyperoxia-treated HPMECs induced by BMSC-EVs-miR-34c-5p. CONCLUSION: BMSC-EVs-miR-34c-5p alleviated lung injury and inflammation in hyperoxia-induced BPD by blocking the OTUD3/PTEN axis.

ING5 overexpression upregulates miR-34c-5p/Snail1 to inhibit EMT and invasion of lung cancer cells.

ING5 belongs to the inhibitor of growth (ING) candidate tumor suppressor family, which is involved in multiple cellular functions, such as cell cycle regulation, apoptosis, and chromatin remodelling. Previously, we reported that ING5 overexpression inhibits EMT by regulating EMT-related molecules, including Snail1, at the mRNA and protein levels. However, the mechanisms remain unclear. In the current study, we identify that ING5 overexpression induces the upregulation of miR-34c-5p. The expression levels of both ING5 and miR-34c-5p in NSCLC tissues from the TCGA database are decreased compared with that in adjacent tissues. Higher expression levels of both ING5 and miR-34c-5p predict longer overall survival (OS). Snail1 is the target gene of miR-34c-5p, as predicted by an online database, which is further verified by a dual-luciferase reporter assay. The expression level of Snail1 in NSCLC cells is markedly reduced following miR-34c-5p overexpression, leading to the inactivation of the Snail1 downstream TGF-ß/Smad3 signaling pathway. The TGF-ß signaling-specific inhibitor LY2157299 reverses the enhanced EMT, proliferation, migration, and invasion abilities induced by the miR-34c-5p inhibitor. Furthermore, tail vein injection of miR-34c-5p agomir inhibits xenografted tumor metastasis. Overall, this study concludes that miR-34c-5p, induced by ING5 overexpression, is a tumor suppressor that targets Snail1 and mediates the inhibitory effects of ING5 on the EMT and invasion of NSCLC cells. These results provide a novel mechanism mediating the antitumor effects of ING5.

Methylation detection of circulating tumor cell miR-486-5p/miR-34c-5p in the progression of colorectal cancer

Aims This study set out to examine the expression and methylation levels of miR-486-5p/miR-34c-5p and its mechanism of action based on the microRNA methylation level of circulating tumor cells (CTCs) in colorectal cancer (CRC) through clinical data and tissue detection. Methods EGFR and EpCAM immunophospholipid magnetic spheres (EpCAM-IML/EGFR-IML) were synthesized by the thin film method to capture CTCs in peripheral blood. The expression of miR-486-5p/miR-34c-5p was detected via real-time fluorescent quantitative PCR (RT-PCR). Methylation-specific PCR was implemented to detect the methylation level of miR-486-5p/miR-34c-5p, and 5-Aza-dC was used for demethylation treatment to detect the effect of changes in methylation levels on the tumor cells development. Cell Counting Kit-8 (CCK-8) analysis, transwell assay, and flow cytometry were used to determine the effects of demethylation and overexpression on the proliferation, invasion, migration, and apoptosis of CRC cells. Results The results showed that the expression and methylation levels of the miR-486-5p/miR-34c-5p isolated from CTCs were low and the methylation level was high in tumor cells and tissues. In CRC cell lines, demethylation and overexpression of miR-486-5p/miR-34c-5p could effectively inhibit the proliferation, invasion and migration of tumor cells, and facilitate tumor apoptosis (p < 0.05). Conclusion The constructed CTCs sorting system has characteristics of high specificity and high sensitivity, is a supplement to tissue samples, and has guiding significance for the clinical rational use of drugs and personalized therapy (AU)

ncRNA-mediated low expression of P2RY14 correlates with poor prognosis and tumor immune infiltration in ovarian carcinoma.

Background: Ovarian cancer (OV) has been puzzling clinicians because of its poor prognosis. More and more evidence show that the G protein coupled receptor P2RY14 plays a key role in the initiation and progression of various types of human cancer. The purpose of our study is to explore the correlation between P2RY14 and the prognosis of ovarian cancer patients and the relevant mechanism. Methods: First, the differentially expressed gene P2RY14 was screened from The Cancer Genome Atlas (TCGA) database. Explored possible P2RY14 related miRNAs and lncRNAs through multiple public databases, predicted and analyzed the expression level of candidate miRNAs and candidate lncRNAs that can bind to candidate miRNAs in OV through StarBase database. The TIMER database was used to comprehensively analyze the expression of tumor infiltrating immune cells, and to analyze the correlation between the expression level of P2RY14 and the level of immune cell infiltration in OV or the expression level of immune checkpoints. Results: Patients with P2RY14 overexpression had better overall survival (OS) and progression-free interval (PFI). In the Targetscan database, 22 upstream miRNAs that may bind to P2RY14 were predicted. According to the regulatory network constructed by the Cytoscape software, correlation analysis and the role of miRNAs in the prognosis of OV, we first determined that the candidate miRNAs were miR-34c-5p. Then, we predicted the upstream lncRNAs of miR-34c-5p in the StarBase database, the expression level of these lncRNAs in OV in the Gene Expression Profiling Interactive Analysis (GEPIA) database, and the role in prognosis. We determined that LINC00665 is the most potential lncRNA upstream of ovarian cancer miRNA (hsa-miR-34c-5p)-P2RY14. Then, we analyzed the results in the Timer database, suggesting that P2RY14 expression was positively correlated with CD8+T Cell, CD4+T Cell, Macrophage, Neutral and Dendritic cells, and negatively correlated with B cells. Meanwhile, P2RY14 was positively correlated with CD274 and PDCD1. Conclusions: P2RY14 can be used as a new predictive biomarker of ovarian cancer. Intervention of P2RY14 can affect the prognosis of ovarian cancer by affecting LINC00665-miR-34c-5p-P2RY14 axis. These findings provide a potential target for the development of anti-cancer strategies for ovarian cancer.

Sperm DNA Fragmentation and Sperm-Borne miRNAs: Molecular Biomarkers of Embryo Development?

The evaluation of morpho-functional sperm characteristics alone is not enough to explain infertility or to predict the outcome of Assisted Reproductive Technologies (ART): more sensitive diagnostic tools are needed in clinical practice. The aim of the present study was to analyze Sperm DNA Fragmentation (SDF) and sperm-borne miR-34c-5p and miR-449b-5p levels in men of couples undergoing ART, in order to investigate any correlations with fertilization rate, embryo quality and development. Male partners (n = 106) were recruited. Semen analysis, SDF evaluation and molecular profiling analysis of miR-34c-5p and miR-449b-5p (in 38 subjects) were performed. Sperm DNA Fragmentation evaluation- a positive correlation between SDF post sperm selection and the percentage of low-quality embryos and a negative correlation with viable embryo were found. SDF > 2.9% increased the risk of obtaining a non-viable embryo by almost 4-fold. Sperm miRNAs profile­we found an association with both miRNAs and sperm concentration, while miR-449b-5p is positively associated with SDF. Moreover, the two miRNAs are positively correlated. Higher levels of miR-34c-5p compared to miR-449b-5p increases by 14-fold the probability of obtaining viable embryos. This study shows that SDF, sperm miR-34c-5p, and miR-449b-5p have a promising role as biomarkers of semen quality and ART outcome.

Methylation detection of circulating tumor cell miR-486-5p/miR-34c-5p in the progression of colorectal cancer.

AIMS: This study set out to examine the expression and methylation levels of miR-486-5p/miR-34c-5p and its mechanism of action based on the microRNA methylation level of circulating tumor cells (CTCs) in colorectal cancer (CRC) through clinical data and tissue detection. METHODS: EGFR and EpCAM immunophospholipid magnetic spheres (EpCAM-IML/EGFR-IML) were synthesized by the thin film method to capture CTCs in peripheral blood. The expression of miR-486-5p/miR-34c-5p was detected via real-time fluorescent quantitative PCR (RT-PCR). Methylation-specific PCR was implemented to detect the methylation level of miR-486-5p/miR-34c-5p, and 5-Aza-dC was used for demethylation treatment to detect the effect of changes in methylation levels on the tumor cells development. Cell Counting Kit-8 (CCK-8) analysis, transwell assay, and flow cytometry were used to determine the effects of demethylation and overexpression on the proliferation, invasion, migration, and apoptosis of CRC cells. RESULTS: The results showed that the expression and methylation levels of the miR-486-5p/miR-34c-5p isolated from CTCs were low and the methylation level was high in tumor cells and tissues. In CRC cell lines, demethylation and overexpression of miR-486-5p/miR-34c-5p could effectively inhibit the proliferation, invasion and migration of tumor cells, and facilitate tumor apoptosis (p < 0.05). CONCLUSION: The constructed CTCs sorting system has characteristics of high specificity and high sensitivity, is a supplement to tissue samples, and has guiding significance for the clinical rational use of drugs and personalized therapy.

Periodontal ligament fibroblasts-derived exosomes induced by PGE2 inhibit human periodontal ligament stem cells osteogenic differentiation via activating miR-34c-5p/SATB2/ERK.

Several studies have confirmed that exosomes containing microRNAs (miRNAs) from the aseptic inflammatory microenvironment play an important role in bone remodeling. But the mechanism that induces changes in the osteogenic ability of periodontal ligament stem cells (PDLSCs) is still unclear. In the present study, the osteogenic function of periodontal ligament fibroblasts-derived exosomes induced by PGE2 on PDLSCs was detected by real-time PCR, alizarin red assay and alkaline phosphatase staining. High-throughput miRNAs sequencing was used to reveal that miR-34c-5p in exosomes-PGE2 was upregulated compared it in exosomes-normal. Real-time PCR and western blotting assay verified that overexpression of miR-34c-5p inhibited osteogenic differentiation, and reduced phosphorylation of ERK1/2. In addition, dual-luciferase reporter assay revealed that miR-34c-5p targeted special AT-rich sequence-binding protein 2 (SATB2). It was shown that exosomal miR-34c-5p inhibited osteogenic differentiation of PDLSCs via SATB2/ERK pathway.

Downregulation of miR-34c-5p alleviates chronic intermittent hypoxia-induced myocardial damage by targeting sirtuin 1.

Numerous microRNAs (miRs) are abnormally expressed in response to hypoxia-induced myocardial damage. Herein, miR-34c-5p as a potential pharmaco-target was investigated in a mouse model of chronic intermittent hypoxia (CIH)-induced myocardial damage. A mouse model of myocardial damage was established using CIH with 7% or 21% O2 alternating 60 s for 12 h/day, 21% O2 for 12 h/day. AntagomiR-34c-5p (20 nM/0.1 ml; once a week for 12 weeks) was used as a miR-34c-5p inhibitor in a mouse model with tail-vein injection. In another experiment, mice were administrated with Sirt1 activator SRT1720 (50 mg/kg/day) by intraperitoneal injection. Gene Expression Omnibus database showed a significant upregulation of miR-34c-5p expression in the ischemic myocardium of male mice. In CIH-stimulated mice, miR-34c-5p expression was also significantly increased compared with normal mice. Treatment of antagomiR-34c-5p significantly restrained CIH-triggered myocardial apoptosis. After administration of antagomiR-34c-5p or Sirt1 activator SRT1720, cardiac hypertrophy and oxidative stress were attenuated in CIH-stimulated mice. We also found sirtuin 1 (Sirt1) as a direct target of miR-34c-5p, which was able to mediate Sirt1 protein expression in cardiomyocytes. AntagomiR-34c-5p injection markedly elevated Sirt1 protein expression in CIH-stimulated mice. AntagomiR-34c-5p or Sirt1 activator SRT1720 administration exhibited the antioxidative activity and cardioprotective roles in CIH-stimulated mice.

Loss of exosomal miR-34c-5p in cancer-associated fibroblast for the maintenance of stem-like phenotypes of laryngeal cancer cells.

BACKGROUND: Cancer-associated fibroblasts (CAFs) reconstitute cancer stemness. This study aims to investigate whether the loss of CAF-derived exosomal miR-34c-5p contributes to the maintenance of stem-like properties of laryngeal squamous cell carcinoma (LSCC). METHODS: Exosomes from primarily cultured CAFs and paired normal fibroblasts (NFs) were collected and identified. The differential expression of exosomal miR-34c-5p between CAFs and NFs was detected by next-generation sequencing. In vitro and in vivo assays were performed to examine the effects of miR-34c-5p on the maintenance of stem-like properties. RESULTS: MiR-34c-5p expression is significantly reduced in CAF-derived exosomes. In vitro and in vivo assays revealed that exosomal miR-34c-5p can regulate the stem-like properties of LSCC cells, such as proliferation, invasion, sphere and plate colony formation, chemoresistance, tumorigenicity in nude mice, as well as the expression of cancer stem cell genes. CONCLUSIONS: Loss of miR-34c-5p in CAF-derived exosomes contributes to the maintenance of stem-like phenotypes of LSCC.

Ursolic acid represses influenza A virus-triggered inflammation and oxidative stress in A549 cells by modulating the miR-34c-5p/TLR5 axis.

BACKGROUND: Ursolic acid (UA) is a pentacyclic triterpenoid compound with a wide range of anti-tumor, anti-inflammatory, hypotensive and other pharmacological effects. Here, the biological roles and regulatory mechanisms of UA in influenza A virus (IAV)-treated A549 cells were investigated. METHOD: The cytotoxic impacts of UA on A549 cells with or without IAV treatment were determined using MTT and LDH assays. The inflammatory responses and oxidative stress of IAV-treated A549 cells were measured by RT-qPCR, ELISA, DCFH-DA probe, and colorimetric assays. A dual luciferase assay was carried out to validate the molecular interaction between miR-34c-5p and TLR5. Promoter methylation was detected by MSP experiment. Methylation-related proteins were quantified by western blot. Virus replication was assessed by TCID50 and western blot assays. RESULTS: UA significantly ameliorated IAV-triggered cell injury and inflammatory response, virus replication and oxidative stress by elevating cell viability, ROS level and the activities of SOD and GSH-Px but reducing the LDH, MDA, and TCID50 values and the expression of virus-related proteins (NP) and cytokines (TNF-α, IL-1ß, IL-6, and IL-18). Moreover, UA promoted miR-34c-5p expression by repressing DNMTs-mediated methylation. TLR5 was verified to be a direct target of miR-34c-5p and could be downregulated by UA. Rescue experiments revealed that silencing miR-34c-5p diminished the regulatory roles of UA in IAV-treated A549 cells. CONCLUSION: Our data elucidated that UA attenuated IAV-triggered inflammatory responses and oxidative stress in A549 cells by regulating the miR-34c-5p/TLR5 axis, suggesting that UA plays a protective role in IAV-induced pneumonia.