Double Luciferase Reporter Assay Research Articles

MiR-124 is upregulated in diabetic mice and inhibits proliferation and promotes apoptosis of high-glucose-induced β-cells by targeting EZH2.

Diabetes is a chronic metabolic disease, and a variety of miRNA are involved in the occurrence and development of diabetes. In clinical studies, miR-124 is highly expressed in the serum of patients with diabetes and in pancreatic islet β-cells. However, few reports exist concerning the role and mechanism of action of miR-124 in diabetes. To investigate the expression of miR-124 in diabetic mice and the potential mechanism of action in islet β-cells. The expression levels of miR-124 and enhancer of zeste homolog 2 (EZH2) in pancreatic tissues of diabetic mice were detected. The targeted relationship between miR-124 and EZH2 was predicted by Targetscan software and verified by a double luciferase reporter assay. Mouse islet β-cells Min6 were grown in a high glucose (HG) medium to mimic a diabetes model. The insulin secretion, proliferation, cell cycle and apoptosis of HG-induced Min6 cells were detected after interference of miR-124a and/or EZH2. The expression of miR-124 was upregulated and EZH2 was downregulated in the pancreatic tissue of diabetic mice compared with control mice, and the expression of miR-124 was negatively correlated with that of EZH2. miR-124 was highly expressed in HG-induced Min6 cells. Inhibition of miR-124 promoted insulin secretion and cell proliferation, induced the transition from the G0/G1 phase to the S phase of the cell cycle, and inhibited cell apoptosis in HG-induced Min6 cells. EZH2 was one of the targets of miR-124. Co-transfection of miR-124 inhibitor and siRNA-EZH2 could reverse the effects of the miR-124 inhibitor in HG-induced Min6 cells. miR-124 is highly expressed in diabetic mice and HG-induced Min6 cells and regulates insulin secretion, proliferation and apoptosis of islet β-cells by targeting EZH2.

MiR-328-3p promotes TGF-β1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway.

Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-β1-induced airway smooth muscle cells (ASMCs). The effect of TGF-β1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-β1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins. The expression of miR-328-3p was up-regulated in TGF-β1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-β1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-β1-induced ASMC's proliferation, migration, and inflammation. In comparison to the knockdown of miR-328-3p alone, the simultaneous knockdown of miR-328-3p with PTEN decreased PTEN protein expression levels and increased p-Akt/Akt ratio in TGF-β1-induced ASMCs. Through regulating the expression of PTEN and the activity of Akt signaling pathway, miR-328-3p promotes TGF-β1-induced proliferation, migration, and inflammation of ASMCs.

Ginsenoside Rg1 attenuates the NASH phenotype by regulating the miR-375-3p/ATG2B/PTEN-AKT axis to mediate autophagy and pyroptosis

BackgroundNonalcoholic steatohepatitis (NASH) is one of the most frequent liver diseases at present, and there is no radical treatment. The consequences of a variety of ginsenoside compounds on this situation have before been reported, however, the specific effect on the monomeric ginsenoside Rg1 (Rg1) and its associated underlying molecular mechanism stay unknown.Material and methodsIn vitro, the cell models were constructed by exposing free fatty acids (FFAs) to HepG2 cells. A methionine and choline deficiency (MCD)-induced NASH mouse model was also established over 5–6 weeks of treatment. Rg1 is a traditional Chinese medicine monomer. These NASH models were treated with Rg1 and analyzed by qRT-PCR, Western Blot, sequencing, Oil red O staining, immunofluorescence, enzyme activity, HE staining, ELISA, double luciferase reporter assay, and immunohistochemistry.ResultsOverexpression of ATG2B, an autophagy-related protein, attenuated lipid droplet accumulation and reduces ALT, AST, inflammatory cytokines, hydrogen peroxide, and pyroptosis in established mouse and cellular models of NASH and increased levels of ATP and autophagy. The binding sites of miR-375-3p and ATG2B were verified by bioinformatic prediction and a dual-luciferase reporter gene. Knockdown of miR-375-3p promoted autophagy and inhibited pyroptosis. ATG2B knockdown substantially attenuated the impact of miR-375-3p on NASH. Rg1 appears to regulate the occurrence and development of NASH inflammation through miR-375-3p and ATG2B in vitro and in vivo, and is regulated by PTEN-AKT pathway.ConclusionsThis study showed that Rg1 participates in autophagy and pyroptosis through the miR-375-3p/ATG2B/PTEN-AKT pathway, thereby alleviating the occurrence and development of NASH, for that reason revealing Rg1 as a candidate drug for NASH.

Epigallocatechin-3-gallate ameliorates inflammatory injury caused by sepsis by regulating the lncRNA PVT1/miR-16-5p/TLR4 axis.

Sepsis is identified as a severe inflammatory disease. Epigallocatechin-3-gallate (EGCG) has been reported to be a powerful anti-inflammatory chemical substance in numerous diseases. However, the underlying mechanism of the anti-inflammatory effects of EGCG in sepsis remains to be elucidated. The surgery for cecal ligation and puncture (CLP) was performed on male C57BL/6J mice aged 8weeks. THP-1 cells were treated with 1μg/ml lipopolysaccharide (LPS) for 24h to imitate sepsis in vitro. Haematoxylene-Eosin (HE) staining of the sections of liver, lung and kidney was performed to evaluate the pathological changes. The inflammatory cytokines were quantitated by ELISA. qRT-PCR was performed to measure the expression levels of PVT1, miR-16-5p, and TLR4. The protein level of TLR4 was also assessed by Western blotting. Double luciferase reporter assay and RIP assay were performed to validate the interactions among PVT1, miR-16-5p, and TLR4. EGCG inhibited the expression levels of PVT1 and TLR4 and enhanced miR-16-5p expression in CLP-operated mice and LPS-treated THP-1 cells. EGCG reduced the levels of inflammatory cytokines, which were restored by PVT1 overexpression. Mechanistically, PVT1 bound with miR-16-5p to activate TLR4 signaling. Further experiments demonstrated that miR-16-5p silencing or TLR4 overexpression antagonized sh-PVT1 or miR-16-5p mimics-mediated inhibitory effects on inflammatory cytokines, respectively. Knockdown of PVT1 alleviated inflammatory injury in CLP-induced sepsis in mice. EGCG may effectively lower the levels of sepsis-induced inflammatory cytokines by targeting the PVT1/miR-16-5p/TLR4 axis.

Circular RNA SMARCA5 inhibits cholangiocarcinoma via microRNA-95-3p/tumor necrosis factor receptor associated factor 3 axis.

Enhancing research indicatedthat circular RNA (circRNA) acted a critical part in cholangiocarcinoma (CHOL) development. This research aims to discover the role of circRNA SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 5 (SMARCA5) in CHOL bio-progression, which has been proved to be downregulated in CHOL tissues. In this study, quantitative reverse transcription polymerase chain reaction was used to reveal the level and linkage of circRNA SMARCA5, miRNA-95-3p and TNF receptor-associated factor 3 gene (TRAF3) in CHOL tissues and cancer cells. The target sites of circRNA SMARCA5 and miRNA-95-3p were forecast by Starbase, and Targetscan was conducted to forecast the potential linkage points of TRAF3 and miRNA-95-3p, and which is affirmed by double luciferase reporter assay. CCK-8 and flow cytometry assay was carried to indicate cell viability. And apoptosis-related protein was counted by caspase3 activity and Western blot assay. CircRNA SMARCA5 was downregulated in CHOL cell lines and cancer samples. Besides, over-expression of SMARCA5 inhibited cell growth and promoted apoptotic rate. Dual-luciferase reporter assays presented that miRNA-95-3p could link with circRNA SMARCA5. Moreover, miRNA-95-3p was discovered highly expressed in CHOL. Interference of miRNA-95-3p repressed cell proliferation and raised the apoptosis. Importantly, TRAF3 was validated to be a downstream of miRNA-95-3p. Strengthen of miRNA-95-3p reversed the inhibitory impact of circRNA SMARCA5-plasmid transfection, and the results of miRNA-95-3p inhibitor were reversed by si-TRAF3. CircRNA SMARCA5 is involved in CHOL development by interosculating miRNA-95-3p/TRAF3 axis and may become a novel approach for treating CHOL.

Alfalfa Xeno-miR168b Target CPT1A to Regulate Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

It was shown that microRNAs (miRNAs) play an important role in the synthesis of milk fat; thus, this manuscript evaluated whether exogenous miRNA (xeno-miRNAs) from alfalfa could influence the milk fat content in dairy cows. At first, mtr-miR168b was screened from dairy cow milk and blood. Then, EdU staining, flow cytometry, Oil Red O staining, qRT-PCR, and WB were applied to explore the effect of xeno-miR168b on the proliferation, apoptosis, and lipid metabolism of bovine mammary epithelial cells (BMECs). Finally, in order to clarify the pathway that regulated the lipid metabolism of BMECs using xeno-miR168b, a double-luciferase reporter assay was used to verify the target gene related to milk fat. These results showed that overexpression of xeno-miR168b inhibited cell proliferation but promoted apoptosis, which also decreased the expression of several lipid metabolism genes, including PPARγ, SCD1, C/EBPβ, and SREBP1, significantly inhibited lipid droplet formation, and reduced triglyceride content in BMECs. Furthermore, the targeting relationship between CPT1A and xeno-miR168b was determined and it was confirmed that CPT1A silencing reduced the expression of lipid metabolism genes and inhibited fat accumulation in BMECs. These findings identified xeno-miR168b from alfalfa as a cross-kingdom regulatory element that could influence milk fat content in dairy cows by modulating CPT1A expression.

Expression of nucleus accumbens-1 in colon cancer negatively modulates antitumor immunity.

Nucleus accumbens-1 (NAC-1) is highly expressed in a variety of tumors, including colon cancer, and is closely associated with tumor recurrence, metastasis, and invasion. To determine whether and how NAC-1 affects antitumor immunity in colon cancer. NAC-1-siRNA was transfected into RKO colon cancer cells to knock down NAC expression; tumor cells with or without knockdown of NAC-1 were treated with CD8+ T cells to test their cytocidal effect. The level of the immune checkpoint programmed death receptor-1 ligand (PD-L1) in colon cancer cells with or without knockdown of NAC-1 was analyzed using Quantitative real-time polymerase chain reaction and Western blotting. A double luciferase reporter assay was used to examine the effects of NAC-1 on the transcription of PD-L1. Mice bearing MC-38-OVA colon cancer cells expressing NAC-shRNA or control-shRNA were treated with OT-I mouse CD8+ T cells to determine the tumor response to immunotherapy. Immune cells in the tumor tissues were analyzed using flow cytometry. NAC-1, PD-L1 and CD8+ T cells in colon cancer specimens from patients were examined using immunohistochemistry staining. Knockdown of NAC-1 expression in colon cancer cells significantly enhanced the cytocidal effect of CD8+ T cells in cell culture experiments. The sensitizing effect of NAC-1 knockdown on the antitumor action of cytotoxic CD8+ T cells was recapitulated in a colon cancer xenograft animal model. Furthermore, knockdown of NAC-1 in colon cancer cells decreased the expression of PD-L1 at both the mRNA and protein levels, and this effect could be rescued by transfection of an RNAi-resistant NAC-1 expression plasmid. In a reporter gene assay, transient expression of NAC-1 in colon cancer cells increased the promoter activity of PD-L1, indicating that NAC-1 regulates PD-L1 expression at the transcriptional level. In addition, depletion of tumoral NAC-1 increased the number of CD8+ T cells but decreased the number of suppressive myeloid-derived suppressor cells and regulatory T cells. Tumor expression of NAC-1 is a negative determinant of immunotherapy.

Paxillin tunes the relationship between cell-matrix and cell-cell adhesions to regulate stiffness-dependent dentinogenesis.

Mechanical stiffness is recognized as a key physical factor and directs cell function via a mechanotransduction process, from extracellular physical cues to intracellular signaling cascades that affect transcriptional activity. Cells continually receive mechanical signals from both the surrounding matrix and adjacent cells. However, how mechanical stiffness cue at cell-substrate interfaces coordinates cell-cell junctions in guiding mesenchymal stem cell behaviors is poorly understood. Here, polydimethylsiloxane substrates with different stiffnesses were used to study mechanosensation/transduction mechanisms in controlling odontogenic differentiation of dental papilla cells (DPCs). DPC phenotypes (morphology and differentiation) changed in response to the applied force derived from stiff substrates. Significantly, higher expression of paxillin on stiffer substrates promoted DPC dentinogenesis. Upon treatment with siRNA to knockdown paxillin, N-cadherin increased mainly in the cytomembrane at the area of cell-cell contacts, whereas β-catenin decreased in the nuclei. The result of a double luciferase reporter assay showed that stiffness promoted β-catenin binding to TCF, which could coactivate the target genes associated with odontogenic differentiation, as evidenced by bioinformatics analysis. Finally, we determined that the addition of a β-catenin inhibitor suppressed DPC mineralization in all the stiffness groups. Thus, our results indicated that a mechanotransduction process from cell-substrate interactions to cell-cell adhesions was required for DPC odontogenic differentiation under the stimulation of substrate stiffness. This finding suggests that stem cell fate specification under the stimulus of stiffness at the substrates is based on crosstalk between substrate interactions and adherens junctions, which provides an essential mechanism for cell-based tissue engineering.

The role of Connexin26 regulated by MiR-2114-3p in the pathogenesis of ovarian cancer

ObjectivesThe purpose of our research was to determine the expression of Cx26 and miR-2114-3p, and their effects on proliferation, migration, and invasion in ovarian cancer and their mechanisms. Materials and methodsTranscriptome sequencing was performed and differentially expressed Cx26 was screened. The mRNA and protein levels of Cx26 in EOC and normal ovarian tissues were verified. The relationship between Cx26 levels and prognostics was analyzed. Cx26 Lentiviral vectors were constructed to detect its effect on ovarian cancer. WB verified that PI3K/AKT pathway was the possible signal pathway regulated by Cx26. The interaction between miR-2114-3p and Cx26 was detected by double luciferase reporter assay and qrt-PCR. CCK8, clone formation, transwell, and flow cytometry assays were conducted in cells transfected miR-2114-3p plasmids. The vivo experiment investigated the effects of Cx26 on subcutaneous tumor growth, PI3K expression, proliferation proteins Ki67 and PCNA. ResultsCx26 was up-regulated in EOC tissue and cell lines, and was associated with poor prognosis of ovarian cancer, while miR-2114-3p was down-regulated in EOC cell lines. Cx26 was a direct target of miR-2114-3p. Cx26 overexpression and miR-2114-3p inhibition promoted the growth, motility, invasiveness, and S phase arrest of EOC cells. Additionally, Cx26 could activated PI3K pathway whatever in vivo and in vitro. ConclusionsDysregulation of Cx26 is critical in EOC patients. Manipulation of this mechanism may influence the survival of EOC patients. MiR-2114-3p regulates the tumor-promoting activity of Cx26 in EOC. By inhibiting the PI3K pathway or knocking down Cx26 effectively inhibits tumor growth in EOC cells and Nude mouse model.

MicroRNA-137 inhibits pituitary prolactinoma proliferation by targeting AKT2.

Prolactinoma is the most common type of pituitary adenoma. Most prolactinoma need medical treatment, but some of them are aggressive and require surgery. In previous decades, some miRNAs have been manifested as oncogenes or tumor suppressors. Consequently, miRNAs' abnormal expression involves tumorigenesis, invasion, and metastasis of different types of tumors, including pituitary tumors. The current study aim to explore the aggressiveness-associated miRNAs in prolactinoma and underlying molecular mechanisms based on the bioinformatic analysis and fundamental experiment studies. GSE46294 miRNA expression profile from the Gene Expression Omnibus (GEO) database was downloaded. Differentially expressed miRNAs (DEMs) were filtered from this data. Subsequently, the target genes of downregulated miRNAs were analyzed by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. RT-qPCR, western blot, and CCK-8 assays were used to validate the effect of miR-137 on the proliferation of MMQ cells through AKT2. Finally, the binding site of rat miR-137 to AKT2were predicted by Targetscan andBibiserv database, and verified by double luciferase reporter assay. Twenty-four changed DEMs (fourteen upregulated and ten downregulated) were identified. Target genes of downregulated DEMs were classified into three groups by GO terms. KEGG pathway enrichment analysis revealed these target genes enriched in the PI3K-Akt pathway. We also confirmed that miR-137 can target AKT2 and inhibit the proliferation of MMQ cells induced by AKT2. MiR-137 suppressed prolactinomas' aggressive behavior by targeting AKT2.

MiR-138-5p inhibits the progression of colorectal cancer via regulating SP1/LGR5 axis.

Colorectal cancer (CRC) is one of the main malignancies that seriously threaten human health. Considering the high mortality and morbidity associated with this disease, even surgical resection and chemotherapy may not be sufficient in certain cases. This study aimed to explore the molecular mechanisms of miR-138-5p in regulating CRC progression. Quantitative reverse transcriptase polymerase chain reaction and western blot were performed to assess the levels of mRNA and proteins, including miR-138-5p, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), SP1, β-catenin, cyclin D1, and c-myc. The bioactivities of LoVo and HCT116 cells were assessed via MTT assay, flow cytometry, and transwell assay. StarBase was used to identify the downstream targets of genes. Double luciferase reporter and RIP assays revealed the direct binding of miR-138-5p to SP1 and of SP1 to LGR5. Our results illustrated that miR-138-5p was downregulated in CRC and its knockdown accelerated CRC progression. Conversely, SP1 was upregulated in CRC and its knockdown inhibited CRC progression. SP1 is also targeted by miR-138-5p and binds to LGR5. This study showed that miR-138-5p inhibits LoVo and HCT116 cell proliferation, migration, and invasion. Overall, miR-138-5p regulates CRC progression and promotes apoptosis via the SP1/LGR5 axis. This study indicates that miR-138-5p is involved in regulating CRC progression.

LINC00958 Inhibits Autophagy of Bladder Cancer Cells via Sponge Adsorption of miR-625-5p to Promote Tumor Angiogenesis and Oxidative Stress.

Objective This study further explored LINC00958's role in promoting tumor angiogenesis (AG) and oxidative stress (OS) development by inhibiting BC cell autophagy through sponge adsorption of miR-625-5p. Methods BC patients and healthy controls who visited our hospital between June 2017 and February 2019 were selected as the research group (RG) and the control group (CG), respectively, with a total of 133 study subjects. Peripheral blood LINC00958 and miR-625-5p in both cohorts of participants were detected. Additionally, human bladder transitional cell carcinoma cells (T24 and J82) and human normal urothelial cells (SV-HUC-1) were purchased. Alterations in cell biological behavior were observed after transfecting miR-625-5p-mimics, miR-625-5p-inhibition, and miR-625-5p-NC sequences into these cells, respectively. Besides, ELISA was performed to quantify inflammatory factors (IFs), AG indicators, and OS indexes in cells. Subsequently, a double luciferase reporter (DLR) assay was performed to verify the targeting relationship between LINC00958 and miR-625-5p. Finally, BALB/c-nu nude mice were purchased, and T24 cells transfected with silenced LINC00958 and miR-625-5p expression sequences were used to establish subcutaneous tumors to observe tumor growth and pathological changes. Results RG exhibited higher LINC00958 and lower miR-625-5p than CG. LINC00958 and miR-625-5p were strongly linked to myometrial invasion (MI), lymph node metastasis (LNM), distant metastasis (DM), and histology in BC patients, and the increase of LINC00958 and the decrease of miR-625-5p predicted an increased risk of prognostic death in such patients. After miR-625-5p inhibition, the capacity of BC cells to proliferate, invade, and migrate enhanced and the AG, inflammatory response, and OS injury increased, while the apoptosis rate and autophagy ability decreased. The DLR assay revealed inhibited LINC00958WT fluorescence activity by miR-625-5p-mimics, while the biological behavior of BC cells cotransfected with sh-LINC00958 and miR-625-5p-inhibition had no difference with the functions of sh-control and miR-625-5p-NC cotransfected cells. Finally, the nude mouse tumorigenesis experiment showed that the tumor mass, volume, and histopathological features of the sh-LINC00958 group were decreased compared with the sh-control group, while those of the miR-625-5p-inhibition group were increased versus miR-625-5p-NC. Conclusions In BC, LINC00958 is highly expressed while miR-625-5p is underexpressed. LINC00958 can inhibit cell autophagy to enhance cell activity; promote OS, inflammation, and AG; and regulate tumor immunity by targeting miR-625-5p, thus participating in the development of BC.

CircRNF144B/miR-342-3p/FBXL11 axis reduced autophagy and promoted the progression of ovarian cancer by increasing the ubiquitination of Beclin-1

Circular RNAs (circRNAs) can regulate autophagy and ovarian cancer (OC) progression. However, autophagy-associated circRNAs involved in OC progression are largely unknown. Bioinformatics, RNA sequencing, and qRT-PCR were conducted to detect circRNF144B expression in OC as well as its relationship with patient prognosis. Functional experiments were used to determine the effects of circRNF144B on the proliferation, mobility and autophagy of OC. Double luciferase reporter assays, immunoprecipitation, and ubiquitination detection were performed to determine the molecular mechanisms of circRNF144B in autophagy and OC progression. CircRNF144B was elevated in OC tissues with low autophagy levels, and associated with poor prognosis. CircRNF144B promoted the malignant biological properties of OC cells, and inhibited the autophagy. Mechanistically, circRNF144B acts as a sponge for miR-342-3p and inhibits miR-342-3p-induced degradation of lysine demethylase 2 A (FBXL11) mRNA, leading to elevated FBXL11 protein levels. Elevated FBXL11 promoted the ubiquitination and degradation of Beclin-1, thus inhibiting autophagy. In conclusion, CircRNF144B increased FBXL11 level by sponging miR-342-3p, whereas elevated FBXL11 promoted the ubiquitination and protein degradation of Beclin-1, thus suppressing autophagy flux and promoting OC progression. Thus, circRNF144B may be an effective target for OC therapy.

MiR-9a-5p alleviates ventilator-induced lung injury in rats by inhibiting the activation of the MAPK signaling pathway via CXCR4 expression downregulation

BackgroundGlobally, Mechanical ventilation is the most commonly used short-term life support technology. Ventilator-induced lung injury (VILI) is an inflammatory injury caused by mechanical ventilation. MicroRNAs (miRNAs) are considered as new gene regulators that play an important role in lung injury and inflammation. However, the role and mechanism of action of miR-9a-5p in VILI remain unclear. MethodsHerein, a rat model of VILI was established. To determine the expression levels of miR-9a-5p and CXCR4 mRNA, real-time quantitative polymerase chain reactions (qRT-PCR) were conducted. As well as western blot (WB) and immunofluorescence analyses, we determined the expression of CXCR4, SDF-1 and MAPK signaling pathway-related kinases. Hematoxylin and eosin (H&E) staining and the wet-dry ratio of the lung tissue were used to evaluate organ injury. An enzyme-linked immunosorbent assay (Elisa) and myeloperoxidase (MPO) activity measurements were performed to evaluate the inflammatory response. In addition, double luciferase reporter assays were used to verify the association between miR-9a-5p and CXCR4. ResultsThe expression of miR-9a-5p was low, whereas that of CXCR4 was high in the lung tissues of VILI rats. The overexpression of miR-9a-5p alleviated the degree of pathological injury in the lung tissues of rats with VILI, downregulating inflammatory cytokine expression and MPO activity. In the VILI rat model, miR-9a-5p targeted the negative regulation of CXCR4, and CXCR4 overexpression to reverse the lung-protective and anti-inflammatory effects of miR-9a-5p overexpression in VILI rats. miR-9a-5p also inhibited the phosphorylation of extracellular signal receptor-activated kinase (ERK), a protein related to the MAPK signaling pathway, by downregulating CXCR4 expression. ConclusionmiR-9a-5p can hinder the activation of the MAPK/ERK signaling pathway and reduce inflammatory reactions and lung injury in VILI rats through the targeted regulation of CXCR4 expression. Therefore, miR-9a-5p could serve as an intervention target to supply a new strategy for the care of VILI.

Protective effect of fluoxetine against oxidative stress induced by renal ischemia-reperfusion injury via the regulation of miR-450b-5p/Nrf2 axis.

The present study was performed to assess the protective effect of fluoxetine (FLX) on renal ischemia-reperfusion injury (IRI) via the regulation of miR-450b-5p/Nrf2 axis in male rats. In vivo, these male rats were randomly divided into different treatment groups. The rats were administered with FLX (20 mg/kg, intraperitoneally) once daily for 3 days before operation. The pathomorphological changes of renal tissues were assessed by histological examination and Masson staining. In vitro, HK-2 cells were used to detect the activity by CCK-8 assay in Hypoxia/Reoxygenation (H/R) group and Hypoxia/Reoxygenation+Fluoxetine (H/R+FLX) group. In addition, the oxidative stress biomarkers were evaluated. Subsequently, Nrf2, NF-κB, and Nrf2-dependent antioxidant enzymes, were detected by Western blot assay. In vivo, the pathological changes and serological renal function were significantly relieved in the rats with the pre-treatment of FLX, compared to IRI group. After FLX stimulation, the expression levels of oxidative stress indices significantly decreased, while tissue antioxidant indices significantly increased, compared to IRI group. The differently expressed miRNAs on renal IRI in male rats were screened out by miRNA microarray, especially showing that miR-450b-5p was selected as the target miRNA. Following miR-450b-5p agomir injection, the pathological changes and oxidative stress biomarkers significantly aggravated, whether in IRI group or IRI+FLX group. Bioinformatics analysis and double-luciferase reporter assay demonstrated that miR-450b-5p directly targeted Nrf2. The expression level of NF-κB significantly increased, while the expression levels of Nrf2 and Nrf2-dependent antioxidant enzymes significantly decreased after miR-450b-5p agomir injection. Furthermore, the expression levels of Nrf2 and it-dependent antioxidant enzymes were apparently increased in ischemic kidney after the transfection of miR-450b-5p mimic+recombination protein Nrf2, as well as the decreased expression levels of intracellular ROS and iNOS. In vitro, FLX significantly increased HK-2 cell viability, and relieved H/R HK-2 cell oxidative injury via down-regulating ROS and iNOS. In addition, H/R-induced oxidative damage was recovered with miR-450b-5p mimic and recombination protein Nrf2. Consequently, FLX played an important protective role in renal IRI-induced oxidative damage by promoting antioxidation via targeting miR-450b-5p/Nrf2 axis.

MiR-672-5p-Mediated Upregulation of REEP6 in Spinal Dorsal Horn Participates in Bortezomib-Induced Neuropathic Pain in Rats.

Evidence shows that miRNAs are deeply involved in nervous system diseases, but whether miRNAs contribute to the bortezomib (BTZ)-induced neuropathic pain remains unclear. We aimed to investigate whether miRNAs contribute to bortezomib (BTZ)-induced neuropathic pain and explore the related downstream cascades. The level of miRNAs in the spinal dorsal horn was explored using miRNA microarray and PCR. MiR-672-5p was significantly downregulated in dorsal horn neurons in the rats with BTZ treatment. Intrathecal injection of miR-672-5p agomir blunted the increase of the amplitude and frequency of sEPSCs in dorsal horn neurons and mechanical allodynia induced by BTZ. In addition, the knockdown of miR-672-5p by intrathecal injection of antagomir increased the amplitude and frequency of sEPSCs in dorsal horn neurons and decreased the mechanical withdrawal threshold in naïve rats. Furthermore, silico analysis and the data from subsequent assays indicated that REEP6, a potential miR-672-5p-regulating molecule, was increased in the spinal dorsal horn of rats with BTZ-induced neuropathic pain. Blocking REEP6 alleviated the mechanical pain behavior induced by BTZ, whereas overexpressing REEP6 induced pain hypersensitivity in naïve rats. Importantly, we further found that miR-672-5p was expressed in the REEP6-positive cells, and overexpression or knockdown of miR-672-5p reversely regulated the REEP6 expression. Bioinformatics analysis and double-luciferase reporter assay showed the existence of interaction sites between REEP6 mRNA and miR-672-5p. Overall, our study demonstrated that miR-672-5p directly regulated the expression of REEP6, which participated in the neuronal hyperexcitability in the spinal dorsal horn and neuropathic pain following BTZ treatment. This signaling pathway may potentially serve as a novel therapeutic avenue for chemotherapeutic-induced mechanical hypersensitivity.

Exosome Derived from Mesenchymal Stem Cells Alleviates Pathological Scars by Inhibiting the Proliferation, Migration and Protein Expression of Fibroblasts via Delivering miR-138-5p to Target SIRT1.

IntroductionThe therapies of using exosomes derived from mesenchymal stem cells (MSC-Exo) for wound healing and scar attenuation and micro RNAs (miRNAs) for regulation of genes by translational inhibition and mRNA destabilization obtained great achievements. Silent information regulator 1 (SIRT1) is the silent information, which has an intricate role in many biological processes. However, the effects of SIRT1 and miR-138-5p loaded in MSC-Exo on pathological scars remain unclear.MethodsMSC-Exo was isolated and identified by ultracentrifugation, transmission electron microscopy, nanoparticle size measuring instrument and Western blot assays. The relationship between SIRT1 and miR-138-5p was verified by a double-luciferase reporter assay. Cell Counting Kit-8, Τranswell, scratch, and Western blot assays were used to evaluate the proliferation and migration of human skin fibroblasts (HSFs), and the protein expression of SIRT1, NF-κB, α-SMA and TGF-β1 in HSFs, respectively. Flow cytometry was used to assess the apoptosis and cell cycle of HSFs affected by SIRT1.ResultsOur study demonstrated that miR-138-5p loaded in MSC-Exo could attenuate proliferation, migration and protein expression of HSFs-derived NF-κB, α-SMA, and TGF-β1 by targeting to SIRT1 gene, which confirmed the potential effects of MSC-Exo in alleviating pathological scars by performing as a miRNA’s delivery vehicle.ConclusionExosomes derived from MSCs acting as a delivery vehicle to deliver miR-138-5p can downregulate SIRT1 to inhibit the growth and protein expression of HSFs and attenuate pathological scars.

Ameliorating mechanism of nuciferine on high-fat diet-induced dyslipidemia and hepatic steatosis by regulating intestinal absorption and serum extracellular vesicles in rats

We investigated the effects of nuciferine rich in Lotus leaf on the amelioration of hepatic steatosis and dyslipidemia of hyperlipidemic rats, and explored the underlying mechanism on miRNAs and genes expression. Serum extracellular vesicles (EVs) from rats were prepared and characterized, and interactions between miRNAs and target mRNAs were validated in 293T cell line with the double luciferase reporter gene assay. Results showed that nuciferine significantly induced the expression of PPARα, ABCA1, CYP7A1, LXRα in liver and ABCG5 in intestine, while NPC1L1 expression was markedly reduced. In vitro, double luciferase reporter assays showed that miRNA-764-5p, miRNA-758-3p, and miRNA-126-5p interacted with the target mRNAs of PPARα, ABCA1, and CYP7A1, respectively. And the effect of nuciferine on lipid metabolism in rats was related to regulate intestinal absorption and serum EVs miRNAs. These miRNAs were able to reduce lipid levels by targeting lipid metabolism genes and up-regulating PPARα-LXRα-ABCA1 pathway and CYP7A1 expression.

Brucea javanica Oil Emulsion Promotes Autophagy in Ovarian Cancer Cells Through the miR-8485/LAMTOR3/mTOR/ATG13 Signaling Axis.

Background: Ovarian cancer is a common malignant tumor of the female reproductive tract, with the highest mortality rate. At present, no effective approaches to improve the survival rate exist. B. javanica Oil Emulsion (BJOE), an extract from B. javanica (L.) Merr. [Simaroubaceae], exhibits antitumor effects and can increase the sensitivity of radiotherapy and chemotherapy in many types of cancers. MiR-8485, a discovered miRNA, has been shown to be involved in the occurrence and development of tumors. The purpose of this study was to investigate the effect of BJOE on the regulation of mammalian rapamycin target protein (mTOR) autophagy signal pathway and related autophagy factors on ovarian cancer cells through miR-8485. Methods: The main chemical constituents of BJOE were determined by UHPLC-MS/MS. Detection of miR-8485 expression in ovarian cancer cells treated with BJOE by quantitative reverse transcription polymerase chain reaction (qRT-PCR). CCK8 experiment and flow cytometry were used to observe the effects of BJOE and overexpression of miR-8485 on cell proliferation and apoptosis. Then, monodansylcadaverine (MDC) fluorescence staining was used to observe the changes of autophagy vesicles before and after the effect of BJOE and overexpressed miR-8485 on cancer cells. Next, the binding sites between miR8485 and mammalian rapamycin target protein activator 3 (LAMTOR3) were detected by double luciferase reporter assay. Furthermore, qRT-PCR and Western blot experiments were used to explore the changes of autophagy-related factors LAMTOR3, mTOR and autophagy-related 13 (ATG13), and microtubule associated protein 1 light chain 3 beta (LC3-Ⅱ) after BJOE and overexpression of miR-8485, in addition to autophagy inhibitor (3-MA) for rescue experiment verification. Results: The qRT-PCR results showed that the expression of miR-8485 increased after BJOE treatment in the SKOV3 cell. The CCK8 assay and flow cytometry analysis revealed that both BJOE and miR-8485 overexpression inhibited the proliferation and promoted the apoptosis of the SKOV3 cell. MDC fluorescence staining showed that BJOE and miR-8485 overexpression led to a significant increase in autophagy vesicles in the SKOV3 cell. Double luciferase reporter assay confirmed the existence of binding sites between miR8485 and LAMTOR3. The results of qRT-PCR and Western blot showed that BJOE and overexpressed miR-8485 downregulated the expression of LAMTOR3 and mTOR and up-regulated the expression of ATG13 and LC3-Ⅱ. Conclusion: 1) MiR-8485 may be the key factor of BJOE in promoting autophagy and apoptosis and inhibiting cell proliferation of ovarian cancer cells; 2) BJOE may play an antitumor role by regulating LAMTOR3/mTOR/ATG13 signaling axis through miR-8485 to promote autophagy in ovarian cancer cells.

LncRNA SNHG6 accelerates hyperoxia-induced lung cell injury via regulating miR-335 to activate KLF5/NF-κB pathway

BackgroundBronchopulmonary dysplasia (BPD) is a common chronic lung disease in premature infants, and its pathogenesis has not been clarified. Long non-coding RNAs (lncRNA) have important functions in cell bioactivity. However, their role in developmental lung disease remains unclear. ObjectiveThe aim of this study was to demonstrate the role of lncRNA SNHG6 (SNHG6) in BPD and its underlying mechanisms. MethodsThe blood of patients with BPD were collected, and BPD model of BEAS-2B cells was established by hyperoxia method. SNHG6, miR-335 and KLF5 mRNA expression were detected by RT-qPCR. Western blot was conducted to measure the levels of apoptosis-related proteins’ expression and NF-κB pathway related proteins. BEAS-2B cell viability and apoptosis were assessed by CCK-8 and flow cytometry, respectively. Assay Kit was applied to detect ROS, MDA and SOD levels, respectively. ELISA was performed to assess the levels of inflammatory factors. The binding site of miR-335 with SNHG6 or KLF5 were predicted by using DIANA or TargetScan, and which was verified by double luciferase reporter assay. ResultsFirstly, SNHG6 was highly expressed and miR-335 was lowly expressed in BPD model, SNHG6 knockdown and miR-335 mimics both alleviated hyperoxia-induced lung cell injury, and SNHG6 targeted miR-335. Subsequently, KLF5 was targeted by miR-335, and KLF5 promoted lung cell injury via activating NF-κB pathway. Furthermore, SNHG6 mediated lung cell injury via regulating the miR-335/KLF5/NF-κB pathway. ConclusionOur research confirmed that SNHG6 mediated hyperoxia-induced lung cell injury via regulating the miR-335/KLF5/NF-κB pathway. These findings suggest that SNHG6 serves as promising targets for the treatment of newborns with BPD.