CRISPR du-HITI an attractive approach to targeting Long Noncoding RNA HCP5 as inhibitory factor for proliferation of ovarian cancer cell.

This research provides a glimmer of hope that the knockout of HCP5 leads to a therapy response to considerably prolong the life of patients with OC. RT-PCR evaluated the expression of lncRNA HCP5 in the ovarian cancer OVCAR-3 cell line. CRISPR knockout cell lines validated by western blot. Small genomic deletions at the targeted locus were induced. CCK-8 colony formation assays were used to analyze the effect of HCP5 knockout on the proliferation capacity of OVCAR-3 cells. Transwell migration and invasion assayed. Furthermore, the Sphere-formation assay isolated the most aggressive population of cancer stem cells. Bioinformatic analysis showed a significant correlation between lncRNA HCP5 up-regulation and OVCAR-3 cell proliferation. The ChIP technique assesses specific sites of interaction between transcription factors and DNA. Real-time PCR assays explored the relationship between HCP5, Hsa-miR-9-5p, CXCR4, CDH1, caspase-3, p53, bcl2 and survivin. PCR carried out amplification of the 448-bp band for sgRNA1 and sgRNA2 after the use of particular primers for HCP5. the number of breast cancer cells that moved to the bottom chamber reduced considerably after transfection with PX461-sgRNA1/2 vectors compared to the Blank control groups (P < 0.05). MTT assay designated growth curves that showed the rate of OVCAR-3 growth was significantly repressed (***P < 0.001) when compared with control OVCAR-3 cells after HCP5 knockdown. Also, the survival results of W.T cells in 24, 48 and 72 h showed 92%, 87% and 85%, respectively. This is while the cells of the CRISPR/Cas9 group in which LncRNA HCP5 was knocked out had 42% (*P < 0.05), 23%(**P < 0.01) and 14% (**P < 0.01) survival, respectively. The expression levels of caspase-3, Hsa-miR-9-5p, P53 genes in the HCP5 deletion of CRISPR/Cas9 group significantly increased than the W.T. control group; the deletion group showed a considerable reduction in HCP5 expression compared to the blank control group (3.6-fold, p < 0.01). Whereas BCL2, SURVIVIN, CXCR4, CDH1 genes expression markedly increased than in HCP5 knockout cells (5.8-fold, p < 0.05). These results indicate that CRISPR/Cas9-mediated HCP5 disruption on OVCAR-3 cell lines promotes anti-tumor biomarkers, suppressing ovarian cancer progression. Consistent with these results, HCP5 is one of the most critical lnc for the efficient proliferation and migration of OVCAR-3 cell lines.

Circular RNA DLGAP4 alleviates sevoflurane-induced neurotoxicity by regulating miR-9-5p/Sirt1/BDNF pathway.

BACKGROUND: Sevoflurane is a widely used anesthetic in infants. However, long and repeated exposure to this drug can cause developmental neurotoxicity. This study aimed to investigate the role and mechanism of circular RNA DLGAP4 (circDLGAP4) in sevoflurane-induced neurotoxicity. METHODS: Neonatal mice and mouse hippocampal neuronal cell line HT22 were used to construct sevoflurane-induced nerve injury models. The role of circDLGAP4 in sevoflurane-induced neurotoxicity was evaluated by gain-and/or loss-of-function methods. Pathological alterations in hippocampus were analyzed by hematoxylin-eosin and Tunel staining. Cell injury was assessed by cell viability and apoptosis, which was detected by CCK-8 and flow cytometry. The expression of circDLGAP4 and miR-9-5p was determined by real-time PCR. Sirt1 and BDNF levels were measured by Western blot. Productions of TNF-α and IL-6 were examined by ELISA. Dual-luciferase reporter assay and/or RNA pull-down assay were used to confirm the direct binding among circDLGAP4, miR-9-5p, and Sirt1. Rescue experiments were used to further verify the mechanism of circDLGAP4. RESULTS: CircDLGAP4 expression was decreased by sevoflurane both in vivo and in vitro. Overexpression of circDLGAP4 elevated cell viability, reduced apoptosis and levels of TNF-α and IL-6, while circDLGAP4 knockdown showed the opposite effects in sevoflurane-induced HT22 cells. Mechanically, circDLGAP4 functioned via directly binding to and regulating miR-9-5p, followed by targeting the Sirt1/BDNF pathway. Additionally, circDLGAP4 upregulation relieved sevoflurane-induced nerve injury, reduced levels of TNF-α, IL-6 and miR-9-5p, but increased the expression of Sirt1 and BDNF in hippocampus. CONCLUSIONS: Our studies found that circDLGAP4 relieved sevoflurane-induced neurotoxicity by sponging miR-9-5p to regulate Sirt1/BDNF pathway.

The miR-9-5p/KLF5/IL-1ß Axis Regulates Airway Smooth Muscle Cell Proliferation and Apoptosis to Aggravate Airway Remodeling and Inflammation in Asthma.

The aim of this study was to investigate the underlying mechanism of miR-9-5p in airway smooth muscle cells (ASMCs) of asthmatic mice. An asthmatic mouse model was established through the intraperitoneal injection of ovalbumin. Histopathological changes in lung tissues of asthmatic mice were observed using HE staining. ASMCs was identified using immunofluorescence staining and cell morphology. The mRNA expressions of miR-9-5p, KLF5, and IL-1ß were measured using RT-qPCR. Additionally, CCK8 assay and flow cytometry were applied for ASMC proliferation and apoptosis, respectively. The protein levels of OPN, KLF5, and IL-1ß were assessed using western blotting. The results showed that miR-9-5p was abnormally downregulated in lung tissues and ASMCs of asthmatic mice. Dual-Luciferase Reporter Assay and Chromatin immunoprecipitation confirmed that miR-9-5p targeted KLF5 that bounds to IL-1ß promoter. Besides, miR-9-5p negatively regulated IL-1ß mRNA and protein level via KLF5. Moreover, miR-9-5p was found to positively regulate ASMC apoptosis, negatively regulate ASMC proliferation and OPN protein expression, albeit with partial reversal by KLF5. Mechanistically, the regulation of ASMC proliferation and apoptosis by miR-9-5p is achieved by targeting KLF5/IL-1ß axis.

Linc01588 deletion inhibits the malignant biological characteristics of hydroquinone-induced leukemic cells via miR-9-5p/SIRT1.

Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9-5p, miR-9-5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9-5p, miR-9-5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9-5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9-5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9-5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL.

miR-9-5p and miR-221-3p Promote Human Mesenchymal Stem Cells to Alleviate Carbon Tetrachloride-Induced Liver Injury by Enhancing Human Mesenchymal Stem Cell Engraftment and Inhibiting Hepatic Stellate Cell Activation.

Mesenchymal stem cells (MSCs) have shown great potential for the treatment of liver injuries, and the therapeutic efficacy greatly depends on their homing to the site of injury. In the present study, we detected significant upregulation of hepatocyte growth factor (HGF) in the serum and liver in mice with acute or chronic liver injury. In vitro study revealed that upregulation of miR-9-5p or miR-221-3p promoted the migration of human MSCs (hMSCs) toward HGF. Moreover, overexpression of miR-9-5p or miR-221-3p promoted hMSC homing to the injured liver and resulted in significantly higher engraftment upon peripheral infusion. hMSCs reduced hepatic necrosis and inflammatory infiltration but showed little effect on extracellular matrix (ECM) deposition. By contrast, hMSCs overexpressing miR-9-5p or miR-221-3p resulted in not only less centrilobular necrosis and venous congestion but also a significant reduction of ECM deposition, leading to obvious improvement of hepatocyte morphology and alleviation of fibrosis around central vein and portal triads. Further studies showed that hMSCs inhibited the activation of hepatic stellate cells (HSCs) but could not decrease the expression of TIMP-1 upon acute injury and the expression of MCP-1 and TIMP-1 upon chronic injury, while hMSCs overexpressing miR-9-5p or miR-221-3p led to further inactivation of HSCs and downregulation of all three fibrogenic and proinflammatory factors TGF-ß, MCP-1, and TIMP-1 upon both acute and chronic injuries. Overexpression of miR-9-5p or miR-221-3p significantly downregulated the expression of α-SMA and Col-1α1 in activated human hepatic stellate cell line LX-2, suggesting that miR-9-5p and miR-221-3p may partially contribute to the alleviation of liver injury by preventing HSC activation and collagen expression, shedding light on improving the therapeutic efficacy of hMSCs via microRNA modification.

The Relevance of Reperfusion Stroke Therapy for miR-9-3p and miR-9-5p Expression in Acute Stroke-A Preliminary Study.

Reperfusion stroke therapy is a modern treatment that involves thrombolysis and the mechanical removal of thrombus from the extracranial and/or cerebral arteries, thereby increasing penumbra reperfusion. After reperfusion therapy, 46% of patients are able to live independently 3 months after stroke onset. MicroRNAs (miRNAs) are essential regulators in the development of cerebral ischemia/reperfusion injury and the efficacy of the applied treatment. The first aim of this study was to examine the change in serum miRNA levels via next-generation sequencing (NGS) 10 days after the onset of acute stroke and reperfusion treatment. Next, the predictive values of the bioinformatics analysis of miRNA gene targets for the assessment of brain ischemic response to reperfusion treatment were explored. Human serum samples were collected from patients on days 1 and 10 after stroke onset and reperfusion treatment. The samples were subjected to NGS and then validated using qRT-PCR. Differentially expressed miRNAs (DEmiRNAs) were used for enrichment analysis. Hsa-miR-9-3p and hsa-miR-9-5p expression were downregulated on day 10 compared to reperfusion treatment on day 1 after stroke. The functional analysis of miRNA target genes revealed a strong association between the identified miRNA and stroke-related biological processes related to neuroregeneration signaling pathways. Hsa-miR-9-3p and hsa-miR-9-5p are potential candidates for the further exploration of reperfusion treatment efficacy in stroke patients.

LncRNA AGAP2 antisense RNA 1 stabilized by insulin-like growth factor 2 mRNA binding protein 3 promotes macrophage M2 polarization in clear cell renal cell carcinoma through regulation of the microRNA-9-5p/THBS2/PI3K-Akt pathway.

BACKGROUND: Increasing evidence highlights the potential role of long non-coding RNAs (lncRNAs) in the biological behaviors of renal cell carcinoma (RCC). Here, we explored the mechanism of AGAP2-AS1 in the occurrence and development of clear cell RCC (ccRCC) involving IGF2BP3/miR-9-5p/THBS2. METHODS: The expressions of AGAP2-AS1, IGF2BP3, miR-9-5p, and THBS2 and their relationship were analyzed by bioinformatics. The targeting relationship between AGAP2-AS1 and miR-9-5p and between miR-9-5p and THBS2 was evaluated with their effect on cell biological behaviors and macrophage polarization assayed. Finally, we tested the effect of AGAP2-AS1 on ccRCC tumor formation in xenograft tumors. RESULTS: IGF2BP3 could stabilize AGAP2-AS1 through m6A modification. AGAP2-AS1 was highly expressed in ccRCC tissues and cells. The lentivirus-mediated intervention of AGAP2-AS1 induced malignant behaviors of ccRCC cells and led to M2 polarization of macrophages. In addition, THBS2 promoted M2 polarization of macrophages by activating the PI3K/AKT signaling pathway. AGAP2-AS1 could directly bind with miR-9-5p and promote the expression of THBS2 downstream of miR-9-5p. These results were further verified by in vivo experiments. CONCLUSION: AGAP2-AS1 stabilized by IGF2BP3 competitively binds to miR-9-5p to up-regulate THBS2, activating the PI3K/AKT signaling pathway and inducing macrophage M2 polarization, thus facilitating the development of RCC.

HPV+ HNSCC-derived exosomal miR-9-5p inhibits TGF-ß signaling-mediated fibroblast phenotypic transformation through NOX4.

Human papillomavirus (HPV) is a significant risk factor for head and neck squamous cell carcinoma (HNSCC). HPV+ HNSCC patients have a higher survival rate, which may be related to its unique tumor microenvironment. Exosomes are emerging as a communication tool between tumor cells and the tumor microenvironment, including cancer-associated fibroblasts (CAFs). In this study, 111 clinical samples tissues and public sequencing data were analyzed. Our study found fewer CAFs infiltrated in HPV+ HNSCC, and poor CAF infiltration level was associated with a good prognosis. HPV+ HNSCC cell-derived exosomes can significantly reduce the phenotypic transformation of fibroblasts. miR-9-5p, as a miRNA enriched in HPV+ HNSCC cell-derived exosomes, can be transferred to fibroblasts. miR-9-5p mimic transfection decreased the expression of NOX4 and the level of intracellular reactive oxygen species (ROS), which inhibited the transforming growth factor beta 1(TGF-ß1)-induced increase of αSMA levels. Therefore, these results indicated that HPV+ HNSCC-derived exosomal miR-9-5p inhibits TGF-ß signaling-mediated fibroblast phenotypic transformation through NOX4, which is related to the excellent prognosis of HPV patients.

Upregulated miR-9-5p inhibits osteogenic differentiation of bone marrow mesenchymal stem cells under high glucose treatment.

INTRODUCTION: Diabetic osteoporosis (DOP) is a chronic diabetic complication, which is attributed to high glucose (HG)-induced dysfunction of bone marrow mesenchymal stem cells (BMSCs). Studies have revealed that microRNAs (miRNAs) play critical roles in osteogenic differentiation of BMSCs in DOP. Here, the role of miR-9-5p in DOP progression was explored. MATERIALS AND METHODS: The rat model of DOP was established by intraperitoneal injection of streptozotocin (STZ). BMSCs were treated with high glucose (HG) to establish in vitro models. Gene expression in BMSCs and bone tissues of rats was tested by RT-qPCR. The degree of osteogenic differentiation of BMSCs was examined by Alizarin Red staining and ALP activity analysis. The protein levels of collagen-I (COL1), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor-2 (RUNX2), and DEAD-Box Helicase 17 (DDX17) in BMSCs were evaluated by western blotting. The interaction between miR-9-5p and DDX17 was identified by luciferase reporter assay. H&E staining was used to test morphological structure of femurs of rats with STZ treatment. RESULTS: MiR-9-5p was overexpressed in HG-treated BMSCs, while DDX17 was downregulated. Functionally, miR-9-5p knockdown promoted BMSCs osteogenic differentiation under HG condition. Mechanically, miR-9-5p targeted DDX17. DDX17 knockdown reversed the effect of miR-9-5p silencing on osteogenic differentiation of HG-treated BMSCs. In in vivo studies, miR-9-5p downregulation ameliorated the DOP condition of rats and miR-9-5p expression was negatively correlated with DDX17 expression in bone tissues of rats with STZ treatment. CONCLUSION: MiR-9-5p knockdown promotes HG-induced osteogenic differentiation BMSCs in vitro and mitigates the DOP condition of rats in vivo by targeting DDX17.

Cadmium regulates FKBP5 through miR-9-5p and induces carp lymphocyte apoptosis.

Cadmium (Cd) is an environmental pollutant produced by industrial activities, which has no known physiological benefits to organisms. In our previous study, the transcriptomic profiles of carp head kidney exposed to Cd was analyzed by genomics technique, and confirmed that miRNAs are important in the head kidney injury of carp induced by Cd, but the specific biological mechanism was unclear. In order to further explore the effect of Cd on carp head kidney lymphocyte damage, we established a model of Cd exposure in vitro. The results showed that Cd could increase the expression of Bax (Bcl-2 associated X protein), Caspase9 (Cysteinyl aspartate specific proteinase 9) and Caspase3 (Cysteinyl aspartate specific proteinase 3), inhibit the expression of Bcl-2 (B cell lymphoma/leukemia 2), and induce apoptosis of carp head kidney lymphocytes. In our previous study, we screened the differentially expressed miRNA in Cd-treated lymphocytes by high-throughput sequencing, and found that there was a significant difference in the expression of miR-9-5p. The expression trend of miR-9-5p in the vitro model was the same as that of high-throughput sequencing. We screened the differentially expressed gene FKBP5 (FK506-binding protein 51) in lymphocytes treated with Cd. It was confirmed by double luciferase reporter gene analysis that FKBP5 was the target gene of miR-9-5p. We established the overexpression/knockdown model of miR-9-5p in carp head kidney lymphocyte in vitro. The results showed that miR-9-5p could inhibit the expression of FKBP5, increase the phosphorylation level of Akt, inhibit apoptosis and improve the cell survival rate in carp head kidney lymphocytes. Together, Cd could down-regulate the expression of miR-9-5p, target up-regulate the expression of FKBP5, inhibit the phosphorylation of Akt, and promote the apoptosis of carp head kidney lymphocytes through mitochondrial pathway.

DPA promotes hBMSCs osteogenic differentiation by miR-9-5p/ERK/ALP signaling pathway.

Docosahexaenoic acid (DHA) has been reported potentiate osteogenic differentiation, while Docosapentaenoic acid (DPA), another Omega-3 fatty acid, its contribution to the osteogenic differentiation of human bone-marrow-derived mesenchymal stromal cells (hBMSCs) is not entirely elucidated. The Alizarin Red S (ARS) staining and the expression of osteogenesis­associated genes were analyzed during osteogenic induction by DPA. Then, bioinformatics analysis and dual luciferase reporter assays were investigated to confirm the interactions between miR-9-5p and alkaline phosphatase (ALP). miR-9-5p mimics / inhibitor were transfected to human hBMSCs and the osteogenic assay above was also performed. Furthermore, DPA significantly promoted the phosphorylation of ERK via miR-9-5p. PD98059, a highly specific and potent ERK1/2 inhibitor, inhibited the activation of ALP and partially reversed the role of DPA during osteogenic differentiation. These data indicated that DPA promoted osteogenic differentiation of hBMSCs potentially through miR-9-5p/ERK/ALP signaling pathway, providing a potentially useful therapeutic strategy for patients to improve bone loss.

Neuron secrete exosomes containing miR-9-5p to promote polarization of M1 microglia in depression.

BACKGROUND: Neuroinflammation is an important component mechanism in the development of depression. Exosomal transfer of MDD-associated microRNAs (miRNAs) from neurons to microglia might exacerbate neuronal cell inflammatory injury. RESULTS: By sequence identification, we found significantly higher miR-9-5p expression levels in serum exosomes from MDD patients than healthy control (HC) subjects. Then, in cultured cell model, we observed that BV2 microglial cells internalized PC12 neuron cell-derived exosomes while successfully transferring miR-9-5p. MiR-9-5p promoted M1 polarization in microglia and led to over releasing of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which exacerbated neurological damage. Furthermore, we identified suppressor of cytokine signaling 2 (SOCS2) as a direct target of miR-9-5p. Overexpression of miR-9-5p suppressed SOCS2 expression and reactivated SOCS2-repressed Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathways. Consistently, we confirmed that adeno-associated virus (AAV)-mediated overexpression of miR-9-5p polarized microglia toward the M1 phenotype and exacerbated depressive symptoms in chronic unpredictable mild stress (CUMS) mouse mode. CONCLUSION: MiR-9-5p was transferred from neurons to microglia in an exosomal way, leading to M1 polarization of microglia and further neuronal injury. The expression and secretion of miR-9-5p might be novel therapeutic targets for MDD.

Downregulation of Talin-1 is associated with the increased expression of miR-182-5p and miR-9-5p in coronary artery disease.

BACKGROUND: Evidence indicates that the dysregulation of extracellular matrix (ECM) components can lead to cardiovascular diseases. The Talin-1 (TLN1) gene is a major component of the ECM, and it mediates integrin adhesion to the ECM. In this study, we aimed to determine microRNAs (miRs) that regulate the expression of TLN1 and determine expression alterations in TLN1 and its targeting miRs in coronary artery disease (CAD). METHODS: Data sets of CAD and normal samples of blood exosomes were downloaded, and TLN1 was chosen as one of the genes with differential expressions in an in silico analysis. Next, miR-182-5p and miR-9-5p, which have a binding site on 3´-UTR of TLN1, were selected using bioinformatics tools. Then, the miR target site was cloned in the psiCHECK-2 vector, and direct interaction between the miR target site and the TLN1 3'-UTR putative target site was investigated by luciferase assay. The expression of miR-182-5p, miR-9-5p, and TLN1 in the serum samples of CAD and non-CAD individuals was assessed via a real-time quantitative polymerase chain reaction. RESULTS: Our data revealed that miR-182-5p directly regulated the expression of TLN1. Moreover, miR-182-5p and miR-9-5p were significantly upregulated in the CAD group. Hence, both bioinformatics and experimental analyses determined the downregulated expression of TLN1 in the CAD samples. CONCLUSIONS: Our findings demonstrated that miR-182-5p and miR-9-5p could play significant roles in TLN1 regulation and participate in CAD development by targeting TLN1. These findings introduce novel biomarkers with a potential role in CAD pathogenesis.

Knockdown of lncRNA SNHG15 Ameliorates Oxygen and Glucose Deprivation (OGD)-Induced Neuronal Injury via Regulating the miR-9-5p/TIPARP Axis.

Stroke is a cerebrovascular disease with impaired nerve function. Long non-coding RNA (lncRNA) is considered to be an important regulator of various diseases. Nevertheless, the role of lncRNA small nucleolar RNA host gene 15 (SNHG15) in cerebral ischemia injury induced by stroke is still unclear. Cell-counting kit 8 assay and flow cytometry were used to detect cell viability and apoptosis, respectively. The caspase3 activity of cells was measured using Caspase3 Activity Assay Kit. Besides, the protein levels of apoptosis markers and TCCD-induced poly (ADP)-ribose polymerase (TIPARP) were determined using western blot analysis. Moreover, quantitative real-time polymerase chain reaction was employed to examine the relative expression of SNHG15 and miR-9-5p. Furthermore, dual-luciferase reporter assay was used to assess the interaction between miR-9-5p and SNHG15 or TIPARP. In addition, biotin-labeled RNA pull-down assay was performed to evaluate the interaction between miR-9-5p and SNHG15 further. Middle cerebral artery occlusion (MCAO) model was constructed to further explore the role of SNHG15 in neuronal injury in vivo. Our data showed that oxygen and glucose deprivation (OGD) could induce N-2a cell injury and enhance SNHG15 expression. Silenced SNHG15 could promote the viability and suppress the apoptosis of OGD-induced N-2a cells. Also, SNHG15 knockdown also could alleviate the neuronal injury of MCAO mice. Mechanistically, SNHG15 could sponge miR-9-5p, and miR-9-5p could target TIPARP. Further experiments revealed that miR-9-5p inhibition or TIPARP overexpression could reverse the suppressive effect of SNHG15 knockdown on OGD-induced N-2a cell injury. Our findings indicated that SNHG15 knockdown inhibited neuronal injury through the miR-9-5p/TIPARP axis, suggesting that SNHG15 might be a potential target for cerebral ischemia injury induced by stroke.

Oxidized low-density lipoprotein contributes to injury of endothelial cells via the circ_0090231/miR-9-5p/TXNIP axis.

Atherosclerosis (AS) has been reported to induce severe clinical complications. Circular RNA (circRNA) circ_0090231 was found to be aberrantly overexpressed in oxidized low-density lipoprotein (ox-LDL)-induced endothelial cells. This study was designed to explore the role and mechanism of circ_0090231 in ox-LDL-triggered endothelial cell injury in AS. Circ_0090231, microRNA-9-5p (miR-9-5p), and thioredoxin interacting protein (TXNIP) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, angiogenesis, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), tube formation, and flow cytometry assay. Bcl-2, Bax, and TXNIP protein levels were gauged by western blot assay. Malondialdehyde (MDA), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activity were determined by special kits. Tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and interleukin 6 (IL-6) levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits. The binding relationship between miR-9-5p and circ_0090231 or TXNIP was predicted by starBase, and then verified by a dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ_0090231 and TXNIP were increased, and miR-9-5p was decreased in ox-LDL-treated HUVECs. Moreover, circ_0090231 knockdown mitigated ox-LDL-induced HUVEC injury by boosting angiogenesis, oxidative stress, and inflammation, and hindering apoptosis. The mechanical analysis revealed that circ_0090231 acted as a sponge of miR-9-5p to regulate TXNIP expression. Circ_0090231 could attenuate ox-LDL-mediated HUVEC damage by the miR-9-5p/TXNIP axis, providing a promising therapeutic strategy for AS treatment.

LINC01116 promotes proliferation and migration of endometrial stromal cells by targeting FOXP1 via sponging miR-9-5p in endometriosis.

Endometriosis is a common multi-factorial gynaecological disease. Recent studies have revealed that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of endometriosis. In the present study, the expression profiles of lncRNAs in 6 pairs of endometriosis ectopic endometrium (ecEM) and eutopic endometrium (euEM) tissues were analysed by RNA sequencing. From the profiles, LINC01116 was found to be up-regulated in ecEM tissues compared to euEM tissues and was verified by quantitative real-time PCR (qRT-PCR). Then, functional experiments demonstrated that LINC01116 promoted the proliferation and migration of ectopic primary endometrial stromal cells (ESCs), while miR-9-5p exerted the opposite effects. Dual-luciferase reporter assays verified that LINC01116 directly sponged miR-9-5p and relieved the suppression of its target, Forkhead box protein P1 (FOXP1). Rescue experiments further demonstrated that LINC01116 could promote proliferation and migration of ESCs by targeting FOXP1 via sponging miR-9-5p. Overall, our study illuminates that LINC01116 promotes the progression of endometriosis through the miR-9-5p/FOXP1 axis. This finding provides a novel therapeutic target for patients with endometriosis.

Silencing circRNA LRP6 down-regulates PRMT1 to improve the streptozocin-induced pancreatic ß-cell injury and insulin secretion by sponging miR-9-5p.

Due to the sedentary lifestyles of people, the number of obese people is increasing alarmingly, which leads to the high prevalence of diabetes mellitus (DM). It was reported that circularRNA (circRNA) LRP6 was upregulated in HG-treated mesangial cells, and it could regulate high glucose-induced cell injury via sponging miR-205. Thus, the aim of this study was to explore the underlying pathogenesis of DM. Streptozocin (STZ) was used to stimulate the in vitro model of pancreatic ß-cell injury. Then, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and methyl thiazolyl tetrazolium (MTT) assay were used to evaluate the expression of circLRP6 and the cell viability in STZ-challenged INS-1 cells, respectively. After knocking down circLRP6, the cell viability and apoptosis were respectively measured by MTT and TdT-mediated dUTP nick-end labeling (TUNEL) staining, and insulin release and oxidative stress were respectively measured by enzyme-linked immunosorbent assay (ELISA) and corresponding kits. After the interactions among circLRP6, PRMT1, and miR-9-5p were predicted and confirmed, the above mentioned assays were conducted again. The expression of circLRP6 was elevated while cell viability was decreased after INS-1 cells were exposed to STZ. Silencing circLRP6 resulted in an increase in the cell viability, a decrease in the cell apoptosis, together with more insulin release. The circLRP6/miR-9-5p/PRMT1 regulatory network was then confirmed, which affected the cell viability, apoptosis, insulin release, and oxidative stress in STZ-challenged INS-1 cells. In conclusion, this study first provides evidence that the circLRP6/miR-9-5p/PRMT1 regulatory network can affect STZ-induced cell viability, oxidative stress, and insulin secretion in INS-l cells, which can further impact the progression of diabetes.

Lithium alleviated spinal cord injury (SCI)-induced apoptosis and inflammation in rats via BDNF-AS/miR-9-5p axis.

Spinal cord injury (SCI) is a major cause of paralysis, disability and even death in severe cases. Lithium has neuroprotective effects on SCI, while the underlying mechanisms remain obscure. In the present study, we established a SCI rat model, which subsequently received lithium treatment. Results displayed that lithium treatment improved the locomotor function recovery and reduced apoptosis by increasing anti-apoptotic molecule expression and decreasing pro-apoptotic factor expression in SCI rats. Furthermore, lithium treatment alleviated the inflammatory response by inactivating the nuclear factor-kappa B (NF-κB) pathway and inhibited the expression of lncRNA brain-derived neurotrophic factor antisense (BDNF-AS) in SCI rats. Subsequent researches indicated that miR-9-5p was targeted and regulated by BDNF-AS. Lithium treatment rescued the upregulation of BDNF-AS expression and downregulation of miR-9-5p expression induced by H2O2 in SH-SY5Y cells. BDNF-AS overexpression or miR-9-5p interference attenuated the anti-apoptotic and anti-inflammatory effects of lithium chloride in SH-SY5Y cells that was damaged by H2O2 induction, revealing that lithium might act through the BDNF-AS/miR-9-5p axis. In vivo studies showed that the injection of BDNF-AS adenovirus vector or miR-9-5p inhibitor reversed the effects of lithium on the histologic morphology of spinal cord, motor function, inflammatory reaction and apoptosis in SCI rats, which was consistent with the results of in vitro studies. In conclusion, our data demonstrated that lithium reduced SCI-induced apoptosis and inflammation in rats via the BDNF-AS/miR-9-5p axis.

miR-9-5p facilitates hepatocellular carcinoma cell proliferation, migration and invasion by targeting ESR1.

The study aimed to explore the relationship between miR-9-5p and ESR1, and clarify the underlying functional mechanism in the occurrence and development of hepatocellular carcinoma (HCC). Expression data including miRNAs and mRNAs of HCC downloaded from TCGA database were processed for differential analysis, and corresponding clinical data were collected for survival analysis to identify the target miRNA miR-9-5p. Bioinformatics databases were applied for predicting downstream target mRNAs of miR-9-5p. qRT-PCR was used to evaluate expression of miR-9-5p. Western blot was used to detect protein expression of ESR1. MTT, wound healing assay and Transwell assay were used to detect HCC cell proliferation, migration and invasion, respectively. Dual-luciferase reporter gene assay was used to identify the targeting relationship between miR-9-5p and ESR1. Research suggested that miR-9-5p was highly expressed in HCC cells but ESR1 was poorly expressed. Overexpression of miR-9-5p could improve the proliferation, invasion and migration of cells. Dual-luciferase reporter assay showed that ESR1 was the downstream target of miR-9-5p in HCC. Overexpression of miR-9-5p markedly reduced ESR1 mRNA and protein levels in HCC cells, whereas inhibition of miR-9-5p expression produced the contrary results. Silencing ESR1 could noticeably reverse the effect of miR-9-5p knockdown on the proliferation, migration and invasion of HCC cells. As an oncogene, miR-9-5p fostered the proliferation, migration and invasion of HCC cells by targeting and inhibiting ESR1 expression.

Overexpression of miRNA-9 enhances galectin-3 levels in oral cavity cancers.

Oral cavity cancer (OCC) is the predominant subtype of head and neck cancer (HNC) and has up to 50% mortality. Genome-wide microRNA (miR) sequencing data indicates overexpression of miR-9-5p in HNC tumours, however, the biological role of miR-9-5p in OCC is complex; it can either act as a tumour suppressor or an oncomir, regulating many target genes at the post-transcriptional level. We have investigated the overexpression of miR-9-5p in three OCC cell lines. We have evaluated its expression levels and Galectin-3 as potential biomarkers in saliva samples collected from controls and OCC patients. We found that over expression of miR-9-5p in OCC cell lines resulted in a significant reduction in cell proliferation and migration, and an increase in apoptosis, which was paralleled by an increase in Galectin-3 secretion and export of Galectin-3 protein. Our data are consistent with miR-9-5p being a modulator of Galectin-3 via the AKT/γ-catenin pathway. In addition, the positive correlation between the levels of miR-9-5p expression and secreted Galectin-3 in saliva reflects a similar relationship in vivo, and supports the utility of their integrative evaluation in OCC. Our findings indicate that both miR-9-5p and Galectin-3 are critical biomolecules in the progression of OCC.