Dual-luciferase Reporter System Research Articles

MiR-367 alleviates inflammatory injury of microglia by promoting M2 polarization via targeting CEBPA

MiR-367 was reported to regulate inflammatory response of microglia. CCAAT/enhancer-binding protein α (C/EBPA) could mediate microglia polarization. In this study, we explored the possible roles of miR-367 and CEBPA in intracerebral hemorrhage (ICH). ICH and normal specimens were obtained from the tissue adjacent to and distant from hematoma of ICH patients, respectively. Microglia were isolated and identified by immunofluorescence. The isolated microglia were treated with erythrocyte lysate and randomly divided into 8 groups using different transfection reagents. The transfection efficiency of miR-367 was determined by qRT-PCR. The expressions of M1 and M2 microglia markers were detected by Western blotting. The relationship between CEBPA and miR-367 was confirmed by dual luciferase reporter system. Flow cytometry was performed to determine the level of apoptosis in the cells transfected with miR-367 and CEBPA in erythrocyte lysate–treated microglia. We found that miR-367 expression level was downregulated in ICH specimens. Erythrocyte lysate–treated microglia was successfully established using erythrocyte lysate, as decreased miR-367 expression was observed. Overexpression of miR-367 could significantly decrease the expressions of MHC-ІІ, IL-1β, and Bax, reduced apoptosis rate, and increased the expressions of CD206, Bal-2, and Arg-1 in erythrocyte lysate–treated microglia. CEBPA was proved to be a direct target for miR-367, which could inhibit microglia M2 polarization and increase apoptosis rate. However, in the presence of both CEBPA and miR-367 mimic, the protein and mRNA expressions of CEBPA were decreased, leading to promoted microglia M2 polarization and a decreased apoptosis rate. MiR-367 regulates microglia polarization by targeting CEBPA and is expected to alleviate ICH-induced inflammatory injury.

Down-regulation of miR-215 attenuates lipopolysaccharide-induced inflammatory injury in CCD-18co cells by targeting GDF11 through the TLR4/NF-kB and JNK/p38 signaling pathways.

Ulcerative colitis (UC) is a risk factor for carcinogenesis of colorectal cancer, which is associated with disruption of the epithelial barrier and disorder of the inflammatory response. It has been reported that the expression of microRNA (miR)-215 is upregulated in patients with long-term UC. The present study aimed to investigate the effects of miR-215 on lipopolysaccharide (LPS)-induced inflammatory injury in CCD-18Co cells, as well as to identify the underlying possible molecular mechanisms. CCD-18Co cells were treated with 1 µg/ml LPS to induce inflammatory injury. Reverse transcription-quantitative PCR was performed to determine the expression of miR-215 in LPS-treated CCD-18Co cells. Moreover, a dual luciferase reporter system assay was used to evaluate the interaction of miR-215 and growth differentiation factor 11 (GDF11) in CCD-18Co cells. The expression of miR-215 was significantly upregulated in LPS-treated CCD-18Co cells. Knockdown of miR-215 significantly alleviated the inflammatory response and oxidative stress in LPS-treated CCD-18Co cells. In addition, GDF11 was identified as a direct binding target of miR-215 in CCD-18Co cells. Knockdown of miR-215 significantly increased the expression of GDF11, but decreased the expression levels of Toll-like receptor (TLR)4, phosphorylated (p)-p65, iNOS, p-p38 and p-JNK in LPS-treated CCD-18Co cells. Collectively, the present findings indicated that knockdown of miR-215 alleviated oxidative stress and inflammatory response in LPS-treated CCD-18Co cells by upregulating GDF11 expression and inactivating the TLR4/NF-κB and JNK/p38 signaling pathways.

Circ_0062020 Knockdown Strengthens the Radiosensitivity of Prostate Cancer Cells.

BackgroundProstate cancer (PCa) is a major contributor to reduce the life quality of males. Circular RNAs were frequently reported to be associated with cancers. In the case of radiotherapy to PCa, the role of circ_0062020 was still inconclusive, which was further explored in this study.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of circ_0062020, miR-615-5p and thyroid hormone receptor interactor 13 (TRIP13) in PCa tissues and cells, as well as in normal tissues and cell. Meanwhile, the proliferation of PCa cells was evaluated by clone formation assay and cell counting kit 8 (CCK8) assay. Moreover, the metastasis of PCa cells was assessed by transwell and wound healing assays. Furthermore, the apoptosis of PCa cells was determined by flow cytometry assay. Besides, dual-luciferase reporter system was applied to verify the correlation between miR-615-5p and circ_0062020 or TRIP13, which was predicted by online tool CircRNA interactome or TargetScan. In addition, the protein expression of TRIP13 was measured by Western blot in PCa tissues and cells and normal tissues and cells. Finally, xenograft tumor assay was performed to further confirming the function of circ_0062020 in PCa in vivo.ResultsCirc_0062020 and TRIP13 were upregulated, while miR-615-5p was downregulated in PCa tissues and cells. Circ_0062020 knockdown or miR-615-5p overexpression inhibited the proliferation and metastasis, and promoted apoptosis, which could be reversed by miR-615-5p inhibitor or pc-TRIP13 in ionizing radiation (IR)-treated PCa cells. As expected, circ_0062020 sponged miR-615-5p to regulate TRIP13 expression in PCa cells. Circ_0062020 knockdown also suppressed PCa tumor growth in vivo.ConclusionCirc_0062020 suppressed the radiosensitivity by miR-615-5p/TRIP13 axis in PCa cells, which might provide insights into the radiotherapy for PCa.

Klotho, PTSD, and advanced epigenetic age in cortical tissue.

This study examined the klotho (KL) longevity gene polymorphism rs9315202 and psychopathology, including posttraumatic stress disorder (PTSD), depression, and alcohol-use disorders, in association with advanced epigenetic age in three postmortem cortical tissue regions: dorsolateral and ventromedial prefrontal cortices and motor cortex. Using data from the VA National PTSD Brain Bank (n = 117), we found that rs9315202 interacted with PTSD to predict advanced epigenetic age in motor cortex among the subset of relatively older (>=45 years), white non-Hispanic decedents (corrected p = 0.014, n = 42). An evaluation of 211 additional common KL variants revealed that only variants in linkage disequilibrium with rs9315202 showed similarly high levels of significance. Alcohol abuse was nominally associated with advanced epigenetic age in motor cortex (p = 0.039, n = 114). The rs9315202 SNP interacted with PTSD to predict decreased KL expression via DNAm age residuals in motor cortex among older white non-Hispanics decedents (indirect β = -0.198, p = 0.027). Finally, in dual-luciferase enhancer reporter system experiments, we found that inserting the minor allele of rs9315202 in a human kidney cell line HK-2 genomic DNA resulted in a change in KL transcriptional activities, likely operating via long noncoding RNA in this region. This was the first study to examine multiple forms of psychopathology in association with advanced DNA methylation age across several brain regions, to extend work concerning the association between rs9315202 and advanced epigenetic to brain tissue, and to identify the effects of rs9315202 on KL gene expression. KL augmentation holds promise as a therapeutic intervention to slow the pace of cellular aging, disease onset, and neuropathology, particularly in older, stressed populations.

IbERF71, with IbMYB340 and IbbHLH2, coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potato (Ipomoea batatas L.).

The transcription factor (TF) IbERF71 forms a novel complex, IbERF71-IbMYB340-IbbHLH2, to coregulate anthocyanin biosynthesis by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Purple-fleshed sweet potato (Ipomoea batatas L.) is very popular because of its abundant anthocyanins, which are natural pigments with multiple physiological functions. TFs involved in regulating anthocyanin biosynthesis have been identified in many plants. However, the molecular mechanism of anthocyanin biosynthesis in purple-fleshed sweet potatoes has rarely been examined. In this study, TF IbERF71 and its partners were screened by bioinformatics and RT-qPCR analysis. The results showed that the expression levels of IbERF71 and partners IbMYB340 and IbbHLH2 were higher in purple-fleshed sweet potatoes than in other colors and that the expression levels positively correlated with anthocyanin contents. Moreover, transient expression assays showed that cotransformation of IbMYB340+IbbHLH2 resulted in anthocyanin accumulation in tobacco leaves and strawberry receptacles, and additional IbERF71 significantly increased visual aspects. Furthermore, the combination of the three TFs significantly increased the expression levels of FvANS and FvGST, which are involved in anthocyanin biosynthesis and transport of strawberry receptacles. The dual-luciferase reporter system verified that cotransformation of the three TFs enhanced the transcription activity of IbANS1. In addition, yeast two-hybrid and firefly luciferase complementationassays revealed that IbMYB340 interacted with IbbHLH2 and IbERF71 but IbERF71 could not interact with IbbHLH2 in vitro. In summary, our findings provide novel evidence that IbERF71 and IbMYB340-IbbHLH2 form the regulatory complex IbERF71-IbMYB340-IbbHLH2 that coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Thus, the present study provides a new regulatory network of anthocyanin biosynthesis and strong insight into the color development of purple-fleshed sweet potatoes.

Ssc-miR-451 Regulates Porcine Primary Adipocyte Differentiation by Targeting ACACA.

Simple SummarymiR-451 has been extensively studied in humans and model animals. However, there are no reports of miR-451 in pigs, and it is unclear whether miR-451 is related to adipose tissue development. In this work, we first reported the effects of ssc-miR-451 on pig adipose development and meat quality. We focused on the expression and functions of miR-451 in porcine primary adipocyte and pork quality. Porcine primary adipocyte transfection test and dual luciferase reporting system suggested that ssc-miR-451 may inhibit lipid deposition by inhibiting Acetyl-CoA carboxylase alpha (ACACA) expression. Correlation analysis negatively correlated miR-451 expression with intramuscular fat content and positively correlated ACACA expression with intramuscular fat content. Further analysis of fatty acid composition revealed that pigs with high expression of ssc-miR-451 had higher monounsaturated fatty acid and lower polyunsaturated fatty acid.miRNA is a small non-coding RNA, which plays an important role in diverse biological processes. In the present study, we explore the effect of ssc-miR-451 on porcine adipose development and meat quality. We observed that ssc-miR-451 was downregulated during porcine primary adipocyte differentiation. Overexpression of ssc-miR-451 inhibited adipogenic differentiation, while inhibition of ssc-miR-451 promoted adipogenic differentiation. The dual luciferase reporter system indicated Acetyl-CoA carboxylase alpha (ACACA) as a target gene of ssc-miR-451. Correlation analysis negatively correlated miR-451 expression with intramuscular fat content (IMF) and positively correlated ACACA expression with IMF. Further analysis of fatty acid composition revealed that pigs with high expression of ssc-miR-451 had higher monounsaturated fatty acid (MUFA) and lower polyunsaturated fatty acid (PUFA). Taken together, our study suggests that ssc-miR-451 regulates lipid deposition and fatty acid composition by targeting ACACA, and ssc-miR-451 may serve as a potential genetic marker to improve pork quality.

MiR‐1696/GPx3 axis is involved in oxidative stress mediated neutrophil extracellular traps inhibition in chicken neutrophils

As an important immune mechanism of neutrophils, the release of Web-like chromatin structures known as neutrophil extracellular traps (NETs) can rapidly locate and capture invading pathogens, which has received sustained attention. There are still some fundamental questions surrounding established studies on the mechanism of balance between reactive oxygen species (ROS) dependent release and neutrophil antioxidant response. Glutathione peroxidase 3 (GPx3) is an important antioxidant protein and has been identified can regulate the immune response. However, the effect of GPx3 on the NETs formation and microRNA in this process remain poorly understood. In the present study, we used chicken peripheral blood neutrophils treated with Phorbol-12-myristate-13-acetate (PMA) for 3 h as NETs formation model. The result of morphological observation showed that GPx3 inactivation compromised the release of NETs. Further analysis revealed that knockdown of GPx3 significantly disturbed oxidative balance by inhibiting antioxidant enzymes activity and increasing H2 O2 content. Quantitative analysis of NETs-related genes found that the phosphorylation level of mitogen-activated protein kinase (MAPK) pathway genes (ERK, JNK, and p38) and expression of phosphoinositide-3-kinase (PI3K)/AKT pathway genes (PI3K and AKT) were suppressed with the downregulation of GPx3. Meanwhile, we identified that miR-1696 can target GPx3 expression by using dual luciferase reporter system. Additionally, overexpression of miR-1696 can not only inhibit the formation of NETs by restraining the expression of GPx3, interfering with the generation of ROS and activation of the MAPK and PI3K/AKT pathways, but also reducing the release of PMA-induced NETs promoted by overexpression of GPx3. These results provide evidence that miR-1696 targeted GPx3 activities in neutrophils could be used to regulate the NETs formation stimulated by PMA.

MicroRNA-204-5p mediates sevoflurane-induced cytotoxicity in HT22 cells by targeting brain-derived neurotrophic factor.

Sevoflurane is widely used as an inhalational anesthetic in clinical practice. However, sevoflurane can cause cytotoxicity and induce learning capacity decline in patients. A previous publication indicated that miR-204-5p might have a close relationship with sevoflurane-induced neurotoxicity. When exposed to sevoflurane, the expression of miR-204-5p in neonatal hippocampus of rats was significantly increased. Hence, we aimed to investigate the role of miR-204-5p in sevoflurane-induced neurotoxicity using a mouse hippocampal neuronal cell line (HT22). The levels of miR-204-5p in HT22 cells were detected by RT-qPCR. In addition, the effects of miR-204-5p on cell viability, apoptosis and proliferation were evaluated by CCK-8, flow cytometric, and immunofluorescence assay, respectively. Western blotting was used to detect expressions of Bax, Bcl-2, active caspase 3, BDNF, TrkB, p-TrkB, Akt and p-Akt in HT22 cells. ELISA assay was used to examine the levels of total superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and reactive oxygen species (ROS) in cells. Meanwhile, the dual luciferase reporter system assay was employed to explore the interaction of miR-204-5p and BDNF in cells. The level of miR-204-5p was increased in sevoflurane-treated HT22 cells. Moreover, downregulation of miR-204-5p inhibited sevoflurane-induced apoptosis and promoted cell proliferation by upregulating the proteins of Bcl-2 and downregulating the expressions of Bax and active caspase-3 in HT22 cells. In addition, inhibition of miR-204-5p alleviated sevoflurane-induced oxidative injuries in HT22 cells via decline of ROS and MDA and upregulation of SOD and GSH. Furthermore, bioinformatics and dual luciferase assay demonstrated that miR-204-5p can inhibit the TrkB/Akt pathway by targeting BDNF. Our findings indicated that downregulation of miR-204-5p can decrease oxidative status in HT22 cells and alleviate sevoflurane-induced cytotoxicity through stimulating the BDNF/TrkB/Akt pathway. Therefore, miR-204-5p might be a potential biomarker and therapeutic target for the treatment of sevoflurane-induced neurotoxicity.

The Expression Pattern and Regulatory Mechanism of the G0/G1 Switch Gene 2 (G0S2) in the Pathogenesis and Treatment of AChR Myasthenia Gravis (MG).

This study is aimed at exploring the expression pattern and methylation level of G0S2 in the peripheral blood mononuclear cells (PBMCs) of myasthenia gravis (MG) patients with positive acetylcholine receptor (AChR) autoantibodies and revealing the relationship between the G0S2 methylation pattern and MG. The relationship between the NFAT family members and G0S2 was explored to reveal the regulatory mechanism of G0S2 in the pathogenesis and treatment of AChR MG. Moreover, we attempted to demonstrate the potential therapeutic mechanism of tacrolimus in AChR MG. The relative G0S2 expression level in the PBMCs of healthy people was compared with that in the PBMCs of AChR MG patients with quantitative real-time PCR (qRT-PCR). The methylation frequency of the G0S2 promoter was detected by bisulfite sequencing PCR (BSP) and pyrosequencing. A dual-luciferase reporter system was used to reveal the relationship between the G0S2 promoter and nuclear factor of activated T cells 5 (NFAT5). The qRT-PCR results showed that G0S2 expression was significantly upregulated in the B cells and CD8+ T cells of AChR MG patients but not in the CD4+ T cells, and these expression differences were significantly associated with a decrease in G0S2 methylation. NFAT5, which was speculated to bind to island 1 (p1) in the G0S2 promoter, may regulate the lymphocyte balance by regulating G0S2 gene expression but failed to affect the methylation of the G0S2 promoter. Tacrolimus therapy-induced methylation and overexpression of NFAT5 could significantly reduce the expression of G0S2 in AChR MG patients. The G0S2 gene was remarkably upregulated in the PBMCs of MG patients. NFAT5 may affect transcription initiation and downregulate G0S2 expression through p1 in the promoter, thus controlling G0S2 gene expression and regulating the lymphocyte balance. Therefore, G0S2 could be an immune regulatory factor in both AChR MG occurrence and treatment with tacrolimus.

A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L.

Radish (Raphanus sativus L.) taproot contains high concentrations of flavonoids, including anthocyanins (ATCs), in red-skinned genotypes. However, little information on the genetic regulation of ATC biosynthesis in radish is available. A genome-wide association study of radish red skin color was conducted using whole-genome sequencing data derived from 179 radish genotypes. The R2R3-MYB transcription factor production of anthocyanin pigment 2 (PAP2) gene was found in the region associated with a leading SNP located on chromosome 2. The amino acid sequence encoded by the RsPAP2 gene was different from those of the other published RsMYB genes responsible for the red skin color of radish. The overexpression of the RsPAP2 gene resulted in ATC accumulation in Arabidopsis and radish, which was accompanied by the upregulation of several ATC-related structural genes. RsPAP2 was found to bind the RsUFGT and RsTT8 promoters, as shown by a dual-luciferase reporter system and a yeast one-hybrid assay. The promoter activities of the RsANS, RsCHI, RsPAL, and RsUFGT genes could be strongly activated by coinfiltration with RsPAP2 and RsTT8. These findings showed the effectiveness of GWAS in identifying candidate genes in radish and demonstrated that RsPAP2 could (either directly or together with its cofactor RsTT8) regulate the transcript levels of ATC-related genes to promote ATC biosynthesis, facilitating the genetic enhancement of ATC contents and other related traits in radish.

A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF.

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5′-untranslated region (5′UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597–599 and 603–605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344–596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome–HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5′UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.

LncRNA DANCR promotes ATG7 expression to accelerate hepatocellular carcinoma cell proliferation and autophagy by sponging miR-222-3p.

LncRNA differentiation antagonizing non-protein coding RNA (DANCR) is an oncogene in various malignant cancers, including hepatocellular carcinoma (HCC). Autophagy is an intracellular self-digestion mechanism that accelerates the progression of HCC via promoting cell survival. However, the role of lncRNA DANCR in HCC, and the mechanism of lncRNA DANCR in the regulation of autophagy in HCC remains unknown. Therefore, the aims of this study are the investigation of the role of lncRNA DANCR in HCC, and the exploration of the molecular mechanism of lncRNA DANCR in regulating autophagy of HCC cells. In this study, the expression of lncRNA DANCR, miR-222-3p, and autophagy-related gene 7 (ATG7) was detected by qRT-PCR. The cell proliferation and colony formation were measured by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. And the autophagic flux was evaluated by mRFP-GFP-LC3B reporter. The autophagy related proteins were analyzed by Western blotting. Besides, the relationship between lncRNA DANCR and miR-222-3p, as well as between miR-222-3p and ATG7, was determined by Dual-Luciferase reporter system. We found high expression of lncRNA DANCR and ATG7, and low expression of miR-222-3p in HCC tissues and cell lines. And lncRNA DANCR positively correlated with poor survival of HCC patients. Moreover, the knockdown of lncRNA DANCR inhibited cell proliferation and autophagy of HCC cells. And we predicted and proved that lncRNA DANCR induced cell proliferation, colony formation and autophagy by increasing ATG7 and suppressing miR-222-3p. Our study demonstrates the promoting role of lncRNA DANCR in HCC, and indicates the regulatory effects of lncRNA DANCR on regulating autophagy of HCC.

LncRNA SNHG3 promotes autophagy-induced neuronal cell apoptosis by acting as a ceRNA for miR-485 to up-regulate ATG7 expression

Long non-coding RNAs (lncRNAs) are bound up with various human diseases. However, their roles in brain ischemia-reperfusion (I/R) injury remain largely unknown. This study aimed to reveal the potential mechanism of LncRNA SNHG3 on autophagy-induced neuronal cell apoptosis in the brain I/R injury. LncRNA SNHG3 and miR-485 or autophagy markers LC3II/I and Beclin-1 expressions were detected by qRT-PCR or Western blot and the apoptosis of N2a cells was analyzed by flow cytometry. Besides, the interactions between LncRNA SNHG3 and miR-485, miR-485 and ATG7 were validated by RNA pull-down and dual-luciferase reporter system assays. After the Oxygen and Glucose Deprivation (OGD) treatment of N2a cells transfected with pcDNA-SNHG3, pcDNA-SNHG3 + miR-485 mimic for 6h, 1mM autophagy inhibitor 3-MA was added and reoxygenated for 24h, the effect of LncRNA SNHG3 on the autophagy-induced neuronal cell apoptosis was measured by Western blot and flow cytometry. LncRNA SNHG3 was highly expressed in the mouse model of transient middle cerebral artery occlusion and cell model of Oxygen and Glucose Deprivation/Reperfusion, while miR-485 was lowly expressed. Furthermore, miR-485 negatively regulated the luciferase activities of LncRNA SNHG3 and ATG7. After the OGD treatment of N2a cells transfected with pcDNA-SNHG3, pcDNA-SNHG3 + miR-485 mimic for 6h, 1mM 3-MA was added and reoxygenated for 24h, the overexpression of LncRNA SNHG3 raised the ratio of LC3-II/LC3-I and Beclin-1 expression and boosted the apoptosis of N2a cells, while these effects were reversed after the transfection of miR-485 mimic. In general, our data expounded that the interference with LncRNA SNHG3 improved brain I/R injury by up-regulating miR-485 and down-regulating ATG7 to restrain autophagy and neuronal cell apoptosis.

LncRNA MALAT1 inhibits apoptosis of endometrial stromal cells through miR-126-5p-CREB1 axis by activating PI3K-AKT pathway.

Endometriosis is a common, chronic and painful disease in women, whose pathogenesis remains not entirely clear. Long non-coding RNA (lncRNA) MALAT1 participates in the development of endometriosis. This study further investigated the regulation of MALAT1-miR-126-5p-CREB1 axis in the pathological process of endometriosis. MALAT1, miR-126-5p, and CREB1 levels in human endometrial stromal cells (HESCs) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Protein levels were determined by Western blotting. Cell viability and apoptosis was assessed by MTT assay and annexin V-FITC staining, respectively. The interactivity between miR-126-5p and MALAT1 (or CREB1) was assessed by dual luciferase reporter system. Knockdown of MALAT1 or CREB1 restrained proliferation and induced apoptosis as confirmed by upregulating cleaved caspase-3 and Bax, and down-regulating Bcl-2 in HESCs, while inhibition of miR-126-5p presented the opposite results. Moreover, silencing of MALAT1 triggered apoptosis of HESCs via targeting miR-126-5p. In addition, miR-126-5p directly regulated CREB1 expression via binding to its 3' non-coding region. Finally, miR-126-5p inhibitor-mediated apoptosis inhibition was restrained by CREB1 silencing via inactivation of PI3K-AKT pathway in HESCs. Taken together, our study firstly demonstrates that MALAT1 regulates apoptosis of HESCs through miR-126-5p/CREB1 axis mediated PI3K/AKT pathway. Our findings explained the pathogenesis of endometriosis and offered promising therapeutic option for endometriosis.

MiR-3194-3p Inhibits Breast Cancer Progression by Targeting Aquaporin1.

Increasing evidence indicates that the Aquaporin1 (AQP1) aberrant expression may be related to a wide variety of human cancers, including breast cancer (BC). In the present study, we explore the effects and possible mechanism of miR-3194-3p on the biological behaviors of BC. At first, miR-3194-3p is found to modulate AQP1 expression targeting the 3′-UTR using miRNA target prediction algorithms. MiR-3194-3p expression is markedly downregulated, and AQP1 expression is upregulated in BC tissues compared with adjacent normal breast tissues. Moreover, the differential expression of miR-3194-3p and AQP1 are observed in four BC cells with different malignancy degree. Meanwhile, a significant negative correlation between AQP1 and miR-3194-3p expressions in tumor tissues from 30 BC patients is revealed. miR-3194-3p mimic remarkably inhibits cell proliferation, migration, and invasion as well as promotes apoptosis in MDA-MB-231 cells while miR-3194-3p inhibitors exert an opposite role in MCF-7 cells. Dual-luciferase reporter system demonstrates that AQP1 is a direct target gene of miR-3194-3p. Overexpression of AQP1 by pBABE-puro-AQP1 vector partially abrogates the effect of miR-3194-3p mimic in MDA-MB-231 cells. In short, our results suggest that miR-3194-3p suppresses BC cell proliferation, migration, and invasion by targeting AQP1, providing a novel insight into BC tumorigenesis and treatment.

<p>LncRNA MAFG-AS1 Accelerates Cell Migration, Invasion and Aerobic Glycolysis of Esophageal Squamous Cell Carcinoma Cells via miR-765/PDX1 Axis</p>

BackgroundLncRNA dysregulation is implicated in esophageal squamous cell carcinoma (ESCC) progression; However, the precise role and function of lncRNA MAFG-AS1 in ESCC remains unknown.Materials and MethodsExpressions of MAFG-AS1, miR-765, PDX1, GLUT1 and LDH-A were detected via qRT-PCR or/and Western blot in ESCC tissues and cell lines. CCK-8, transwell and glycolysis assays were used to investigate the effects of MAFG-AS1 on ESCC cell proliferation, migration, invasion and aerobic glycolysis after knockdown or overexpression of MAFG-AS1, and bioinformatics analyses, RNA pull-down and dual luciferase reporter systems were applied to investigate the interaction between MAFG-AS1, miR-765 and PDX1.ResultsMAFG-AS1 was significantly up-modulated in ESCC tissues and cell lines. MAFG-AS1 significantly accelerated ESCC cell proliferation, migration, invasion and aerobic glycolysis. MAFG-AS1 competitively adsorbed miR-765, while miR-765 negatively modulated the expression of PDX1. miR-765 and PDX1 participated in the promotive effects of MAFG-AS1 on cell migration, invasion and aerobic glycolysis in ESCC cells.ConclusionOur research indicates that the MAFG-AS1/miR-765/PDX1 axis accelerates ESCC cell proliferation, migration, invasion and aerobic glycolysis.

Circular RNA circEXOC6B Inhibits the Progression of Ovarian Cancer by Sponging miR-421 and Regulating RUS1 Expression.

BackgroundEvidence has been shown that circular RNAs (circRNAs) play a vital role during the development of ovarian cancer. However, the mechanism by which circEXOC6B regulates tumorigenesis of ovarian cancer remains unknown. Thus, this study aimed to investigate the role of circEXOC6B during the progression of ovarian cancer.Materials and MethodsThe dual-luciferase reporter system assay was used to determine the interaction between circEXOC6B, miR-421 and RUS1 in ovarian cancer, respectively. CCK8 and colony formatting were used to evaluate cell proliferation. Meanwhile, the expressions of RSU1, PINCH1 and ILK in SKOV3 cells were detected with Western blot.ResultsDownregulation of circEXOC6B markedly promoted the proliferation and invasion in A2780 cells. In contrast, upregulation of circEXOC6B significantly inhibited the proliferation and invasion in SKOV3 cells. Moreover, overexpression of circEXOC6B obviously induced the apoptosis of SKOV3 cells. Furthermore, luciferase reporter assay identified that miR-421 was the potential miRNA binding of circEXOC6B, and RUS1 was the potential binding target of miR-421. Mechanism analysis indicated that upregulation of circEXOC6B increased the level of RUS1 by acting as a competitive “sponge” of miR‐421.ConclusionIn this study, we found that circEXOC6B suppressed the growth of ovarian cancer cells through upregulating RSU1 partially via sponging miR-421. Therefore, circEXOC6B might be a potential target for the treatment of ovarian cancer.

Epigenetic restoration of voltage-gated potassium channel Kv1.2 alleviates nerve injury-induced neuropathic pain.

Voltage-gated potassium channels (Kv) are important regulators of neuronal excitability for its role of regulating resting membrane potential and repolarization. Recent studies show that Kv channels participate in neuropathic pain, but the detailed underlying mechanisms are far from being clear. In this study, we used siRNA, miR-137 agomir, and antagomir to regulate the expression of Kv1.2 in spinal cord and dorsal root ganglia (DRG) of naïve and chronic constriction injury (CCI) rats. Kv currents and neuron excitability in DRG neurons were examined by patch-clamp whole-cell recording to verify the change in Kv1.2 function. The results showed that Kv1.2 was down-regulated in DRG and spinal dorsal horn (SDH) by CCI. Knockdown of Kv1.2 by intrathecally injecting Kcna2 siRNA induced significant mechanical and thermal hypersensitivity in naïve rats. Concomitant with the down-regulation of Kv1.2 was an increase in the expression of the miR-137. The targeting and regulating of miR-137 on Kcna2 was verified by dual-luciferase reporter system and intrathecal injecting miR-137 agomir. Furthermore, rescuing the expression of Kv1.2 in CCI rats, achieved through inhibiting miR-137, restored the abnormal Kv currents and excitability in DRG neurons, and alleviated mechanical allodynia and thermal hyperalgesia. These results indicate that the miR-137-mediated Kv1.2 impairment is a crucial etiopathogenesis for the nerve injury-induced neuropathic pain and can be a novel potential therapeutic target for neuropathic pain management.

Long non‑coding RNA SNHG12 regulates cell proliferation, invasion and migration in endometrial cancer by targeting miR‑4429.

Long non-coding RNA small nucleolar RNA host gene 12 (SNHG12) has been demonstrated to be oncogenic. The aim of the present study was to examine the effects of SNHG12 on the progression of endometrial cancer (EC). The expression levels of SNHG12 and microRNA (miR)-4429 were assessed in EC cell lines by reverse transcription-quantitative PCR. Plasmids, including SNHG12 short hairpin RNAs (shRNAs), shRNA negative control (NC), SNHG12 overexpression (OV), OV-NC, miR-4429 mimic and mimic-NC, were transfected into RL95-2 cells. Post-transfection, Cell Counting Kit-8, Transwell Matrigel and wound-healing assays were performed to assess cell proliferation, invasion and migration, respectively. Cell cycle phase distribution was assessed by flow cytometry. The protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 were detected by western blotting. miR-4429 target genes were predicted by bioinformatics analysis using target prediction online tools; the findings of this analysis were verified using a dual-luciferase reporter system. Identified as a target of miR-4429, SNHG12 was overexpressed in EC cell lines with decreased expression of miR-4429. Further experiments demonstrated that SNHG12 silencing and overexpression of miR-4429 markedly suppressed proliferation, migration and invasion of RL95-2 cells, arrested cells in the G1 phase, and markedly downregulated the expression of MMP2 and MMP9. The opposite effects were observed in miR-4429 mimic-transfected RL95-2 cells after SNHG12 was overexpressed. The findings of the present study established the role of SNHG12 and miR-4429 in EC. Therefore, targeting the SNHG12/miR-4429 axis could serve as a potential future therapeutic target for treatment of EC.

Effects of miR-152-Mediated Targeting of SOCS3 on Hepatic Insulin Resistance in Gestational Diabetes Mellitus Mice

BackgroundThe present study explored the effects of miR-152-mediated targeting of suppressor of cytokine signaling 3 (SOCS3) on hepatic insulin resistance (HIR) in mice with gestational diabetes mellitus (GDM). Healthy SPF C57BL/6J mice were selected to establish a GDM model. MethodsMice were divided into seven groups as follows: Normal group, Model group, NC-mimic group, miR-152 mimic group, NC-pcDNA3.0 group, pcDNA3.0-SOCS group, and miR-152 mimic + pcDNA3.0-SOCS3 group. The relationship between miR-152 and SOCS3 expression was analyzed by a dual-luciferase reporter system. Islet cell morphology and expression of miR-152 and SOCS3 mRNA and protein in the islet tissue were detected by hematoxylin and eosin (HE) staining, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and western blot analysis. Fasting blood glucose (FBG) level was measured by a glucose meter while triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and insulin levels were determined by an automatic biochemical analyzer. Insulin resistance index (HOMA-IR) was calculated by the formula FBG × FINS/22.5. ResultsThe results showed that the levels of miR-152, SOCS3, FBG, fasting insulin (FINS), TG, and TC increased, and HDL-C content decreased in other groups as compared with those in the Normal group (all p < 0.05). Oral glucose tolerance test (OGTT), FBG, FINS, TG, TC, and HDL-C values showed an opposite trend in miR-152 mimic and pcDNA3.0-SOCS3 groups as compared with the Model group (all p < 0.05). ConclusionsmiR-152 can inhibit HIR in GDM mice by downregulating the expression of SOCS3.