miR-16-5p specifically inhibits IFN-γ-regulated memory T helper cell differentiation in malignant pleural effusion of non-small cell lung cancer.

Background: The mechanism for memory T helper (Th) cell differentiation in malignant pleural effusion (MPE) of non-small cell lung cancer (NSCLC) is poorly understood. MicroRNAs (miRNAs), as small non-coding RNA that regulate gene expression, play a crucial role in the regulation of memory Th cell differentiation. However, whether miRNAs can inhibit the differentiation of memory Th cells in MPE of NSCLC has not been reported. This study aimed to explore miR-16-5p specifically inhibits interferon-gamma (IFN-γ)-regulated memory Th cell differentiation in MPE of NSCLC. Methods: A total of 30 patients with NSCLC and 30 age- and sex-matched patients, who were clinically diagnosed as benign pleural effusion (BPE) of lung disease and had not received any intervention, were collected. The expression of nucleic acids, miRNAs, and cytokines was detected by polymerase chain reaction (PCR), miRNA microarray, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and western blotting. Results: The expression of CD4+CD69+ T cells in NSCLC with MPE was lower than that in lung disease BPE. CD4+CD69+ T cells highly express CD45RO+ and mainly secrete anti-tumor cytokines IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α). The expression of miR-16-5p in CD4+CD69+ CD45RO+ T cells in MPE was higher than that in BPE. Moreover, miR-16-5p can bind to both IFN-γ promoter and its 5'untranslated region (5'-UTR), suggesting that IFN-γ may be the target gene directly affected by miR-16-5p. IFN-γ also affects the differentiation of memory CD4+ T cells by regulating T-bet. Conclusions: We believe that miR-16-5p may regulate the decrease of differentiation of naïve CD4+ T cells into memory CD4+CD69+ T cells through its target gene IFN-γ in MPE, thus reducing the number of cytokines that produce anti-tumor effects. It may be the main reason for the low response rate of lung cancer with MPE immunotherapy.

Functional mechanism and clinical implications of LINC00339 in delayed fracture healing.

OBJECTIVE: Delayed fracture healing is a common complication of fractures that significantly impacts human health. This study aimed to explore the role of LINC00339 (lncRNA) in delayed fracture healing to provide new directions for its treatment. METHODS: This study included 82 patients with fractures healing in a normal manner and 90 patients experiencing delayed fracture healing. Levels of LINC00339, miR-16-5p, and osteogenic marker-related mRNAs were measured using RT-qPCR. The predictive potential of LINC00339 for delayed fracture healing was validated using ROC curve analysis. The interaction between LINC00339 and miR-16-5p was validated using dual-luciferase reporter assays and RIP experiments. CCK-8 was used to assess cell proliferation, and apoptosis rates were measured by flow cytometry. RESULTS: LINC00339 was significantly upregulated in delayed fracture healing patients and exhibited strong predictive ability for this condition. Overexpression of LINC00339 inhibited osteoblast proliferation, promoted apoptosis, and reduced mRNA levels of osteogenic markers (P < 0.05). miR-16-5p was recognized as a target mRNA of LINC00339, with LINC00339 exerting negative regulation on miR-16-5p, while overexpression of miR-16-5p mitigated the inhibitory effects of LINC00339 on fracture healing (P < 0.05). CONCLUSION: This research indicated that LINC00339 may serve as a diagnostic marker for delayed fracture healing and revealed the function of the LINC00339/miR-16-5p axis on fracture healing by regulating osteoblasts.

The mechanism and therapeutic potential of lncRNA MIR497HG/miR-16-5p axis in breast cancer.

BACKGROUND: Breast cancer has become a major public health problem in the current society, and its incidence rate ranks the first among Chinese female malignant tumors. This paper once again confirmed the efficacy of lncRNA in tumor regulation by introducing the mechanism of the diagnosis of breast cancer by the MIR497HG/miR-16-5p axis. METHODS: The abnormal expression of MIR497HG in breast cancer was determined by RT-qPCR method, and the correlation between MIR497HG expression and clinicopathological characteristics of breast cancer patients was analyzed via Chi-square test. To understand the diagnostic potential of MIR497HG in breast cancer by drawing the receiver operating characteristic curve (ROC). The overexpressed MIR497HG (pcDNA3.1-MIR497HG) was designed and constructed to explore the regulation of elevated MIR497HG on biological function of BT549 and Hs 578T cells through Transwell assays. Additionally, the luciferase gene reporter assay and Pearson analysis evaluated the targeting relationship of MIR497HG to miR-16-5p. RESULTS: MIR497HG was decreased in breast cancer and had high diagnostic function, while elevated MIR497HG inhibited the migration and invasion of BT549 and Hs 578T cells. In terms of functional mechanism, miR-16-5p was the target of MIR497HG, and MIR497HG reversely regulated the miR-16-5p. miR-16-5p mimic reversed the effects of upregulated MIR497HG on cell biological function. CONCLUSIONS: In general, MIR497HG was decreased in breast cancer, and the MIR497HG/miR-16-5p axis regulated breast cancer tumorigenesis, providing effective insights for the diagnosis of patients.

Novel circular RNA Sestrin1 promotes chicken myoblast proliferation and differentiation via circSesn1/miR-16-5p/SESN1 pathway.

1. The development of chicken skeletal muscle is directly relevant to poultry husbandry production. Numerous studies have suggested that circular RNA play pivotal roles in muscle development. However, the functions and mechanisms of most circRNA in chicken myogenesis remain largely unknown.2. This study identified a novel circSESN1 based on existing sequencing data and examined its authenticity and subcellular localisation by enzyme digestion and RNA fluorescence in situ hybridisation. Additionally, there was a positive correlation between the expression levels of circSESN1 and the developmental stage of chicken muscle.3. Mechanistically, knockdown or overexpression of circSESN1 was performed in primary myoblasts to validate its function. The interactions between circSESN1, miR-16-5p, and the target gene sestrin 1 (SESN1) were investigated using bioinformatics analysis and a dual fluorescein reporter system. Real-time qPCR, a cell proliferation assay, and immunofluorescence staining techniques were used to investigate the promotion effect of circSESN1 on myoblast proliferation and differentiation by miR-16-5p/SESN1 pathway.4. The results demonstrated that the newly identified chicken circSESN1 directly sponges gga-miR-16-5p to regulate SESN1 gene expression, promoting myoblast proliferation and differentiation.

Elucidating thoracic aortic dissection pathogenesis: The interplay of m1A-related gene expressions and miR-16-5p/YTHDC1 Axis in NLRP3-dependent pyroptosis.

The underlying molecular mechanisms of thoracic aortic dissection (TAD) remain incompletely understood. Recent insights into RNA methylation and microRNA-mediated gene regulation offer new avenues for exploring how these processes contribute to the pathophysiology of TAD, particularly through the modulation of pyroptosis and smooth muscle cell viability. This research aimed to elucidate the interplay of m1A-related gene expressions and miR-16-5p/YTHDC1 Axis in NLRP3-dependent pyroptosis, a mechanism implicated in the pathogenesis of TAD. We collected tissue samples from 28 human TAD patients and 8 healthy aortic group, as well as utilized a mouse model to replicate the disease. A combination of computational, in vitro, and in vivo methods was applied, including CIBERSORTx analysis, Pearson correlation, gene transfection using antagomiR-16-5p, siRNA, and several staining as well as cell culture techniques. Our analysis indicated two differentially expressed genes, ALKBH2 and YTHDC1. We found significant upregulation of has-miR-16-5p and downregulation of YTHDC1 at mRNA level in AD samples. Immune cell infiltration in TAD samples was examined using the CIBERSORTx database. We confirmed that YTHDC1 was a target of miR-16-5p, as evidenced by an inhibitory effect on luciferase activity. Inhibition of miR-16-5p enhanced SMC proliferation and promoted cell viability whilst downregulating NLRP3-pyroptosis. YTHDC1 expression was increased, and NLRP3-pyroptosis expressions were inhibited, suggesting miR-16-5p/YTHDC1 axis may involve the NLRP3-pyroptosis of the SMC. In vivo analyses confirmed the prevention of NLRP3-pyroptosis in middle layer of the thoracic aorta, implying that the miR-16-5p/YTHDC1 axis regulated SMC proliferation via NLRP3-pyroptosis signaling. Our findings underscored the anti-pyroptotic role of miR-16-5p/YTHDC1 axis in the pathogenesis of TAD, suggesting a potential therapeutic strategy via targeting YTHDC1 and suppressing miR-16-5p to inhibit NLRP3-dependent pyroptosis. Although further investigation is needed, these results relating to SMC proliferation are a significant step forward in understanding TAD.

hUCMSC-derived exosomes protect against GVHD-induced endoplasmic reticulum stress in CD4+ T cells by targeting the miR-16-5p/ATF6/CHOP axis.

Exosomes generated from mesenchymal stem cells (MSCs) are thought to be a unique therapeutic strategy for several autoimmune deficiency illnesses. The purpose of this study was to elucidate the protective effects of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-Exo) on CD4+ T cells dysfunction during graft-versus-host disease (GVHD) and to identify the underlying processes involved. Here, we showed that hUCMSC-Exo treatment can effectively attenuate GVHD injury by alleviating redox metabolism disorders and inflammatory cytokine bursts in CD4+ T cells. Furthermore, hUCMSC-Exo ameliorate ER stress and ATF6/CHOP signaling-mediated apoptosis in CD4+ T cells and promote the development of CD4+IL-10+ T cells during GVHD. Moreover, downregulating miR-16-5p in hUCMSC-Exo impaired their ability to prevent CD4+ T cells apoptosis and weakened their ability to promote the differentiation of CD4+IL-10+ T cells. Collectively, the obtained data suggested that hUCMSC-Exo suppress ATF6/CHOP signaling-mediated ER stress and apoptosis in CD4+ T cells, enhance the differentiation of CD4+IL-10+ T cells, and reverse the imbalance of immune homeostasis in the GVHD process by transferring miR-16-5p. Our study provided further evidence that GVHD patients can benefit from hUCMSC-Exo-mediated therapy.

miR-16-5p aggravates sepsis-associated acute kidney injury by inducing apoptosis.

Sepsis-associated acute kidney injury (S-AKI) is a common disease in pediatric intensive care units (ICU) with high morbidity and mortality. The newly discovered results indicate that microRNAs (miRNAs) play an important role in the diagnosis and treatment of S-AKI and can be used as markers for early diagnosis. In this study, the expression level of miR-16-5p was found to be significantly upregulated about 20-fold in S-AKI patients, and it also increased by 1.9 times in the renal tissue of S-AKI mice. Receiver operating characteristic (ROC) curve analysis showed that miR-16-5p had the highest predictive accuracy in the diagnosis of S-AKI (AUC = 0.9188). In vitro, the expression level of miR-16-5p in HK-2 cells treated with 10 µg/mL lipopolysaccharide (LPS) increased by more than 2 times. In addition, LPS-exposed renal tissue and HK-2 cells lead to upregulation of inflammatory cytokines IL-6, IL-1ß, TNF-a, and kidney damage molecules kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL). However, inhibition of miR-16-5p significantly mitigated LPS expose-mediated kidney injury and inflammation. Furthermore, LPS-exposed HK-2 cells increased more than 1.7-fold the expression levels of Bax and caspase-3, decreased 3.2-fold the expression level of B-cell lymphoma-2 (Bcl-2), and significantly promoted the occurrence of apoptosis. MiR-16-5p mimic further increased LPS-induced apoptosis in HK-2 cells. Nevertheless, inhibition of miR-16-5p significantly attenuated this effect. In summary, up-regulation of miR-16-5p expression can significantly aggravate renal injury and apoptosis in S-AKI, which also proves that miR-16-5p can be used as a potential biomarker to promote early identification of S-AKI.

Hub genes, key miRNAs and interaction analyses in type 2 diabetes mellitus: an integrative in silico approach.

Diabetes is a rising global metabolic disorder and leads to long-term consequences. As a multifactorial disease, the gene-associated mechanisms are important to know. This study applied a bioinformatics approach to explore the molecular underpinning of type 2 diabetes mellitus through differential gene expression analysis. We used microarray datasets GSE16415 and GSE29226 to identify differentially expressed genes between type 2 diabetes and normal samples using R software. Following that, using the STRING database, the protein-protein interaction network was constructed and further analyzed by Cytoscape software. The EnrichR database was used for Gene Ontology and pathway enrichment analysis to explore key pathways and functional annotations of hub genes. We also used miRTarBase and TargetScan databases to predict miRNAs targeting hub genes. We identified 21 hub genes in type 2 diabetes, some showing more significant changes in the PPI network. Our results revealed that GLUL, SLC32A1, PC, MAPK10, MAPT, and POSTN genes are more important in the PPI network and can be experimentally investigated as therapeutic targets. Hsa-miR-492 and hsa-miR-16-5p are suggested for diagnosis and prognosis by targeting GLUL, SLC32A1, PC, MAPK10, and MAPT genes involved in the insulin signaling pathway. Insight: Type 2 diabetes, as a rising global and multifactorial disorder, is important to know the gene-associated mechanisms. In an integrative bioinformatics analysis, we integrated different finding datasets to put together and find valuable diagnostic and prognostic hub genes and miRNAs. In contrast, genes, RNAs, and enzymes interact systematically in pathways. Using multiple databases and software, we identified differential expression between hub genes of diabetes and normal samples. We explored different protein-protein interaction networks, gene ontology, key pathway analysis, and predicted miRNAs that target hub genes. This study reported 21 significant hub genes and some miRNAs in the insulin signaling pathway for innovative and potential diagnostic and therapeutic purposes.

Long noncoding RNA KCNQ1OT1 aggravates cerebral infarction by regulating PTBT1/SIRT1 via miR-16-5p.

Cerebral infarction (CI) is one of the leading causes of disability and death. LncRNAs are key factors in CI progression. Herein, we studied the function of long noncoding RNA KCNQ1OT1 in CI patient plasma samples and in CI models. Quantitative real-time PCR and Western blotting tested gene and protein expressions. The interactions of KCNQ1OT1/PTBP1 and miR-16-5p were analyzed using dual-luciferase reporter and RNA immunoprecipitation assays; MTT assays measured cell viability. Cell migration and angiogenesis were tested by wound healing and tube formation assays. Pathological changes were analyzed by triphenyltetrazolium chloride and routine staining. We found that KCNQ1OT1 and PTBP1 were overexpressed and miR-16-5p was downregulated in CI patient plasma and in oxygen-glucose deprived (OGD) induced mouse brain microvascular endothelial (bEnd.3) cells. KCNQ1OT1 knockdown suppressed pro-inflammatory cytokine production and stimulated angiogenic responses in OGD-bEnd.3 cells. KCNQ1OT1 upregulated PTBP1 by sponging miR-16-5p. PTBP1 overexpression or miR-16-5p inhibition attenuated the effects of KCNQ1OT1 knockdown. PTBP1 silencing protected against OGD-bEnd.3 cell injury by enhancing SIRT1. KCNQ1OT1 silencing or miR-16-5p overexpression also alleviated ischemic injury in a mice middle cerebral artery occlusion model. Thus, KCNQ1OT1 silencing alleviates CI by regulating the miR-16-5p/PTBP1/SIRT1 pathway, providing a theoretical basis for novel therapeutic strategies targeting CI.

Evaluation of miRNA Expression in Patients with Gestational Diabetes Mellitus: Investigating Diagnostic Potential and Clinical Implications.

Purpose: Gestational diabetes mellitus (GDM) is common pregnancy complication (8%), characterized by hyperglycemia resulting from pathological homeostatic mechanisms. There's a concerning trend of increasing GDM prevalence. New markers, particularly epigenetic ones, are sought for early detection and enhanced care. miRNA are small non-coding RNA molecules. The main goal was to investigate the potential role of miRNA (miR-16-5p, miR-222-3p, miR-21-5p) in GDM and their association with clinical features. Patients and Methods: The study included 72 pregnant patients, with 42 having GDM and 30 in the control group. miRNA expression was measured using ELISA. Results: There were no significant differences in miR-222-3p expression between GDM patients and the control group. The GDM group exhibited a positive correlation between miR-16-5p expression and miR-21-5p expression as well as between miR-16-5p expression and insulin resistance. In the GDM group, a positive correlation was observed between miR-21-5p expression and fasting glucose levels. Conclusion: Results do not confirm the role of miR-222-3p in GDM pathogenesis or as a diagnostic marker. Additionally, a role for miR-16-5p in GDM pathogenesis was observed. Furthermore, a potential role for miR-21-5p in monitoring GDM treatment is indicated.

Extracellular Vesicles from Neural Stem Cells Carry microRNA-16-5p to Reduce Corticosterone-induced Neuronal Injury in Depression Rats.

OBJECTIVE: Depression is a common mental illness. Neural stem cell-derived extracellular vesicles (NSC-EVs) are involved in repairing neuronal injury. We estimated the mechanism of miR-16-5p in depression rats. METHODS: EVs were extracted from NSCs. The depression rat model was established by corticosterone (CORT) induction and treated with NSC-EVs. The depression behavioral/pathological changes in rats were assessed using forced swimming test, open field test, sucrose consumption test and western blotting. The neuronal apoptosis in hippocampal tissue were detected. CORT-induced PC12 cell model was established. EV uptake by PC12 cells was measured and PC12 cell apoptosis was detected. The downstream targets of miR-16-5p were predicted and verified. The expressions of miR-16-5p and MYB in rats, PC12 cells, and EVs were measured. Functional rescue experiments were conducted to verify the role of miR-16-5p and MYB in PC12 cell apoptosis. RESULTS: CORT induction increased neuronal apoptosis in hippocampal tissue and induced depression-like behaviors in rats, while NSC-EV treatment improved depression-like behaviors and apoptosis in rats. In PC12 cells, NSC-EVs decreased CORT-induced PC12 cell apoptosis. NSC-EVs carried miR-16-5p into PC12 cells. miR-16-5p knockdown in EVs partially reversed the inhibitory effects of NSC-EVs on CORT-induced PC12 cell apoptosis. miR-16-5p targeted to inhibit MYB to repress CORT-induced PC12 cell apoptosis. In vivo experiments further verified that NSC-EVs reduced neuronal injury in CORT-induced depression rats via the miR-16-5p/MYB axis. CONCLUSION: NSC-EVs-mediated alleviation on neuronal injury by carrying miR-16-5p to target MYB was highly likely one of the mechanisms by which NSC-EVs mediated miR-16-5p in neuroprotection of depression rats.

Regulatory mechanism of GPER in the invasion and migration of ectopic endometrial stromal cells in endometriosis.

Endometriosis (EMS) is a chronic inflammatory disorder of high incidence that causes serious reproductive consequences. High estrogen production is a consistently observed endocrine feature of EMS. The present study aims to probe the molecular mechanism of G protein-coupled estrogen receptor 1 (GPER) in the invasion and migration of ectopic endometrial stromal cells (Ect-ESCs) and provides a new rationale for EMS treatment. Eutopic and ectopic endometrial tissues were collected from 41 EMS patients, and primary ESCs were separated. GPER, miR-16-5p, and miR-103a-3p levels in cells and tissues were determined by qRT-PCR or Western blot assay. Cell viability, proliferation, invasion, and migration were evaluated by CCK-8, colony formation, and Transwell assays. The upstream miRNAs of GPER were predicted by databases, and dual-luciferase assay was performed to validate the binding of miR-16-5p and miR-103a-3p to GPER 3'UTR. GPER was highly expressed in EMS tissues and Ect-ESCs. Inhibition of GPER mitigated the proliferation, invasion, and migration of Ect-ESCs. GPER was regulated by miR-16-5p and miR-103a-3p. Overexpression of miR-16-5p and miR-103a-3p negatively regulated GPER expression and inhibited the invasion and migration of Ect-ESC. In conclusion, GPER promoted the invasion and migration of Ect-ESCs, which can be reversed by upstream miR-16-5p and miR-103a-3p.

circ_SPEF2 Regulates the Balance of Treg Cells by Regulating miR-16-5p/BACH2 in Lymphoma and Participates in the Immune Response.

BACKGROUND: This study aims to explore the potential mechanism of action of the newly discovered hsa_circ_0128899 (circSPEF2) in diffuse large B-cell lymphoma (DLBCL). METHODS: circSPEF2, miR-16-5p and BTB and CNC homologue 2 (BACH2) expression patterns in DLBCL patients and cell lines were studied by RT-qPCR. The biological function of circSPEF2 in vitro and in vivo was investigated by function acquisition experiments. The proliferation activity of lymphoma cells was detected by MTT. Bax, Caspase-3, and Bcl-2 were determined by Western Blot. Apoptosis and the ratio of CD4 to Treg of immune cells in the co-culture system were analyzed by flow cytometry. The mechanism of action of circSPEF2 in DLBCL progression was further investigated by RIP and dual luciferase reporter experiments. RESULTS: circSPEF2 was a circRNA with abnormally down-regulated expression in DLBCL. Increasing circSPEF2 expression inhibited the proliferative activity and induced apoptosis of lymphoma cells in vitro and in vivo, as well as increased CD4+T cells and decreased Treg cell proportion of immune cells in the tumor microenvironment. Mechanically, circSPEF2 was bound to miR-16-5p expression, while BACH2 was targeted by miR-16-5p. circSPEF2 overexpression-mediated effects on lymphoma progression were reversible by upregulating miR-16-5p or downregulating BACH2. CONCLUSIONS: circSPEF2 can influence DLBCL progression by managing cellular proliferation and apoptosis and the proportion of immune cells Treg and CD4 through the miR-16-5p/BACH2 axis.

Loss of ACOX1 in clear cell renal cell carcinoma and its correlation with clinical features.

Clear cell renal cell carcinoma (ccRCC) is a major pathological type of kidney cancer with a poor prognosis due to a lack of biomarkers for early diagnosis and prognosis prediction of ccRCC. In this study, we investigated the aberrant expression of Acyl-coenzyme A oxidase 1 (ACOX1) in ccRCC and evaluated its potential in diagnosis and prognosis. ACOX1 is the first rate-limiting enzyme in the peroxidation ß-oxidation pathway and is involved in the regulation of fatty acid oxidative catabolism. The mRNA and protein levels of ACOX1 were significantly downregulated in ccRCC, and its downregulation was closely associated with the tumor-node-metastasis stage of patients. The ROC curves showed that ACOX1 possesses a high diagnostic value for ccRCC. The OS analysis suggested that lower expression of ACOX1 was closely related to the worse outcome of patients. In addition, gene set enrichment analysis suggested that expression of ACOX1 was positively correlated with CDH1, CDH2, CDKL2, and EPCAM, while negatively correlated with MMP9 and VIM, which strongly indicated that ACOX1 may inhibit the invasion and migration of ccRCC by reversing epithelial-mesenchymal transition. Furthermore, we screened out that miR-16-5p is upregulated at the mRNA transcript level in ccRCC and negatively correlated with ACOX1. In conclusion, our results showed that ACOX1 is abnormally low expressed in ccRCC, suggesting that it could serve as a diagnostic and prognostic biomarker for ccRCC. Overexpression of miR-16-5p may be responsible for the inactivation of ACOX1.

Potential of miRNAs in Plasma Extracellular Vesicle for the Stratification of Prostate Cancer in a South African Population.

Prostate cancer (PCa) is the most common cause of cancer death among African men. The analysis of microRNAs (miRNAs) in plasma extracellular vesicles (EVs) can be utilized as a non-invasive tool for the diagnosis of PCa. In this study, we used small RNA sequencing to profile miRNAs cargo in plasma EVs from South African PCa patients. We evaluated the differential expression of miRNAs between low and high Gleason scores in the plasma EVs of South African patients and in the prostatic tissue from data available in the Cancer Genome Atlas (TCGA) Data Portal. We identified 7 miRNAs differently expressed in both EVs and prostatic tissues. We evaluated their expression using qPCR in a larger cohort of 10 patients with benign prostatic hyperplasia (BPH) and 24 patients with PCa. Here, we reported that the ratio between two of these miRNAs (i.e., miR-194-5p/miR-16-5p) showed a higher concentration in PCa compared to BPH and in metastatic PCa compared to localized PCa. We explored for the first time the profiling of miRNAs cargo in plasma EVs as a tool for the identification of putative markers in the South African population. Our finding indicated the ratio miR-194-5p/miR-16-5p as a non-invasive marker for the evaluation of PCa aggressiveness in this population.

Overexpression of Circ_0005585 Alleviates Cerebral Ischemia Reperfusion Injury via Targeting MiR-16-5p.

Circular RNAs are implicated in the pathogenesis of ischemic stroke. In this work, we explored the modulation and potential mechanisms of action of circ_0005585 in ischemic stroke. Expression of circ_0005585 and miR-16-5p was assessed by quantitative real-time reverse transcription PCR. Ischemic stroke was modeled in mice by middle cerebral artery occlusion (MCAO). The infarct volume was assessed by triphenyl tetrazolium chloride staining. Neurological deficits were evaluated according to Neurological Severity Score. The permeability of the blood-brain barrier was assessed by Evan's blue leakage and brain water content. Apoptosis in brain tissues was measured by the TUNEL test. Relative expression of apoptosis-related proteins was evaluated by Western blotting. The direct interaction between circ_0005585 and miR-16-5p was verified by dual-luciferase reporter assay. The expression of circ_0005585 was lower in mice with MCAO. Lentivirus-mediated overexpression of circ_0005585 ameliorated the neurological deficits and decreased the infarction volume in MCAO mice. The brain water content and Evan's blue leakage through the blood-brain barrier were reduced. In addition, overexpression of circ_0005585 inhibited apoptosis in the cerebral tissues. Our results revealed direct interaction between circ_0005585 and miR-16-5p. Hence, circ_0005585 protects mouse brain during ischemic stroke by targeting miR-16-5p, which uncovers the pathogenesis of this pathology and opens new vitas for its therapy.

miR-16-5p enhances sensitivity to RG7388 through targeting PPM1D expression (WIP1) in Childhood Acute Lymphoblastic Leukemia.

Aim: Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation, the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia (chALL). Methods: miRTarBase and miRDB, along with KEGG and STRING databases, were used to predict miR-16-5p target genes and explore protein-protein interaction networks, respectively. B- and T-lymphoblastic cell lines, in addition to patient primary cells, were treated with RG7388. Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR. Cell viability was evaluated using the MTT assay. Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells. Paired sample t-test was employed for statistical analyses. Results: MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells. However, CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance. Western blot analyses validated the bioinformatics results, confirming the downregulation of WIP1, p53 stabilization, as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p. Moreover, enhanced sensitivity to RG7388 was observed in the transfected cells. Conclusion: This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist, RG7388. These findings might be useful for researchers and clinicians to pave the way for better management of chALL.

LncRNA SNHG1 knockdown inhibits hyperglycemia induced ferroptosis via miR-16-5p/ACSL4 axis to alleviate diabetic nephropathy.

BACKGROUND: Hyperglycemia accelerates the development of diabetic nephropathy (DN) by inducing renal tubular injury. Nevertheless, the mechanism has not been elaborated fully. Here, the pathogenesis of DN was investigated to seek novel treatment strategies. METHODS: A model of diabetic nephropathy was established in vivo, the levels of blood glucose, urine albumin creatinine ratio (ACR), creatinine, blood urea nitrogen (BUN), malondialdehyde (MDA), glutathione (GSH), and iron were measured. The expression levels were detected by qRT-PCR and Western blotting. H&E, Masson, and PAS staining were used to assess kidney tissue injury. The mitochondria morphology was observed by transmission electron microscopy (TEM). The molecular interaction was analyzed using a dual luciferase reporter assay. RESULTS: SNHG1 and ACSL4 were increased in kidney tissues of DN mice, but miR-16-5p was decreased. Ferrostatin-1 treatment or SNHG1 knockdown inhibited ferroptosis in high glucose (HG)-treated HK-2 cells and in db/db mice. Subsequently, miR-16-5p was confirmed to be a target for SNHG1, and directly targeted to ACSL4. Overexpression of ACSL4 greatly reversed the protective roles of SNHG1 knockdown in HG-induced ferroptosis of HK-2 cells. CONCLUSIONS: SNHG1 knockdown inhibited ferroptosis via the miR-16-5p/ACSL4 axis to alleviate diabetic nephropathy, which provided some new insights for the novel treatment of diabetic nephropathy.

E2F2 serves as an essential prognostic biomarker and therapeutic target for human renal cell carcinoma by presenting "E2F2/miR-16-5p/SPTLC1" schema.

BACKGROUND: Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system with high mortality and morbidity. Although E2F2, a classical transcription factor implicated in cell cycle, has been shown to foster tumorigenesis in several human cancers, it could not draw a satisfy answer referring to precise downstream signaling axis in RCC development yet. METHODS: Based on the publicly available data from TCGA database, expression patterns of E2F2, SPTLC1 and miR-16-5p were identified, either with the ability to predict the prognosis of patients with RCC, which was further validated in 38 paired RCC tissues and matched adjacent tissues by RT-qPCR and Western blot, respectively. Their cellular biofunctions were evaluated using MTT, EdU, Colony formation and transwell assays. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay were employed to certain the exquisite core transcription regulatory circuitry of E2F2/miR-16-5p/SPTLC1 in RCC progression, which was also determined in xenograft tumor model. RESULTS: Consistent with the public TCGA database, E2F2 was significantly increased in RCC tissues and cells, indicating shorter overall survival. Mechanistically, E2F2 served as a transcriptional activator of miR-16-5p, thus accounting for its negative regulation on SPTLC1 expression. E2F2 knockdown-mediated suppressive biofunctions on RCC cells were rescued by miR-16-5p mimics, while this effect was abolished again by SPTLC1 overexpression. Role of E2F2 on RCC tumorigenesis via the miR-16-5p/SPTLC1 axis was verified both in vitro and in vivo. CONCLUSION: E2F2 promoted RCC progression via the miR-16-5p/SPTLC1 axis, which may represent a novel prognostic and therapeutic biomarker for RCC.

BMSC-derived extracellular vesicles promoted osteogenesis via Axin2 inhibition by delivering MiR-16-5p.

Osteoporosis (OP) is a systemic bone disease caused by an imbalance in osteogenesis and osteoclastic resorption. Extracellular vesicles (EVs)-encapsulated miRNAs from bone mesenchymal stem cells (BMSCs) have been reported to participate in osteogenesis. MiR-16-5p is one of the miRNAs that regulates osteogenic differentiation; however, studies have shown that its role in osteogenesis is controversial. Thus, this study aims to investigate the role of miR-16-5p from BMSC-derived extracellular vesicles (EVs) in osteogenic differentiation and uncover the underlying mechanisms. In this study, we used an ovariectomized (OVX) mouse model and an H2O2-treated BMSCs model to investigate the effects of BMSC-derived EVs and EV-encapsulated miR-16-5p on OP and the underlying mechanisms. Our results proved that the miR-16-5p level was significantly decreased in H2O2-treated BMSCs, bone tissues of OVX mice, and lumbar lamina tissues from osteoporotic women. EVs-encapsulated miR-16-5p from BMSCs could promote osteogenic differentiation. Moreover, the miR-16-5p mimics promoted osteogenic differentiation of H2O2-treated BMSCs, and the effects exerted by miR-16-5p were mediated by targeting Axin2, a scaffolding protein of GSK3ß that negatively regulates the Wnt/ß-catenin signaling pathway. This study provides evidence that EVs-encapsulated miR-16-5p from BMSCs could promote osteogenic differentiation by repressing Axin2.