Targeting Relationship Research Articles

Silencing lncRNA GABPB1-AS1 alleviates cerebral ischemia reperfusion injury through the miR-641/NUCKS1 axis.

To investigate the possible mechanism of lncRNA GA binding protein transcription factor beta subunit 1 antisense RNA 1 (GABPB1-AS1) in cerebral ischemia/reperfusion (CI/R) injury. RT-qPCR was applied to determine GABPB1-AS1 expression in oxygen-glucose deprivation/reoxygenation (OGD/R) cells. The targeting relationships between GABPB1-AS1 and miR-641, as well as between miR-641 and nuclear casein and cyclin-dependent kinase substrate 1 (NUCKS1) were examined by dual luciferase reporter assay. The protein expression of caspase-3, Bax, Bcl-2 and NUCKS1 was examined by western blot. Cell apoptosis was measured by flow cytometry (FCM) and western blot. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GABPB1-AS1 was significantly elevated in SH-SY5Y cells under OGD/R. Downregulation of GABPB1-AS1 accelerated cell viability and suppressed cell apoptosis. GABPB1-AS1 silencing reduced ROS and MDA levels in OGD/R-treated cells. Furthermore, miR-641 inhibitor aggravated damage from OGD/R, but GABPB1-AS1 silencing notably attenuated this effect. NUCKS1 was proven to be a target gene of miR-641. GABPB1-AS1 silencing alleviated CI/R injury through the miR-641/NUCKS1 axis, indicating that GABPB1-AS1 might serve as a therapeutic target for CI/R injury.

The RNA m6A writer RBM15 contributes to the progression of esophageal squamous cell carcinoma by regulating miR-3605-5p/KRT4 pathway

Cancer progression can be modulated by N6-methyladenosine (m6A) modification. RNA binding motif protein 15 (RBM15) is an essential RNA m6A writer that influences carcinogenesis, however its significance in esophageal squamous cell carcinoma (ESCC) is uncertain. This research is intended to examine how RBM15 regulates the development of ESCC. We performed qRT-PCR analysis to evaluate the expression of RBM15, microRNA (miR-3605-5p) as well as keratin 4 (KRT4) in ESCC. Target relationship between miR-3605-5p and KRT4 was validated by dual luciferase reporter assay. Western blotting analyzed the protein levels of KRT4, p53, and p21. To demonstrate that RBM15 is responsible for the m6A alteration of miR-3605-5p, RIP and Me-RIP experiments were carried out concurrently. m6A content was measured by m6A quantification assay. Cell growth and migration were assessed using the CCK-8 and transwell assays. In addition, the role of RBM15 in vivo was examined using a mouse tumor xenograft model. RBM15 and miR-3605-5p were both substantially expressed in ESCC, however KRT4 was not expressed highly. Overexpressed RBM15 triggered cell proliferation and migration in ESCC. Besides, RBM15/m6A could mediate pri-3605-5p to form the mature miR-3605-5p, and miR-3605-5p further targeted KRT4. Further investigations showed that upregulation of KRT4 overturned the promoting impact of RBM15 overexpression on cell proliferation as well as on cell migration in ESCC by activating p53 signaling pathway. This work implied the carcinogenic activity of RBM15/m6A in ESCC via miR-3605-5p/KRT4 pathway, providing a novel m6A modification pattern in the tumorigenesis of ESCC.

LncRNA AGAP11 Suppresses Lung Adenocarcinoma Progression by miR-494-3p and Predicts Prognosis.

Lung adenocarcinoma (LUAD) is a subtype of lung cancer that occurs frequently and results in high mortality and morbidity, comprising almost 50% of all cases with the disease. Previously, long non-coding RNAs (lncRNAs) was evidenced to be helpful in the diagnosis and prognosis of LUAD. lncRNA AGAP11 was identified as a dysregulated lncRNA in LUAD. Whether AGAP11 is linked to the progression and prognosis of LUAD has not been known. The purpose was to probe the action of AGAP11 in the LUAD progression together with its intrinsic mechanism, with a view to supplying a perspective biomarker and therapeutic target for LUAD. AGAP11 expression in LUAD was analyzed by searching in the GEPIA database and conducting RT-qPCR. The significance of AGAP11 for the prognosis of LUAD was assessed by statistical analyses. The targeting relationship between AGAP11 and miR-494-3p was corroborated with Dual-luciferase reporter assay. The role of AGAP11 on cellular processes in LUAD cells was evaluated by CCK-8 and Transwell assays. AGAP11 was markedly down-regulated in LUAD and tightly correlated with TNM stage, lymph node metastasis, and tumor differentiation degree of patients. Down-regulation of AGAP11 was found to predict a dismal prognosis of LUAD. AGAP11 negatively modulated miR-494-3p expression by interacting with it. The growth, migration, and invasion of LUAD cells could be impaired by AGAP11 overexpression, which would be attenuated by the enhanced miR-494-3p expression. AGAP11 acted as a predictor for prognosis and curbed LUAD progression through modulating miR-494-3p.

Actin filament-associated protein 1-antisense RNA1 promotes the development and invasion of tongue squamous cell carcinoma via the AFAP1-AS1/miR-133a-5p/ZIC2 axis.

The present study aimed to explore the biological role and underlying mechanism of the long non-coding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) in the progression of tongue squamous cell carcinoma (TSCC). A quantitative reverse transcriptase-PCR (RT-qPCR) was conducted to assess relative levels of the miR-133a-5p, lncRNAs AFAP1-AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1-AS1, miR-133a-5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial-mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR-133a-5p, as well as between miR-133a-5p and lncRNA AFAP1-AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1-AS1. The potential biological pathway for AFP1-AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1-AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1-AS1. In TSCC cell lines and specimens, lncRNA AFAP1-AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1-AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1-AS1 could upregulate ZIC2 by competitively inhibiting miR-133a-5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1-AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1-AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1-AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator-activated receptor signaling pathway was significantly correlated with low expression of AFP1-AS1. Finally, the results of drug prediction indicated that the group with high AFAP1-AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib. The upregulation of lncRNA AFAP1-AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1-AS1/miR-133a-5p/ZIC2 axis, aids in the progression of TSCC.

Clinical Value and Mechanism Exploration of Serum miR-379 in Obesity-Polycystic Ovary Syndrome.

As a common endocrine and metabolic disorder, polycystic ovary syndrome (PCOS) is mostly associated with an obese phenotype. The present research focuses on the clinical significance of miR-379 in obesity-PCOS and attempts to elucidate its potential mechanisms. Healthy individuals (n = 46), obesity-PCOS (n = 92), and non-obesity PCOS (n = 31) subjects were enrolled. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the level of serum miR-379. The receiver operating characteristic (ROC) curve and logistic regressions were applied to reveal the diagnostic significance. Dual luciferase reporters were performed to validate the targeting relationships. And cell count kit (CCK-8) assay was used to detect cell proliferation. Serum miR-379 was highly expressed in PCOS patients (P < 0.05), in especially obesity-PCOS patients. Higher miR-379 was associated with greater body mass index (BMI), higher bioavailable testosterone (bT), and greater insulin resistance (IR). Additionally, miR-379 was an independent risk factor for the development of obesity-PCOS. The sensitivity of miR-379 in identifying patients with obesity-PCOS from healthy or non-obesity-PCSO patients was 81.52% and 72.83%, and the specificity was 86.96% and 80.65%. Semaphorin 3 A (SEMA3A) was identified as a target of miR-379 and was reduced in the patients with obesity PCOS (P < 0.05). Inhibition of miR-375 reduced KGN proliferation, but this reduction was partially restored by silencing of SEMA3A (P < 0.05). Elevated miR-379 assists the diagnosis of obesity-PCOS and regulates the proliferation of KGN by targeting SEMA3A engaged in disease development.

PepT1-targeted nanodrug based on co-assembly of anti-inflammatory peptide and immunosuppressant for combined treatment of acute and chronic DSS-induced ColitiS.

Inflammatory bowel disease (IBD), as a chronic and recurrent inflammatory bowel diseases with limited therapeutic outcomes, is characterized by immune disorders and intestinal barrier dysfunction. Currently, the most medications used to cure IBD in clinic just temporarily induce and maintain remission with poor response rates and limited outcomes. Therefore, it is urgently necessary to develop an appropriate therapeutic candidate with preferable efficacy and less adverse reaction for curing IBD. Five groups of mice were utilized: control that received saline, DSS group (mice received 2.5% DSS or 4% DSS), KPV group (mice received KPV), FK506 group (mice received FK506) and NPs groups (mice received NPs). The effect of NP on the inflammatory factors of macrophage was evaluated using CCK-8, quantitative polymerase chain reaction (PCR), Elisa and Western blot (WB). Immunofluorescent staining revealed the targeting relationship between NP and Petp-1. Immunohistochemistry staining showed the effect of NP on tight junction proteins. Moreover, in vivo animal experiments confirmed that NPs reduced inflammatory levels in IBD. After administering with NPs, mice with DSS-induced acute or chronic colitis exhibited significant improvement in body weight, colon length, and disease activity index, decreased the level of the factors associated with oxidative stress (MPO, NO and ROS) and the inflammatory cytokines (TNF-α, IL-1β and IL-6), which implied that NPs could ameliorate murine colitis effectively. Furthermore, treating by NPs revealed a notable reduction of the expressions of CD68 and CD3, restoring the expression levels of tight junction proteins (Claudin-5, Occludin-1, and ZO-1) were significantly restored, surpassing those observed in the KPV and FK506 groups. which indicated that NPs can reduce inflammation and enhance epithelial barrier integrity by decreasing the infiltration of macrophages and T-lymphocytes. Collectively, those results demonstrated the effectively therapeutic outcome after using NPs in both acute and chronic colitis, suggesting that the newly co-assembled of NPs can be as a potential therapeutic candidate for colitis.

Systematic identification and characterization of compounds with reliable activity against multiple target proteins from different classes

Polypharmacology provides a basis for the treatment of diseases originating from perturbed target and pathway networks. It depends on the use of compounds with suitable multi-target activity and pharmacological effects resulting from multi-target engagement. In addition to their central role for polypharmacology, multi-target compounds are also of interest for basic research, for example, to better understand their binding characteristics or interaction patterns. While it is not unlikely that active compounds might bind to closely related targets, we have been interested in identifying compounds that are active against distantly or unrelated proteins, representing more unexpected instances of multi-target activity. Therefore, we have carried out a systematic search for compounds with activity against proteins from different classes, termed multi-class compounds (MC-CPDs). To this end, currently available compound activity data from medicinal chemistry were carefully curated. Subsequent analysis of ∼120,000 qualifying compounds with high-confidence target annotations identified 689 MC-CPDs with activity against at least three targets from two different protein classes. The target distribution of these compounds was determined and structural relationships were explored together with activity profiles. MC-CPDs covered many target combinations and established a wealth of compound-based target relationships, some of which could be rationalized by considering ligand binding characteristics of distinct targets. As a part of our study, the collection of newly identified MC-CPDs and associated target information is made publicly available.

MiR-497-5p promoted neuronal injury in ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt pathway.

The aim of this study was to investigate the molecular mechanism by which miR-497-5p regulates neuronal injury after ischemic stroke through the BDNF/TrkB/Akt signaling pathway. PC12 cells were used to construct a stroke injury model by oxygen-glucose deprivation/reoxygenation (OGD/R). The expression level of miR-497-5p was measured by RT-qPCR. CCK-8 kit was used to detect cell viability. Cell apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. MDA and SOD detection kits were used to detect MDA content and SOD activity. A double luciferase reporter system was used to verify the targeting relationship between miR-497-5p and BDNF. The expression of BDNF, TrkB, p-TrkB, Akt and p-Akt was detected by Western blot. We have found that miR-497-5p expression was inhibited after treatment with OGD/R. Simultaneously, cell apoptosis, MDA content and ROS were upregulated, while cell viability and SOD were significantly decreased in PC12 cells. The effects of OGD/R on PC12 cells were reversed with the downregulation of miR-497-5p. A double luciferase reporter assay demonstrated that miR-497-5p negatively targets BDNF. BDNF inhibited cell apoptosis and oxidative stress injury in PC12 cells. These findings suggest that miR-497-5p aggravates neuronal injury in experimental model of ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt signaling pathway.

MiR-34b-5p suppresses the epithelial-mesenchymal transition and metastasis in endometrial cancer AN3CA cells by targeting ZEB1.

Tumor metastasis is a primary cause of recurrence and mortality in endometrial cancer. miR-34b-5p is abnormally expressed in various cancers and participates in tumor cell progression and metastasis. The objective of this study was to elucidate the biological functions and molecular mechanisms of miR-34b-5p in regulating the epithelial-mesenchymal transition (EMT) and metastasis in AN3CA endometrial cancer cells. The expression levels of miR-34b-5p and zinc finger E-box-binding homeobox 1 (ZEB1) in endometrial cancer cells were analyzed by qRT-PCR, and ZEB1 expression in endometrial cancer tissues was examined by immunohistochemistry. Proliferation, migration, and invasion of endometrial cancer AN3CA cells were evaluated using CCK8, scratch, and transwell assays, respectively. Bioinformatic analysis and dual-luciferase reporter gene assays were used to validate the targeting relationship between miR-34b-5p and ZEB1. Western blotting was performed to analyze the expression levels of ZEB1 and EMT-related proteins. miR-34b-5p was significantly downregulated in endometrial cancer AN3CA cells. Overexpression of miR-34b-5p significantly inhibited proliferation, invasion, migration, and the EMT of endometrial cancer AN3CA cells. ZEB1, which was identified as a direct target gene of miR-34b-5p, exhibited high expression in endometrial cancer cells and tissues. Additionally, ZEB1 upregulation partially reversed the inhibitory effects of miR-34b-5p on proliferation, migration, invasion, and the EMT of endometrial cancer AN3CA cells. miR-34b-5p suppresses the EMT and metastasis in endometrial cancer AN3CA cells by targeting ZEB1, indicating that the miR-34b-5p-ZEB1-EMT axis may be a therapeutic target for endometrial cancer.

MiR-451a suppresses the proliferation and migration of high-grade serous ovarian cancer by targeting RAB5A through the Ras/Raf/MEK/ERK pathway.

Ovarian cancer is one of the most common cancers in women. Profiles changes of microRNAs (miRNAs) are closely linked to malignant tumors. In the present study, we investigated expression of miR-451a in high-grade serous ovarian cancer (HGSOC). We also investigated the potential pathological roles and the likely mechanism of miR-451a in the development of HGSOC using animal models and cell lines. Using bioinformatics techniques and a real-time PCR, we analyzed differently expressed miRNAs in HGSOC compared to normal tissue. MTT (i.e. 3-[4, 5-dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide), EDU (i.e. 5-ethynyl-2'-deoxyuridine) and transwell assays were performed to investigate the effect of miR-451a on the proliferation and migration of HGSOC SKOV-3 cells. A dual luciferase reporter assay was performed to verify the targeting relationship of miR-451 and RAB5A (one of the Rab GTPase proteins that regulates endocytosis and vesicle transport). Also, we analyzed levels of the RAB5A mRNA and protein by real-time PCR, western blotting and immunohistochemistry assays in HGSOC cells and tissues. Finally, we performed in vivo experiments using HGSOC mice. miR-451a was substantially upregulated in HGSOC and associated with favorable clinical characteristics. miR-451a knockdown significantly increased growth and metastasis of HGSOC cell line SKOV-3 through Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. In addition, RAB5A, an early endosome marker, was shown to be a direct target of miR-451a. Moreover, RAB5A is correlated with unfavorable clinical features and shows independent prognostic significance in HGSOC. We found that the miR-451a/RAB5A axis is associated with tumorigenesis and progression through the Ras/Raf/MEK/ERK pathway, providing prognostic indicators and therapeutic targets for patients with HGSOC.

LncRNA Gm28382 promotes lipogenesis by interacting with miR-326-3p to regulate ChREBP signaling pathway in NAFLD

Long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, their biological roles and function mechanisms in NAFLD remain largely unknown. In this study, we found that Gm28382 may be a potential pathogenic lncRNA of NAFLD and highly expressed in NAFLD through RNA-seq. Overexpression of Gm28382 significantly enhanced the lipid accumulation in AML12 cells, whereas Gm28382 silencing reduced lipogenesis both in palmitic acid (PA)-induced AML12 cells and high fat diet (HFD)-induced mice. Then, bioinformatics were employed to speculate the potential interacting genes of Gm28382, and found that Gm28382 may regulate ChREBP expression through binding with miR-326-3p. Fluorescence in situ hybridization (FISH), dual luciferase reporter assay, immunofluorescence RNA pull-down and RNA immunoprecipitation (RIP) assays were used to validate the binding and targeting relationship of these genes, and we confirmed that Gm28382 competitively binds to miR-326-3p to increase ChREBP expression as a ceRNA. Mechanistically, overexpression of Gm28382 upregulated the ChREBP-mediated lipid synthesis signaling pathway, but the function was sabotaged by miR-326-3p deletion or ChREBP overexpression. Furthermore, in PA-challenged AML12 cells or HFD-induced mice, silencing of Gm28382 reversed the aberrant ChREBP signaling pathway and lipid accumulation, whereas ChREBP overexpression or liver-specific silencing of miR-326-3p blocked this function of Gm28382. Collectively, these findings reveal a critical role of Gm28382 in the promotion of lipogenesis in NAFLD by regulating the ChREBP signaling pathway through interaction with miR-326-3p, which could serve as a potential therapeutic target for NAFLD treatment.

CircMAP3K4 Suppresses H2O2-Induced Human Lens Epithelial Cell Injury by miR-630/ERCC6 Axis in Age-Related Cataract

Background Dysregulated circular RNAs (circRNAs) is involved in the pathogenesis of age-related cataract (ARC). Here, this study aimed to explore the function and mechanism of circMAP3K4 in ARC. Methods Human lens epithelial cells were exposed to hydrogen peroxide (H2O2) for functional experiments. qRT-PCR and western blotting analyses were used for the expression detection of genes and proteins. Cell proliferation was tested using cell counting kit-8 and EdU. Flow cytometry was applied to analyze cell apoptosis and cell cycle. The oxidative stress was evaluated by detecting the production of malondialdehyde (MDA), reactive oxygen species (ROS), and superoxide dismutase (SOD). The target relationship between miR-630 and circMAP3K4 or Excision repair cross-complementing group 6 (ERCC6) was analyzed by dual-luciferase reporter assay and RIP assay. Results CircMAP3K4 was lowly expressed in ARC patients and H2O2-induced HLECs. Functionally, forced expression of circMAP3K4 protected HLECs against H2O2-evoked proliferation inhibition, cell cycle arrest and the promotion of cell apoptosis and oxidative stress. Mechanistically, circMAP3K4 acted as a sponge for miR-630 to regulate the expression of its target ERCC6. MiR-630 was highly expressed while ERCC6 was lowly expressed in ARC patients and H2O2-induced HLECs. Up-regulation of miR-630 could reverse the protective effects of circMAP3K4 on HLECs under H2O2 treatment. In addition, inhibition of miR-630 suppressed H2O2-induced HLEC injury, which was abolished by ERCC6 silencing. Conclusion Forced expression of circMAP3K4 protected HLECs against H2O2-evoked apoptotic and oxidative injury via miR-630/ERCC6 axis, suggesting that circMAP3K4 may function as a potential therapeutic target for ARC.

Downregulating miR-184 relieves calcium oxalate crystal-mediated renal cell damage via activating the Rap1 signaling pathway.

Renal calculi are a very prevalent disease with a high incidence. Calcium oxalate (CaOx) is a primary constituent of kidney stones. Our paper probes the regulatory function and mechanism of miR-184 in CaOx-mediated renal cell damage. CaOx was used to treat HK2 cells and human podocytes (HPCs) to simulate kidney cell damage. The qRT-PCR technique checked the profiles of miR-184 and IGF1R. The examination of cell proliferation was conducted employing CCK8. TUNEL staining was used to monitor cell apoptosis. Western blot analysis was used to determine the protein profiles of apoptosis-concerned related proteins (including Mcl1, Bcl-XL, and Caspase-3), the NF-κB, Nrf2/HO-1, and Rap1 signaling pathways. ELISA confirmed the levels of the inflammatory factors IL-6, TNF-α, MCP1, and ICAM1. The targeting relationship between miR-184 and IGF1R was validated by dual luciferase assay and RNA immunoprecipitation assay. Glyoxylate-induced rat kidney stones model and HK2 and HPC cells treated with CaOx demonstrated an increase in the miR-184 profile. Inhibiting miR-184 relieved CaOx-mediated renal cell inflammation, apoptosis and oxidative stress and activated the Rap1 pathway. IGF1R was targeted by miR-184. IGF1R activation by IGF1 attenuated the effects of miR-184 on renal cell damage, and Hippo pathway suppression reversed the inhibitory effect of miR-184 knockdown on renal cell impairment. miR-184 downregulation activates the Rap1 signaling pathway to ameliorate renal cell damage mediated by CaOx.

Circ_CHMP5 aggravates oxidized low-density lipoprotein-induced damage to human umbilical vein endothelial cells through miR-516b-5p/TGFβR2 axis.

Atherosclerosis (AS) was one of the main causes of death in the elderly, and lesions in human umbilical vein endothelial cells (HUVECs) could lead to AS. CircRNA-charged multivesicular body protein 5 (circ_CHMP5) was reported to participate in the progression of AS. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the levels of circ_CHMP5, miR-516b-5p, and transforming growth factor beta receptor 2 (TGFβR2) in AS patients or ox-LDL-induced HUVECs. 5-Ethynyl-2'-deoxyuridine and cell counting kit-8 assays were performed to detect cell proliferation. Proteins expression was assessed by western blot assay. Cell apoptosis was examined by flow cytometry. Tube formation assay was utilized to measure the tube formation ability of HUVCEs. The targeting relationships between miR-516b-5p and circ_CHMP5 or TGFβR2 were confirmed by dual-luciferase reporter assay and RNA-pull down assay. Circ_CHMP5 was enhanced in the serum of AS patients and ox-LDL-exposure HUVECs. Ox-LDL blocked proliferation and tube formation of HUVECs and induced cell apoptosis, and circ_CHMP5 knockdown reversed these effects. In addition, circ_CHMP5 regulated the growth of ox-LDL-induced HUVECs through miR-516b-5p and TGFβR2. Moreover, the effects of circ_CHMP5 knockdown on ox-LDL-induced HUVECs were obviously recovered by downregulation of miR-516b-5p, and overexpression of TGFβR2 restored the effects of miR-516b-5p upregulation on ox-LDL-stimulated HUVECs. Silence of circ_CHMP5 overturned ox-LDL-treated inhibition of HUVECs proliferation and angiogenesis by miR-516b-5p and TGFβR2. These results provided new solutions for the treatment of AS.

Circ_0000115 Protects Against Cerebral Ischemia Injury by Suppressing Neuronal Apoptosis, Oxidative Stress and Inflammation by miR-1224-5p/Nos3 Axis In Vitro.

Cerebral ischemia is a severe neurological disability related to neuronal apoptosis and cellular stress response. Circular RNAs (circRNAs) are emerging regulators of cerebral ischemia. Herein, this study proposed to probe the action of circ_0000115 in cerebral ischemia injury. The mouse neuroblastoma cells N2a and HT22 underwent oxygen-glucose deprivation (OGD) were used as a model of in vitro cerebral ischemia. Levels of genes and proteins were detected by qRT-PCR and western blotting. Cell proliferation and apoptosis were determined by EdU assay and flow cytometry. Western blotting was used to detect the protein level of pro-inflammatory factors. The oxidative stress injury was evaluated by detecting reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) generation. Dual-luciferase reporter and RIP assays were used to confirm the target relationship between miR-1224-5p and circ_0000115 or nitric oxide synthase 3 (NOS3). OGD exposure decreased circ_0000115 and NOS3 expression, and increased miR-1224-5p in N2a and HT22 cells in a time-dependent manner. Circ_0000115 silencing attenuated OGD-induced apoptosis, oxidative stress and inflammation in N2a and HT22 cells. Mechanistically, circ_0000115 directly sponged miR-1224-5p, which targeted NOS3. Furthermore, rescue experiments showed that miR-1224-5p overexpression abolished the neuroprotective effect of circ_0000115 in N2a and HT22 cells under OGD treatment. Besides that, silencing of miR-1224-5p protected N2a and HT22 cells against OGD-evoked injury, which was counteracted by NOS3 knockdown. Circ_0000115 protects N2a and HT22 cells against OGD-evoked neuronal apoptosis, inflammation, and oxidative stress via the miR-1224-5p/NOS3 axis, providing an exciting view of the pathogenesis of cerebral ischemia.

Mechanism of NO2-induced migraine in rats: The exploration of the role of miR-653-3p/IGF1 axis

Migraine is a severely disabling primary neurological disorder. Although some studies have confirmed that nitrogen dioxide (NO2) pollution increases the risk of migraine, and our previous study demonstrated the role of the channel protein transient receptor potential cation channel subfamily V member 1 (TRPV1) in NO2-induced migraine, the underlying mechanisms have not been fully elucidated. This study aimed to explore the intrinsic toxicity mechanism of NO2-induced migraines using transcriptome sequencing. First, the differentially expressed genes in NO2-induced migraine, insulin-like growth factor 1 (IGF1) and miRNA miR-653-3p were identified using RNA and small RNA sequencing, and a protein interaction network was constructed using STRING to explore the possible mechanisms. Next, the targeting relationship between miR-653-3p and IGF1 was determined. NO2-induced migraine was verified by silencing miR-653-3p and IGF1, independently or in combination to regulate the protein kinase B (AKT)/TRPV1 signalling pathway through the miR-653-3p/IGF1 axis. These results indicate that the key molecular mechanism of NO2-induced migraine may be that the miR-653-3p/IGF1 axis regulates the AKT/TRPV1 signalling pathway to induce migraine. The findings of this study will further elucidate the neurotoxic mechanism of NO2-induced migraines and lay a new experimental foundation for implementing migraine-related preventive and therapeutic control measures.

MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.

The regulatory effect of lncRNA LINC00943 on the progression of hepatocellular carcinoma and its relationship with clinicopathological features

BackgroundThe risk factors for the pathogenesis of HCC are highly variable, and the prognosis of patients is very unsatisfactory. In this study, we investigated the regulatory effect of LINC00943 on HCC progression and its relationship with clinicopathological features. MethodsLINC00943 level in HCC tissues and cell specimens was verified by RT-qPCR. The pathologic significance of LINC00943 in the prognosis of HCC was analyzed by Kaplan-Meier and Cox regression analyses. The behavioral function of LINC00943 in HCC cells was evaluated via CCK-8 and Transwell assays. The specific targeting relationship between LINC00943 and miR-195–5p was investigated by luciferase activity assay. ResultsLINC00943 was highly expressed in HCC tissues and cell specimens. Clinical data analysis showed that elevated LINC00943 indicated poor prognosis in patients with HCC and was related to TNM stage and lymph node metastasis. Cell experiments demonstrated that silencing LINC00943 sponge miR-195–5p suppressed the proliferation, migration and invasion of HCC cells. Mechanistically, miR-195–5p inhibitor remedied the suppressive effect of silencing LINC00943 on the biological functions of HCC cells. ConclusionLINC00943 may be an independent prognostic factor of HCC, which provides new thinking for the prognosis and treatment of HCC patients.

Astragaloside IV-induced BMSC exosomes promote neovascularization and protect cardiac function in myocardial infarction mice via the miR-411/HIF-1α axis

This study focused on investigating the mechanism of the astragaloside IV-induced bone marrow mesenchymal stem cell exosome (AS-IV-MSC-exo)/microRNA(miR)-411/HIF-1α axis in affecting vascular neovascularization and protecting cardiac function in myocardial infarction (MI) mice. Exosomes (MSC-exo and AS-IV-MSC-exo) were separated by differential centrifugation and then characterized. MI mouse models were established by left anterior descending coronary artery ligation. Echocardiography was used to evaluate cardiac function. HE staining and Masson staining were performed to observe myocardial histopathology. Capillary density in the myocardium via immunohistochemistry and quantified the expression of vascular endothelial growth factor (VEGF) via RT-qPCR. The expression of miR-411 and HIF-1α was tested by RT-qPCR and western blot and the targeting relationship of miR-411 and HIF-1α was verified by bioinformatics website and dual luciferase reporter gene assay. Exosomes with lipid bi-layer membrane structure, expressing exosomal surface marker proteins, and being taken up by cardiomyocytes could be successfully isolated utilizing ultracentrifugation. Intramyocardial injection of MSC-exo could restore cardiac function, decrease myocardial pathological changes and collagen deposition, and promote neovascularization in MI mice; the effect of AS-IV-MSC-exo was more significant. The ability of AS-IV-MSC-exo to restore cardiac function, lower myocardial pathological changes and collagen deposition, and promote neovascularization in MI mice was diminished when miR-411 expression in AS-IV-MSC-exo was reduced. Mechanistically, miR-411 was found to target and inhibit HIF-1α expression. Overexpression of HIF-1α impaired the impact of AS-IV-MSC-exo on improving cardiac function and promoting neovascularization in MI mice. AS-IV-MSC-exo improves cardiac function and promoted neovascularization via the miR-411/HIF-1α axis, thereby ameliorating MI.

Metformin promotes the normalization of abnormal blood vessels after radiofrequency ablation deficiency in hepatocellular carcinoma by microRNA-302b-3p targeting thioredoxin-interacting protein

Metformin has shown great promise in the treatment of HCC. Radiofrequency ablation (RFA) deficiency results in recurrence and metastasis of remaining HCC tumors. Here, we aimed to investigate the role and mechanism of metformin in HCC after RFA deficiency. HCC cell line Hep-G2 was selected to simulate RFA deficiency and named HepG2-H cells. After treating cells with different concentrations of metformin (2.5, 5, 10 μM) or transfecting related plasmids, cell proliferation, migration, invasion, apoptosis and angiogenesis were detected, in vitro permeability test was performed, and an angiogenesis-related protein VEGFA was analyzed. The residual HCC model after RFA deficiency was established in mice. Metformin was administered by gavage to detect changes in tumor volume and weight, and CD31 staining was used to observe microvessels. The targeting relationship between miR-302b-3p and TXNIP was demonstrated by the bioinformatics website, dual-luciferase reporter assay, and RNA pull-down assay. The results found that metformin inhibited RFA deficiency-induced growth and angiogenesis of HCC cells in vitro. miR-302b-3p counteracted the therapeutic effect of metformin on RFA deficiency. miR-302b-3p targeted regulation of TXNIP. The up-regulation of TXNIP reversed the effects of overexpression of miR-302b-3p on RFA-deficient HCC cells. Metformin inhibited RFA-deficiency-induced HCC growth and tumor vascular abnormalities in vivo. Overall, metformin promotes the normalization of abnormal blood vessels after RFA deficiency in HCC by miR-302b-3p targeting TXNIP, which can be used to prevent the progression of HCC after RFA.