Senescence induces miR-409 to down-regulate CCL5 and impairs angiogenesis in endothelial progenitor cells.

This study explores the impact of senescence on autocrine C-C motif chemokine ligand 5 (CCL5) in human endothelial progenitor cell (EPCs), addressing the poorly understood decline in number and function of EPCs during ageing. We examined the effects of replication-induced senescence on CCL5/CCL5 receptor (CCR5) signalling and angiogenic activity of EPCs in vitro and in vivo. We also explored microRNAs controlling CCL5 secretion in senescent EPCs, its impact on EPC angiogenic activity, and validated our findings in humans. CCL5 secretion and CCR5 levels in senescent EPCs were reduced, leading to attenuated angiogenic activity. CCL5 enhanced EPC proliferation via the CCR5/AKT/P70S6K axis and increased vascular endothelial growth factor (VEGF) secretion. Up-regulation of miR-409 in senescent EPCs resulted in decreased CCL5 secretion, inhibiting the angiogenic activity, though these negative effects were counteracted by the addition of CCL5 and VEGF. In a mouse hind limb ischemia model, CCL5 improved the angiogenic activity of senescent EPCs. Analysis involving 62 healthy donors revealed a negative association between CCL5 levels, age and Framingham Risk Score. These findings propose CCL5 as a potential biomarker for detection of EPC senescence and cardiovascular risk assessment, suggesting its therapeutic potential for age-related cardiovascular disorders.

Long non-coding RNA Small Nucleolar RNA Host Gene 4 ameliorates cigarette smoke-induced proliferation, apoptosis, inflammation, and airway remodeling in alveolar epithelial cells through the modulation of the mitogen-activated protein kinase signaling pathway via the microRNA-409-3p/Four and a Half LIM Domains 1 axis.

The long non-coding RNA (lncRNA) Small Nucleolar RNA Host Gene 4 (SNHG4) has been demonstrated to be significantly downregulated in various inflammatory conditions, yet its role in chronic obstructive pulmonary disease (COPD) remains elusive. This study aims to elucidate the biological function of SNHG4 in COPD and to unveil its potential molecular targets. Our findings reveal that both SNHG4 and Four and a Half LIM Domains 1 (FHL1) were markedly downregulated in COPD, whereas microRNA-409-3p (miR-409-3p) was upregulated. Importantly, SNHG4 exhibited a negative correlation with inflammatory markers in patients with COPD, but a positive correlation with forced expiratory volume in 1s percentage (FEV1%). SNHG4 distinguished COPD patients from non-smokers with high sensitivity, specificity, and accuracy. Overexpression of SNHG4 ameliorated cigarette smoke extract (CSE)-mediated inflammation, apoptosis, oxidative stress, and airway remodeling in 16HBE bronchial epithelial cells. These beneficial effects of SNHG4 overexpression were reversed by the overexpression of miR-409-3p or the silencing of FHL1. Mechanistically, SNHG4 competitively bound to miR-409-3p, mediating the expression of FHL1, and consequently improving inflammation, apoptosis, oxidative stress, and airway remodeling in 16HBE cells. Additionally, SNHG4 regulated the miR-409-3p/FHL1 axis to inhibit the activation of the mitogen-activated protein kinase (MAPK) pathway induced by CSE. In a murine model of COPD, knockdown of SNHG4 exacerbated CSE-induced pulmonary inflammation, apoptosis, and oxidative stress. In summary, our data affirm that SNHG4 mitigates pulmonary inflammation, apoptosis, and oxidative damage mediated by COPD through the regulation of the miR-409-3p/FHL1 axis.

CircAGFG1 Promotes Ovarian Cancer Progression Through the miR-409-3 p/ZEB1 Axis.

OBJECTIVES: Circular RNAs (circRNAs) serve a crucial regulatory role in ovarian cancer (OC). Circular RNA ArfGAP with FG repeats 1 (circAGFG1) has been shown to be involved in promoting the progression of several cancers, containing triple-negative breast cancer, esophageal cancer and colorectal cancer. However, the function of circAGFG1 in OC is unclear. METHODS: Quantitative real-time reverse transcription PCR (RT-qPCR) was conducted to determine the expression levels of circAGFG1 and miR-409-3p. The proliferation and metastasis of cells were determined by colony formation assays, EdU assays, transwell assays and wound healing assays. In addition, a dual-luciferase reporter assay was performed to validate the mechanism between circAGFG1, miR-409-3p, and ZEB1. RESULTS: Our data suggested that circAGFG1 was significantly overexpressed in OC tissues compared to normal ovarian epithelial tissues. Overexpression of circAGFG1 was correlated with intraperitoneal metastasis, tumor recurrence and advanced stage. Additionally, circAGFG1 overexpression revealed a poor prognosis in OC patients. Knockdown of circAGFG1 suppressed the proliferation, invasion and migration of OC cells. Mechanistically, circAGFG1 acted as a sponge of miR-409-3p to enhance the expression level of zinc finger E-box binding homeobox 1 (ZEB1), thereby conferring OC cell proliferation, invasion and migration. Importantly, re-expression of ZEB1 effectively reversed the effects of circAGFG1 knockdown on OC cells. CONCLUSIONS: In summary, our study indicated that circAGFG1 may act as a prognostic biomarker and potential therapeutic target for patients with OC.

Investigating the molecular mechanisms of microRNA­409­3p in tumor progression: Towards targeted therapeutics (Review).

MicroRNAs (miRNAs) are a group of non­coding RNAs that exert master regulatory functions in post­-transcriptional gene expression. Accumulating evidence shows that miRNAs can either promote or suppress tumorigenesis by regulating different target genes or pathways and may be involved in the occurrence of carcinoma. miR­409­3p is dysregulated in a variety of malignant cancers. It plays a fundamental role in numerous cellular biological processes, such as cell proliferation, apoptosis, migration, invasion, autophagy, angiogenesis and glycolysis. In addition, studies have shown that miR­409­3p is expected to become a non­invasive biomarker. Identifying the molecular mechanisms underlying miR­409­3p­mediated tumor progression will help investigate miR­409­3p­based targeted therapy for human cancers. The present review comprehensively summarized the recently published literature on miR­409­3p, with a focus on the regulation and function of miR­409­3p in various types of cancer, and discussed the clinical implications of miR­409­3p, providing new insight for the diagnosis and treatment of cancers.

Morroniside improves the symptoms of post-stroke depression in mice through the BDNF signaling pathway mediated by MiR-409-3p.

BACKGROUND: Post-stroke depression (PSD) is a common psychiatric symptom after a stroke. Morroniside, an iridoid glycoside found in Cornus officinalis, has garnered significant attention for its potential to alleviate symptoms associated with depression. PURPOSE: This study aims to highlight the potential use of morroniside in the treatment of PSD and elucidate the underlying molecular mechanisms. METHODS: To establish a reliable PSD model, male C57BL/6 mice were subjected to brief MCAO in conjunction with CUMS. Post-morroniside administration, neuronal viability, and hippocampal cell apoptosis were evaluated by Nissl staining and TUNEL detection, respectively. Depression-like behaviors were evaluated using SPT, TST, and FST. The Longa score and cylinder test were used to evaluate the effect of morroniside on motor function. Furthermore, to investigate the underlying molecular mechanisms, bioinformatic analysis and the dual luciferase assay were performed to investigate the MiR-409-3p-BDNF interaction. In addition, subsequent to MiR-409-3p overexpression via AAV virus, we assessed mRNA expression and protein levels of key components within the BDNF/TrkB signaling pathway using RT-qPCR, immunohistochemistry, and western blot analysis. RESULTS: The observed decrease in apoptosis and amelioration of depression-like behaviors strongly indicate the potential of morroniside as a therapeutic agent for PSD. Furthermore, the upregulation of key proteins within the BDNF/TrkB signaling pathway in the cortex suggests that morroniside activates this pathway. Through bioinformatics analysis, MiR-409-3p was identified and found to bind to the BDNF gene, resulting in the inhibition of BDNF expression. Importantly, we demonstrate that morroniside mitigates this inhibitory effect of MiR-409-3p on BDNF, thereby facilitating the activation of the BDNF/TrkB signaling pathway. CONCLUSION: The findings suggest that morroniside demonstrates the ability to improve PSD symptoms through the BDNF/TrkB signaling pathway mediated by MiR-409-3p. These results emphasize the importance of the BDNF signaling pathway in improving PSD symptoms and provide a possible mechanism for morroniside to treat PSD.

circFOXK2 Regulates Proliferation,Migration and Invasion of Lung Cancer Cells by Targeting miR-409-3p / 华中科技大学学报(医学版)

Objective To investigate the effect of circFOXK2 on the proliferation,migration and invasion of lung cancer cells and its possible mechanism.Methods The cancer tissues and adjacent tissues of 45 patients with lung cancer were collect-ed,and the expression of circFOXK2 and miR-409-3p in the tissues were detected by qRT-PCR.Lung cancer cell A549 and H1299 was cultured in vitro,and the dual-luciferase reporter assay was used to verify the regulatory relationship between circ-FOXK2 and miR-409-3p.The CCK-8,clone formation and Transwell assays were used to detect cell proliferation,migration and invasion.Western blot was used to detect the protein expression of E-cadherin and N-cadherin in cells.Results The expression of circFOXK2 was significantly higher(P＜0.05),but the expression of miR-409-3p was significantly lower(P＜0.05)in lung cancer tissues.circFOXK2 could target miR-409-3p and negatively regulate its expression in lung cancer cells.The proliferation,migration and invasion were reduced in circFOXK2(all P＜0.05),N-cadherin level was decreased(P＜0.05),but E-cadherin level was increased(P＜0.05)in circFOXK2 knockdown or miR-409-3p overexpressed A549 and H1299 cells.The inhibitory effects of circFOXK2 knockdown on the proliferation,migration and invasion of lung cancer cells could be reversed by miR-409-3p inhibitor.Conclusion The circFOXK2 was upregulated in lung cancer,which promoted the proliferation,migration and inva-sion of lung cancer cells by targeting miR-409-3p.

circMSH3 is a potential biomarker for the diagnosis of colorectal cancer and affects the distant metastasis of colorectal cancer.

Objectives: To identify the most significantly differentially expressed circular RNAs (circRNAs) in colorectal cancer (CRC) tissues in terms of their expression levels and circularity, and to analyze the relationship between their expression levels and the clinical characteristics of patients. Methods: circRNA RNA-seq technology was used to screen differentially expressed circRNAs in CRC. Sanger sequencing was used to identify circRNA back-splice junction sites. The relative expression levels of hsa_circ_0003761 (circMSH3) in CRC tissues and cell lines were detected by quantitative real-time fluorescence PCR technology. An RNA-protein pull-down assay was used to detect protein binding to circRNAs. Dual-luciferase reporter gene vectors were constructed to verify that circRNAs bind to microRNAs. Results: Four hundred twenty circRNAs were found to be upregulated, and 616 circRNAs were downregulated. circMSH3 was derived from the MutS homolog 3 (MSH3) gene and was formed by a loop of exons 9, 10, 11, and 12. In 110 pairs of CRC and adjacent tissues, circMSH3 expression was 4.487-fold higher in CRC tissues. circMSH3 was also highly expressed in the HT-29 and LOVO CRC cell lines. The expression level of circMSH3 was associated with distant metastasis in CRC patients (P = 0.043); the area under the curve (AUC) of circMSH3 for CRC diagnosis was 0.75, with a sensitivity and specificity of 70.9% and 66.4%, respectively. circMSH3 could bind to a variety of proteins, mainly those involved in RNA transcription, splicing, cell cycle, and cell junctions. Furthermore, circMSH3 could bind to miR-1276, miR-942-5p, and miR-409-3p. Conclusion: circMSH3 is a potential biomarker for the diagnosis of CRC and affects the distant metastasis of CRC. Multiple RNA-binding protein binds to circMSH3, and circMSH3 binds to miR-1276, miR-942-5p, and miR-409-3p, thereby affecting the expression of circMSH3.

Circ_0099630 knockdown alleviates lipopolysaccharide-induced injuries of human periodontal ligament cells through the inhibition of TLR4 by releasing miR-409-3p.

BACKGROUND: Periodontitis triggers tooth loss and affects the health of population worldwide. Emerging evidence hints that circular RNAs (circRNAs) are involved in various diseases, including periodontitis. This study aimed to investigate the role of circ_0099630 in the progression of periodontitis. METHODS: Periodontitis cell model was constructed by treating human periodontal ligament cells (HPDLCs) with lipopolysaccharide (LPS). Quantitative real-time PCR was used to analyze the expression of circ_0099630, microRNA-409-3p (miR-409-3p) and toll-like receptor 4 (TLR4) mRNA. Western blot was used for detecting protein levels of TLR4, cleaved-caspase 3, Bcl-2, CyclinD1 and NF-κB signaling markers. For function analyses, cell proliferation was assessed by CCK-8 assay and EdU assay. The releases of pro-inflammation factors were monitored by ELISA kits. The potential relationship between miR-409-3p and circ_0099630 or TLR4 was verified by dual-luciferase reporter assay, RIP assay and pull-down assay. RESULTS: The expression of circ_0099630 and TLR4 was elevated in periodontitis patients and LPS-treated HPDLCs. LPS induced HPDLC proliferation inhibition, apoptosis and inflammatory responses, while circ_0099630 knockdown or TLR4 knockdown alleviated these injuries. Besides, TLR4 overexpression reversed the inhibitory effect of circ_0099630 knockdown on LPS-induced HPDLC injuries. Mechanism analysis showed that circ_0099630 positively regulated TLR4 expression by acting as miR-409-3p sponge. MiR-409-3p restoration largely ameliorated LPS-induced HPDLC injuries by depleting TLR4. Moreover, LPS activated the NF-κB signaling pathway, while circ_0099630 knockdown inhibited the activity of NF-κB signaling via the miR-409-3p/TLR4 axis. CONCLUSION: Circ_0099630 knockdown relieved LPS-induced HPDLC injury by miR-409-3p/TLR4 axis, suggesting that circ_0099630 might be a potential target for periodontitis treatment.

MiR-409-3p regulates the proliferation and apoptosis of THP-1 through targeting Rab10.

Acute myeloid leukemia cytogenetics and molecular subtypes are connected with microRNAs, although it is unclear how miRNAs affect AML pathogenesis. miR-409-3p expression is downregulated in bone marrows, as we have previously demonstrated in our team. Nevertheless, the tumor-suppressing activities and molecular mechanisms of miR-409-3p remain unknown. Hence, in this study, we investigate at the functional significance of miR-409-3p in the development of AML. We found that a significant decrease in miR-409-3p expression was observed in THP-1 cell. The expression of miR-409-3p was altered in THP-1 by transfecting with agomiR-409-3p and agomiR-409-3p NC. A series of experiments showed that overexpression of miR-409-3p expression significantly suppressed proliferation and increased the apoptosis of THP-1. Moreover, Rab10 was confirmed as a direct target gene of miR-409-3p and was negatively modulated by miR-409-3p. Rab10 downregulation imitated the suppressed proliferation and increased the apoptosis of THP-1. Furthermore, miR-409-3p overexpression or Rab10 knockdown obviously down-regulated the expression levels of Bcl-2, but up-regulated Bax expression. In a xenograft mouse model, miR-409-3p-overexpressed THP-1 cells resulted in much less tumor weight and size in the mice bearing the cells as compared to the mock-transfected mice. Collectively, our findings demonstrated that miR-409-3p exerted tumor suppressor gene effects in AML by directly targeting Rab10, which might provide a promising therapeutic target for AML.

LINC00886 Facilitates Hepatocellular Carcinoma Tumorigenesis by Sequestering microRNA-409-3p and microRNA-214-5p.

Purpose: As the major subtype of liver cancer, hepatocellular carcinoma (HCC) suffers from high mortality and is prone to recurrence. Long non-coding RNAs (lncRNAs) are well characterized to be pivotal players contributing to HCC pathogenesis and progression. Therefore, this study intended to probe the biological functions of LINC00886 in hepatocarcinogenesis. Patients and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to analysis of LINC00886, microRNA-409-3p (miR-409-3p), microRNA-214-5p (miR-214-5p), RAB10 and E2F2 expression. Subcellular localization of LINC00886 was identified through a fluorescent in situ hybridization (FISH) kit and a subcellular assay. Additionally, proliferated cells were determined with EdU as well as cell counting kit-8 (CCK-8) assays. Scratch and Transwell assays were applied to detect migratory and invasive cells. Apoptotic cells were measured via TUNEL staining assay. Furthermore, targeted binding between LINC00886 and miR-409-3p or miR-214-5p was validated utilizing dual-luciferase reporter assays. RAB10, E2F2 and NF-κB signaling-associated protein levels were evaluated utilizing Western blot. Results: LINC00886, RAB10 and E2F2 levels were aberrantly increased, with the abnormal expressed decline of miR-409-3p and miR-214-5p, in HCC tissues, cells and peripheral blood mononuclear cells (PBMCs). Silencing LINC00886 attenuated the proliferative, migratory, invasive, and anti-apoptotic potential of HCC cells, while LINC00886 overexpression proceeded in the contrary direction. Mechanistically, miR-409-3p and miR-214-5p were validated as binding targets for LINC00886 and inverted the biological functions of LINC00886 during HCC progression. Furthermore, the LINC00886-miR-409-3p/miR-214-5p axis could regulate RAB10 and E2F2 expression via mediating NF-κB pathway activation in hepatocarcinogenesis. Conclusion: Our findings indicated that LINC00886 facilitated HCC progression via absorbing miR-409-3p or miR-214-5p to upregulate RAB10 and E2F2 through activation of NF-κB pathway, offering a promising novel target for HCC therapy.

BRF2 is mediated by microRNA-409-3p and promotes invasion and metastasis of HCC through the Wnt/ß-catenin pathway.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Its invasiveness and ability to metastasize contributes to an extremely high patient mortality. However, the molecular mechanisms that underlie the characteristics of HCC progression are not well understood. BRF2 has been shown to be an oncogene in a number of tumors; however, its role in HCC has not yet been thoroughly examined. In this study, we identified and validated BRF2 as an oncogene in HCC, providing a new insight into HCC pathogenesis and therapeutic possibilities. We showed that BRF2 expression was significantly upregulated in HCC cell lines and tissues, while BRF2 depletion suppressed HCC metastasis and invasion. We then examined the upstream regulation of BRF2 and identified miR-409-3p as being predicted to bind to the 3' UTR of BRF2. We used a luciferase activity assay and functional verification to show that BRF2 is downregulated by miR-409-3p. Finally, we used bioinformatic analysis to show that BRF2 may be related to early HCC development through the Wnt/ß-catenin signaling pathway.

Hsa-miR-409-3p regulates endothelial progenitor senescence via PP2A-P38 and is a potential ageing marker in humans.

We explored the roles of hsa-microRNA (miR)-409-3p in senescence and signalling mechanism of human endothelial progenitor cells (EPCs). Hsa-miR-409-3p was found upregulated in senescent EPCs. Overexpression of miRNA mimics in young EPCs inhibited angiogenesis. In senescent EPCs, compared to young EPCs, protein phosphatase 2A (PP2A) was downregulated, with activation of p38/JNK by phosphorylation. Young EPCs treated with siPP2A caused inhibited angiogenesis with activation of p38/JNK, similar to findings in senescent EPCs. Time series analysis showed, in young EPCs treated with hsa-miR-409-3p mimics, PP2A was steadily downregulated for 72 h, while p38/JNK was activated with a peak at 48 hours. The inhibited angiogenesis of young EPCs after miRNA-409-3p mimics treatment was reversed by the p38 inhibitor. The effect of hsa-miR-409-3p on PP2A signalling was attenuated by exogenous VEGF. Analysis of human peripheral blood mononuclear cells (PBMCs) obtained from healthy people revealed hsa-miR-409-3p expression was higher in those older than 65 years, compared to those younger than 30 years, regardless of gender. In summary, hsa-miR-409-3p was upregulated in senescent EPCs and acted as a negative modulator of angiogenesis via targeting protein phosphatase 2 catalytic subunit alpha (PPP2CA) gene and regulating PP2A/p38 signalling. Data from human PBMCs suggested hsa-miR-409-3p a potential biomarker for human ageing.

The Effect of MicroRNA-409-3p for Treatment and Response to Tumor Proliferation of Lung Cancer Cell Lines (In Vitro).

OBJECTIVE: Monitoring the result of miR 409 3p and its response to tumor proliferation and its mechanism of action on some types of lung cancer in vitro (A549 cell line). METHODS: Two A549 cell line group negative control group with oligonucleotide cultured under conventional conditions and transfected with positive control nucleotides. Experiments Based on the control group, chemically synthesized miR 409 3p mimics were used in the positive group with liposome transfection to construct A549 cells with high miR-409-3p expression. RESULT: miR4093p expression was estimated using the qPCR method in the two groups after 48 h. Later, the miR-409-3p expression in A549 cells obviously increased significantly with a positive attitude in the positive control group that was transfected by miR-409-3p (mimics) (P<0.20). As a result of this investigation, a significant increase in the percentage of total cell apoptosis was significantly increased in the positive group compared to the control group (22.68% ± 4.62%), (7.79%±1.94%) respectively, (P<0.05). However, in terms of the G1 phase, the rate is obviously low compared to the control group (40.22%±5.36%); (56.08%±5.21%) (P<0.05). endogenous ELF2 was considerably reduced after overexpression of the miR-409-3p mimic (P<0.05). CONCLUSION: miR-409-3p may prevent non-small cell lung cancer (NSCLC) by affecting ELF2 transcription and other cellular regulators to regulate A549 cell division and induce apoptosis.

Knockdown circTRIM28 enhances tamoxifen sensitivity via the miR-409-3p/HMGA2 axis in breast cancer.

BACKGROUND: Tamoxifen (TAM) is a frequently-used treatment for breast cancer (BC). But the TAM resistance seriously affects the patient therapeutic effect. Previous research indicated that circular RNAs (circRNAs) might participate in the regulatory processes of BC. Here, we discovered the parts of circular RNA tripartite motif-containing 28 (circTRIM28) in BC. METHODS: CircTRIM28, microRNA-409-3p (miR-409-3p), and high mobility group AT-hook 2 (HMGA2) levels were perceived by qRT-PCR and western blot. Moreover, the biological functions of the cells were examined. Furthermore, dual-luciferase report was employed to reconnoiter the targeted relationship between miR-409-3p and circTRIM28 or HMGA2. RESULTS: CircTRIM28 and HMGA2 were augmented, and the miR-409-3p was repressed in BC. Silencing circTRIM28 enhanced tamoxifen sensitivity and cell apoptosis, whereas hampered cell development in BC cells. In mechanism, circTRIM28 could sponge miR-409-3p to increase HMGA2. In addition, silencing circTRIM28 impeded tumor growth. CONCLUSION: CircTRIM28 facilitated the BC via miR-409-3p/HMGA2.

Hsa_circ_0079530/AQP4 Axis Is Related to Non-Small Cell Lung Cancer Development and Radiosensitivity.

BACKGROUND: Circular RNAs are associated with non-small cell lung cancer (NSCLC) development and radiosensitivity. Nevertheless, the function and regulation mechanism of hsa_circ_0079530 (circ_0079530) in NSCLC development and radiosensitivity are largely unknown. METHODS: The abundances of circ_0079530, microRNA (miR-409-3p), aquaporin 4 (AQP4), E-cadherin, intercellular adhesion molecule-1, vitronectin, proliferating cell nuclear antigen, and matrix metalloproteinase 9 were determined via quantitative reverse transcription polymerase chain reaction or western blotting. Cell proliferation, survival fraction, cycle process, migration, invasion, and in vivo growth were examined by cell counting kit-8, colony formation, flow cytometry, transwell, and xenograft analyses. The binding relationship was assessed via dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Circ_0079530 expression was increased in NSCLC tissues and radioresistant samples. Circ_0079530 knockdown restrained cell proliferation, migration, and invasion, and facilitated radiosensitivity. Circ_0079530 silence decreased tumor growth with or without radiation treatment. Circ_0079530 was verified as a miR-409-3p sponge, and miR-409-3p downregulation mitigated the effects of circ_0079530 interference on NSCLC cell malignancy and radiosensitivity. AQP4 was directly targeted by miR-409-3p. MiR-409-3p restrained cell proliferation, migration, and invasion, and enhanced radiosensitivity by decreasing AQP4 expression. Notably, circ_0079530 silence decreased AQP4 expression by regulating miR-409-3p expression. CONCLUSION: Circ_0079530 silence repressed cell proliferation, migration, and invasion, and facilitated radiosensitivity in NSCLC cells by mediating miR-409-3p/AQP4 axis.

Post-Transcriptional Effects of miRNAs on PCSK7 Expression and Function: miR-125a-5p, miR-143-3p, and miR-409-3p as Negative Regulators.

The regulatory mechanism of PCSK7 gene is still unknown, although its encoded protein PC7 is the most ancient and highly conserved of all proprotein convertases and exhibits enzymatic and non-enzymatic functions in liver triglyceride regulation. Bioinformatics algorithms were used to predict regulatory microRNAs (miRNAs) of PCSK7 expression. This led to the identification of four miRNAs, namely miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p, with potential binding sites on the 3'-untranslated region (3'-UTR) of human PCSK7 mRNA. The expression patterns of these miRNAs and PCSK7 mRNA were assessed in three different cell lines with quantitative polymerase chain reaction (qPCR), which revealed reciprocal expression patterns between the expression levels of the four selected miRNAs and PCSK7. Next, the interactions and effects of these miRNAs on PCSK7 expression levels were investigated via cell-based expression analysis, dual-luciferase assay, and Western blot analysis. The data revealed that PCSK7 mRNA levels decreased in cells transfected with vectors overexpressing miR-125a-5p, miR-143-3p, and miR-409-3p, but not miR-320a-3p. The dual-luciferase assay demonstrated that the above three miRNAs could directly interact with putative target sites in PCSK7 3'-UTR and regulate its expression, whereas miR-320-3p exhibited no interaction. Western blot analysis further revealed that the overexpression of miR-125a-5p in Huh7 cells inhibits the expression and ability of PC7 to cleave human transferrin receptor 1. Our results support a regulatory role of these miRNAs on PCSK7 expression and function and open the way to assess their roles in the regulation of PC7 activity in vivo in the development of hepatic steatosis.

Identification and Validation of miR-222-3p and miR-409-3p as Plasma Biomarkers in Gestational Diabetes Mellitus Sharing Validated Target Genes Involved in Metabolic Homeostasis.

Gestational diabetes mellitus (GDM) causes both maternal and fetal adverse outcomes. The deregulation of microRNAs (miRNAs) in GDM suggests their involvement in GDM pathogenesis and complications. Exosomes are extracellular vesicles (EVs) of endosomal origin, released via exocytosis into the extracellular compartment. Through EVs, miRNAs are delivered in distant target cells and are able to affect gene expression. In this study, miRNA expression was analyzed to find new miRNAs that could improve GDM classification and molecular characterization. MiRNA were profiled in total plasma and EVs in GDM patients and normal glucose tolerance (NGT) women. Samples were collected at third trimester of gestation from two diabetes centers. MiRNA expression was profiled in a discovery cohort using the multiplexed NanoString nCounter Human v3 miRNA. Validation analysis was performed in a second independent cohort using RT-qPCR. A set of miRNAs resulted to be differentially expressed (DE) in total plasma and EVs in GDM. Among them, total plasma miR-222-3p and miR-409-3p were validated in the independent cohort. MiR-222-3p levels correlated with fasting plasma glucose (FPG) (p < 0.001) and birth weight (p = 0.012), whereas miR-409-3p expression correlated with FPG (p < 0.001) and inversely with gestational age (p = 0.001). The major validated target genes of the deregulated miRNAs were consistently linked to type 2 diabetes and GDM pathophysiology. MiR-222-3p and miR-409-3p are two circulating biomarkers that could improve GDM classification power and act in the context of the molecular events leading to the metabolic alterations observed in GDM.

LINC00630 as a miR-409-3p sponge promotes apoptosis and glycolysis of colon carcinoma cells via regulating HK2.

Long-chain non-coding RNAs (lncRNAs) belong to the family of non-coding RNAs and contain more than 200 nucleotides. They are involved in the growth, apoptosis, and glycolysis of carcinoma cells. A newly discovered lncRNA, LINC00630, has been reported in colon carcinoma. In this study, we found that the expression of LINC00630 was remarkably upregulated in colon carcinoma tissues and cell lines compared with that in adjacent tissues and the NCM-460 cell lines. Knocking out LINC00630 resulted in inhibition of proliferation and glycolysis but increase in apoptosis. In addition, we confirmed the direct interaction between LINC00630 and miR-409-3p in colon carcinoma cells using bioinformatics methods and dual luciferase reporter gene assay. Finally, we demonstrated that LINC00630 could promote cell growth and glycolysis and inhibit apoptosis by functioning as a miR-409-3p sponge, and further regulate hexokinase 2 (HK2) in colon carcinoma cells. Our results confirmed that LINC00630 regulates proliferation, glycolysis, and apoptosis mainly through targeting the miR-409-3p/HK2 axis, which may explain the progression of colon carcinoma and provide a potential target for the treatment of colon carcinoma.

Hsa_circ_0000418 promotes the progression of glioma by regulating microRNA-409-3p / pyruvate dehydrogenase kinase 1 axis.

Glioma is the commonest intracranial malignancy, and circRNAs are important regulatory factors which are implicated in the development of glioma. Nonetheless, the role of circRNAs in glioma is largely unknown. The research is performed to elaborate on the biological role of has_circ_0000418 (circ_0000418) in glioma progression and its potential molecular mechanism. The differentially expressed circRNAs in glioblastoma patient derived cells and neural progenitor cells were analyzed based on the microarray data of GSE146463. Additionally, qRT-PCR and Western blot experiments were conducted to measure the expression of circ_0000418, microRNA-409-3p (miR-409-3p) and pyruvate dehydrogenase kinase 1 (PDK1) in glioma tissues/cells. Cell growth and cell cycle distribution were monitored using CCK-8 assay, BrdU assay and flow cytometry. Bioinformatics prediction, dual-luciferase reporter gene experiment and RIP assay were conducted to verify the targeting relationship between circ_0000418 and miR-409-3p, miR-409-3p and PDK1 3'UTR. In this work, we observed that, circ_0000418 expression level was significantly up-regulated in glioma tissues and cell lines. Circ_0000418 overexpression facilitated glioma cell growth and accelerated cell cycle progression, while knockdown of circ_0000418 produced the opposite effects. Circ_0000418 specifically combined with miR-409-3p, and circ_0000418 negatively modulated the expression of miR-409-3p. PDK1 acted as a target gene of miR-409-3p, and PDK1 could be positively and indirectly modulated by circ_0000418 in glioma cells. In summary, circ_0000418 enhances glioma cell growth and accelerates cell cycle progression by regulating miR-409-3p/PDK1 axis.

Administration of rTMS Alleviates Stroke-Induced Cognitive Deficits by Modulating miR-409-3p/CTRP3/AMPK/Sirt1 Axis.

Cognitive deficit is a typical complication induced by stroke injuries. Repetitive transcranial magnetic stimulation (rTMS) is a technique that can both attenuate neuropsychiatric disorders and influence miR levels. We attempted to assess effects of rTMS on post-stroke cognitive deficit (PSCD) by focusing on the activity of miR-409-3p/CTRP3/AMPK/Sirt1 axis. PSCD was induced in rats using middle cerebral artery occlusion (MCAO) method and handled with rTMS. MiRs responding to rTMS administration were determined using microarray method. Changes in cognitive function, brain histological feature, neuron apoptosis, and activity of miR-409-3p/CTR3/AMPK/Sirt1 axis were detected. The interaction between of miR-409-3p and rTMS was verified by inducing its level in MCAO rats. rTMS influenced levels of miRs in MCAO rats, with 104 miRs being upregulated and 249 s miR being downregulated, contributing to the function changes in multiple biological processes. Moreover, the technique improved brain function and structure in model rats. At the molecular level, rTMS inhibited miR-409-3p and activated CTRP3/AMPK/Sirt1 pathway. After the induction of miR-409-3p, effects of rTMS were counteracted, which were represented by the impaired cognitive function and neuron viability in model rats. Collectively, rTMS could protect against stroke-induced cognitive deficits, which depended on the inhibition of miR-409-3p level.