Dual-luciferase Reporter Analysis Research Articles

Verbascoside inhibits oral squamous cell carcinoma cell proliferation, migration, and invasion by the methyltransferase 3-mediated microRNA-31-5p/homeodomain interacting protein kinase 2 axis

ObjectiveThe study aimed to investigate the effects of verbascoside on oral squamous cell carcinoma (OSCC) cellular behaviors and underlying molecular mechanisms. DesignFor this purpose, SCC9 and UM1 cell lines were treated with verbascoside, and their biological behaviors, including proliferation, migration, and invasion, were evaluated using cell counting kit-8, 5-Ethynyl-2′-deoxyuridine, and Transwell assays. The expression of methyltransferase-3 (METTL3), microRNA (miR)− 31–5p, and homeodomain interacting protein kinase-2 (HIPK2) were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between METTL3 and miR-31–5p was evaluated by RNA immunoprecipitation and methylated RNA immunoprecipitation, while the interaction between miR-31–5p and HIPK2 was evaluated by dual-luciferase reporter analysis. ResultsThe results indicated inhibition of OSCC cell proliferation, migration, and invasion post verbascoside treatment. Similarly, METTL3 was upregulated in OSCC cells and was inhibited post-verbascoside treatment. Overexpressing METTL3 promoted the cellular processes. Moreover, miR-31–5p was upregulated in OSCC cells, where METTL3 facilitated the processing of miR-31–5p in an N6-methyladenosine (m6A)-dependent manner. The HIPK2 served as miR-31–5p target, where overexpressing miR-31–5p or HIPK2 knockdown reversed the suppression of verbascoside-induced biological behaviors. ConclusionsVerbascoside inhibited the progression of OSCC by inhibiting the METTL3-regulated miR-31–5p/HIPK2 axis. These findings suggested that verbascoside might be an effective drug for OSCC therapy.

Hsa_circ_0087862 contributes to the progression of colorectal cancer through regulating miR-512-3p/HK2 axis

BackgroundColorectal cancer (CRC) theratened thousands of people every year. Emerging evidences suggested that circular RNAs (circRNAs) were involved in CRC malignancies. However, the underlying mechanisms have yet not been revealed. MethodsQuantitative real-time PCR (qRT-PCR) was used to determine the expression of circ_0087862 and microRNA-512–3p (miR-512–3p). Western blot was performed to measure the protein expression of hexokinase 2 (HK2), B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax) and BCL2 antagonist/killer 1 (Bak). Moreover, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay, colony formation and 5-ethynyl-2’-deoxyuridine (EdU) assay were employed to assess CRC cell proliferation. Also, migration/invasion abilities and apoptosis rates were investigated by transwell assay and flow cytometry. Glucose consumption, lactate production and ATP production were detected using the corresponding kits. Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) experiments were utilized to analyze the target association of miR-512–3p and circ_0087862 or HK2. Finally, xenograft assay was carried out to analyze the function of circ_0087862 in tumor growth in vivo. ResultsCirc_0087862 expression was elevated in CRC tissues and cells. Circ_0087862 silencing repressed cell viabilities, proliferation, migration/invasion and glycolysis, and reinforced cell apoptosis. However, HK2 could weaken these impacts. Additionally, miR-512–3p targeted HK2, and circ_0087862 could regulate HK2 expression by miR-512–3p. Furthermore, circ_0087862 silencing decreased CRC cell xenograft tumor growth. ConclusionCollectively, our data suggested that circ_0087862 knockdown impeded cell viabilities, proliferation, and glycolysis, and contributed to cell apoptosis in CRC, indicating circ_0087862 as a promising tumor promoter.

A novel long non-coding RNA SLNCR1 promotes proliferation, migration, and invasion of melanoma via transcriptionally regulating SOX5

Steroid receptor RNA activator (SRA)-like non-coding RNA (SLNCR1) has been implicated in various tumorigenic processes, but the precise regulatory role in melanoma progression remains uncertain. We performed a comprehensive analysis to investigate the prognostic value of SLNCR1 expression in patients with melanoma by TCGA database and melanoma tissue samples via the Kaplan–Meier method. Subsequently, we conducted qRT-PCR and Fluorescence in Situ Hybridization (FISH) assays to identify SLNCR1 expression levels and localization in tissues and cells, respectively. Loss-of-function assays utilizing shRNAs vectors were used to investigate the potential impact of SLNCR1. Our data showed that SLNCR1 is significantly up-regulated in human malignant melanoma tissues and cell lines and functions as an oncogene. Silencing of SLNCR1 suppressed melanoma cell proliferation, migration, invasion, and inhibited tumorigenesis in a mouse xenograft model. Additionally, we employed bioinformatic predictive analysis, combined with dual-luciferase reporter analysis and functional rescue assays, to elucidate the mechanistic target of the SLNCR1/SOX5 axis in melanoma. Mechanistically, we discovered that SLNCR1 promotes EMT of human melanoma by targeting SOX5, as downregulation of SLNCR1 expression leads to a decrease in SOX5 protein levels and inhibits melanoma tumorigenesis. Our research offers promising insights for more precise diagnosis and treatment of human melanoma.

WRKY6 transcription factor modulates root potassium acquisition through promoting expression of AKT1 in Arabidopsis.

Potassium (K+ ), being an essential macronutrient in plants, plays a central role in many aspects. Root growth is highly plastic and is affected by many different abiotic stresses including nutrient deficiency. The Shaker-type K+ channel Arabidopsis (Arabidopsis thaliana) K+ Transporter 1 (AKT1) is responsible for K+ uptake under both low and high external K+ conditions. However, the upstream transcription factor of AKT1 is not clear. Here, we demonstrated that the WRKY6 transcription factor modulates root growth to low potassium (LK) stress in Arabidopsis. WRKY6 showed a quick response to LK stress and also to many other abiotic stress treatments. The two wrky6 T-DNA insertion mutants were highly sensitive to LK treatment, whose primary root lengths were much shorter, less biomass and lower K+ content in roots than those of wild-type plants, while WRKY6-overexpression lines showed opposite phenotypes. A further investigation showed that WRKY6 regulated the expression of the AKT1 gene via directly binding to the W-box elements in its promoter through EMSA and ChIP-qPCR assays. A dual luciferase reporter analysis further demonstrated that WRKY6 enhanced the transcription of AKT1. Genetic analysis further revealed that the overexpression of AKT1 greatly rescued the short root phenotype of the wrky6 mutant under LK stress, suggesting AKT1 is epistatic to WRKY6 in the control of LK response. Further transcriptome profiling suggested that WRKY6 modulates LK response through a complex regulatory network. Thus, this study unveils a transcription factor that modulates root growth under potassium deficiency conditions by affecting the potassium channel gene AKT1 expression.

Implications of IFNγ SNP rs2069705 in primary Sjögren's syndrome: transcriptional activation and B cell infiltration.

Primary Sjögren's syndrome (pSS) is characterized by its autoimmune nature. This study investigates the role of the IFNγ SNP rs2069705 in modulating the susceptibility to pSS. Differential expression of IFNγ and BAFF was analyzed using the GEO database's mRNA microarray GSE84844. Genotyping of the IFNγ SNP rs2069705 was conducted via the dbSNP website. The JASPAR tool was used for predicting transcription factor bindings. Techniques such as dual-luciferase reporter assays, Chromatin immunoprecipitation, and analysis of a pSS mouse model were applied to study gene and protein interactions. A notable increase in the mutation frequency of IFNγ SNP rs2069705 was observed in MNCs from the exocrine glands of pSS mouse models. Bioinformatics analysis revealed elevated levels of IFNγ and BAFF in pSS samples. The model exhibited an increase in both CD20+ B cells and cells expressing IFNγ and BAFF. Knocking down IFNγ resulted in lowered BAFF expression and less lymphocyte infiltration, with BAFF overexpression reversing this suppression. Activation of the Janus kinase (JAK)/STAT1 pathway was found to enhance transcription in the BAFF promoter region, highlighting IFNγ's involvement in pSS. In addition, rs2069705 was shown to boost IFNγ transcription by promoting interaction between its promoter and STAT4. SNP rs2069705 in the IFNγ gene emerges as a pivotal element in pSS susceptibility, primarily by augmenting IFNγ transcription, activating the JAK/STAT1 pathway, and leading to B-lymphocyte infiltration in the exocrine glands.NEW & NOTEWORTHY The research employed a combination of bioinformatics analysis, genotyping, and experimental models, providing a multifaceted approach to understanding the complex interactions in pSS. We have uncovered that the rs2069705 SNP significantly affects the transcription of IFNγ, leading to altered immune responses and B-lymphocyte activity in pSS.

Xinfeng Capsule alleviates interleukin-1β-induced chondrocyte inflammation and extracellular matrix degradation by regulating the miR-502-5p/TRAF2/NF-κB axis

To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule (XFC) on interleukin (IL)-1β-induced impairment of chondrocytes. XFC-medicated serum was collected from SD rats with XFC gavage, and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry. Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2. In cultured human chondrocytes induced with IL-1β, the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum, alone or in combination, on expression levels of IL-1β, tumor necrosis factor-α (TNF-α), IL-4, and IL-10 were examined with ELISA, and the changes in the expressions of collagen type Ⅱ alpha 1 (COL2A1), matrix metalloproteinase 13 (MMP13), adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR, Western blotting, and immunofluorescence assay. In cultured human chondrocytes, treatment with IL-1β significantly decreased the cell viability, increased cell apoptosis rate, lowered miR-502-5p, IL-4, IL-10, and COL2A1 expressions, and enhanced IL-1β, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 expressions (P < 0.05), and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20% (P < 0.05). Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1β, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 and lowered expressions of miR-502-5p, IL-4, IL-10, and COL2A1, and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor. XFC can inhibit IL-1β-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Exosome microRNA-22 inhibiting proliferation, migration and invasion through regulating Twist1/CADM1 axis in osteosarcoma

This study aims to the function of miR-22 original mesenchymal stem cells (MSC) on osteosarcoma (OS) proliferation, migration and invasion. Bio-informatics analysis including GEO2R analysis, Gene Ontology analysis, integration analysis were used to confirmed the target genes (miR-22, Twist1, CADM1) in OS. RT-qPCR and western blotting confirmed the different expression of miR-22, Twist1, CADM1 in OS tissues, MG63 and Saos cell lines. MTS assay, CCK8 assay, colony forming assay, EdU assay were performed to detect the proliferation effect of miR-22 on MG63. Transwell migration assay, transwell invasion assay, wound healing assay were used to verify the migration and invasion effect of miR-22 on MG63. Luciferase reporter assay confirm the binding sites between miR-22 and Twist1. RT-qPCR confirmed miR-22 and CADM1 downregulated and Twist1 upregulated in OS tissues, MG63 and Saos. Exosome original MSC labeled with PKH-26 could be uptake by MG63, which upregulated the expression of miR-22 in MG63. High expression of miR-22 in MG63 inhibited proliferation, migration and invasion, which could be rescued by Twist1. Dual luciferase reporter analysis confirmed Twist1 was a target of miR-22. Exosome modified with miR-22 mimic inhibit proliferation, migration and invasion more efficient than exosome original MSC. miR-22 cargo in exo-MSC could uptake by MG63 and supply MG63 with miR-22, which inhibit MG63 proliferation, migration and invasion through targeting Twist1.

Sweet cherry AP2/ERF transcription factor, PavRAV2, negatively modulates fruit size by directly repressing PavKLUH expression.

For sweet cherry, fruit size is one of the main targets in breeding programs owing to the high market value of larger fruits. KLUH/CYP78A5 is an important regulator of seed/fruit size in several plant species, but its molecular mechanism is largely unknown. In this study, we characterized the function of PavKLUH in the regulation of sweet cherry fruit size. The ectopic overexpression of PavKLUH in Arabidopsis increased the size of its siliques and seeds, whereas virus-induced gene silencing of PavKLUH in sweet cherry significantly decreased fruit size by restricting mesocarp cell expansion. We screened out an AP2/ERF transcription factor containing a B3-like domain, designated as PavRAV2, which was able to physically interact with PavKLUH promoter in a yeast one-hybrid (Y1H) system. In Y1H assays, electrophoretic mobility shift assays, and dual-luciferase reporter analyses, PavRAV2 directly bound to the promoter of PavKLUH in vitro and in vivo, and suppressed PavKLUH expression. Silencing of PavRAV2 resulted in enlarged fruit as a result of enhanced mesocarp cell expansion. Together, our results provide new insights into signaling pathways related to fruit size, and outline a possible mechanism for how the RAV transcription factor directly regulates CYP78A family members to influence fruit size and development.

LINC01176 Hinders Thyroid Cancer Progression by Sponging miR-146b-5p to Enhance SGIP1.

Long non-coding RNA (lncRNAs) plays a crucial role in tumor pathogenesis. However, the function of most of these genes remains unclear. In the present study, we aimed to unveil LINC01176's role in thyroid cancer. Western blotting and qRT-PCR were applied for the analysis of the expressions of LINC01176, miR-146b-5p, and SH3GL interacting endocytic adaptor 1 (SGIP1). Proliferative and migratory capabilities were assessed using the CCK-8 assay and wound-healing experiments, respectively. Apoptosis of the cells was studied by quantifying the apoptosis-related markers Bcl-2 and Bax by western blotting. Animal models were established using nude mice to determine the role of LINC01176 in tumorigenesis. MiR-146b-5p's putative binding to LINC01176 and SGIP1 was validated using dual-luciferase reporter and RIP analyses. LINC01176 expression was downregulated in the thyroid cancer cell lines and tissues. LINC01176 overexpression represses cancer cell proliferation and migration but induces apoptosis. Elevated LINC01176 expression hampers tumorigenesis in animal models. LINC01176 targeted miR-146b-5p and negatively regulated its expression. Enrichment of miR-146b-5p counteracted the functional effects of LINC01176 overexpression. Additionally, miR-146b-5p interacted with SGIP1 and negatively regulated its expression. Thus, miR-146b-5p attenuates the anti-cancer effects of SGIP1. LINC01176 negatively regulates the expression miR-146b-5p and upregulates SGIP1 expression. Hence, LINC01176 blocks the malignant progression of thyroid cancer.

Rho GTPase-activating protein 1 promotes hepatocellular carcinoma progression via modulation by CircPIP5K1A/MiR-101-3p.

There has been an increased focus on regulating cell function with Rho family GTPases, including proliferation, migration/invasion, polarity, and adhesion. Due to the challenges involved in targeting Rho family GTPases directly, it may be more effective to target their regulators, such as Rho GTPase-activating protein 1 (ARHGAP1). This present research was performed to define the clinical significance of ARHGAP1 expression, as well as its regulatory mechanisms in hepatocellular carcinoma. ARHGAP1 and miR-101-3p expression of liver cancer patients, and their relevance with clinicopathological characteristics and prognosis were analyzed by the Cancer Genome Atlas sequencing data, and verified using samples of hepatocellular carcinoma patients. The interactions between miR-101-3p and ARHGAP1 or circPIP5K1A were validated by bioinformatic analyses, as well as confirmed by quantitative reverse transcription polymerase chain reaction, western blotting, and dual-luciferase reporter analysis. Plate clonality assays, cell adhesion and migration experiments, and proliferation experiments were used for assessing the participation of the circPIP5K1A/miR-101-3p/ARHGAP1 pathway in cell proliferation and motility. Elevated ARHGAP1 and reduced miR-101-3p expression are related to poorer survival. MiR-101-3p targets ARHGAP1 to suppress hepatocellular carcinoma cell colony formation and invasion, whereas miR-101-3p inhibitor reverses liver cancer proliferation and metastasis suppression caused by ARHGAP1 knockdown. In addition, circPIP5K1A, which is mainly distributed in the cytosol, showedcarcinogenic effects by sponging miR-101-3p, thus regulating ARHGAP1 expression. ARHGAP1 serves as an oncogenic gene in liver cancer, and the expression thereof is regulated by circPIP5K1A through sponging miR-101-3p.

MYCT1 attenuates renal fibrosis and tubular injury in diabetic kidney disease

Tubulointerstitial abnormalities contribute to the progression of diabetic kidney disease (DKD). However, the underlying mechanism of the pathobiology of tubulointerstitial disease is largely unknown. Here, we showed that MYCT1 expression was downregulated in invitro and invivo DKD models. Adeno-associated virus (AAV)-Myct1 significantly attenuated renal dysfunction and tubulointerstitial fibrosis in diabetic db/db mice and downregulated Sp1 transcription and TGF-β1/SMAD3 pathway activation. In human proximal tubular epithelial cells, high glucose-induced high expression of SP1 and TGF-β1/SMAD3 pathway activation as well as overaccumulation of extracellular matrix (ECM) were abrogated by MYCT1 overexpression. Mechanistically, the binding of VDR to the MYCT1 promoter was predicted and confirmed using dual-luciferase reporter and ChIP analysis. VDR transcriptionally upregulates MYCT1. Our data reveal MYCT1 as a new and potential therapeutic target in treating DKD.

CircECE1 promotes osteosarcoma progression through regulating RAB3D by sponging miR-588

BackgroundCircular RNAs (circRNAs) have been confirmed to be involved in cancer pathogenesis. However, the underlying mechanism of circRNA endothelin converting enzyme 1 (circECE1) in osteosarcoma (OS) development is still not understood.MethodsThe expression levels of circECE1, microRNA-588 (miR-588) and RAB3D, member RAS oncogene family (RAB3D) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. OS cell proliferation was assessed by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2’-deoxyuridine (EdU) assay. OS cell apoptosis rate and metastasis were identified by flow cytometry and transwell assay. Dual-luciferase reporter analysis and RNA immunoprecipitation (RIP) assay were performed to confirm the interactions among circECE1, miR-588 and RAB3D. Xenograft tumor models were established to explore circECE1 function in vivo. Immunohistochemistry (IHC) assay was applied to analyze RAB3D level after circECE1 knockdown.ResultsIn OS, circECE1 expression was higher than that in normal chondroma tissues. High levels of circECE1 were positively linked to OS cell viability, proliferation, migration and invasion, and negatively linked to OS cell apoptosis rate. It was found that circECE1 was a miR-588 sponge, and miR-588 inhibitor abrogated the influence of si-circECE1 on OS cells. MiR-588 targeted RAB3D to further regulate the pathological process of OS. Moreover, silencing circECE1 blocked OS tumor growth in vivo.ConclusionWe elucidated the function of a novel circECE1/miR-588/RAB3D axis in OS progression.

Hypoxia Activates HIF-1α and Affects Gene Expression and Transcriptional Regulation of PHD in Tegillarca granosa

Hypoxia is a common challenge faced by mollusks, and the role of hypoxia-inducible factor-1α (HIF-1α) in regulating related target genes under hypoxia in Tegillarca granosa (Tg) remains unclear. In this study, we identified gene HIF-1α and further explored its function. qRT-PCR was performed to determine the mRNA expression of HIF-1α, prolyl 4-hydroxylases (PHD), hemoglobin (Hb), and myoglobin (Mb) in response to hypoxia. Dual-luciferase reporter analysis was used to assess the transcriptional activity of HIF-1α on the PHD and Hb genes during hypoxia. Results showed that the expression levels of HIF-1α and PHD mRNAs were highest in the gill and lowest in the adductor muscle. Under hypoxic conditions, HIF-1α and PHD mRNAs were both induced, and their expression levels increased significantly, peaking at 8 h and gradually decreasing thereafter. The transcription of Tg-PHD was induced by hypoxia and was Tg-HIF-1α dependent. Notably, the expression of Hb decreased in hemocytes (p &lt; 0.01) after 8 h at 0.5 mg/L, while the expression of MbI and MbII increased (p &lt; 0.01) in the hepatopancreas after 24 h and 120 h, respectively. Moreover, Tg-HIF-1α could transactivate the PHD promoter but not that of Hb. These findings provide valuable insights into the regulatory role of Tg-HIF-1α on Tg-PHD, safeguarding it from degradation by PHD and offering significant contributions to the understanding of HIF-1α’s function. Nonetheless, the expression of Tg-HIF-1α protein was not detected under hypoxic conditions, and additional investigations are required to quantify Tg-HIF-1α protein dynamics and establish the correlation between mRNA expression and protein levels.

Circular RNA circSLC7A11 contributes to progression and stemness of laryngeal squamous cell carcinoma via sponging miR-877-5p from LASP1

BackgroundLaryngeal squamous cell carcinoma (LSCC) belongs to tumors of head and neck. Circular RNA circSLC7A11 functions as oncogenes in various tumors. However, the role of circSLC7A11 in LSCC remains largely unknown. Here, we aimed to clarify the circSLC7A11 function in LSCC. MethodsRelevance between circSLC7A11 expressions and LSCC clinicopathological was checked using chi-square. Relevance between circSLC7A11 expressions and LSCC patients' survival time was validated using Kaplan-Meier analysis. CircSLC7A11 expressions in LSCC tissues and cells were determined using quantitative real-time PCR. CircSLC7A11 functions in LSCC were examined by Cell Counting Kit-8, EdU analysis, Western blot, flow cytometry, sphere formation assay, and Transwell analysis. Meanwhile, circSLC7A11 mechanism in LSCC was determined using dual-luciferase reporter analysis, RNA pull-down, RNA Immunoprecipitation. ResultsCircSLC7A11 was highly expressed in LSCC, and high circSLC7A11 expressions were interrelated to the TNM stage. Also, LSCC patients with high circSLC7A11 owned shorter overall survival. Functional studies revealed that circSLC7A11 knockdown reduced LSCC cell proliferation, migration, invasion, stemness characteristics, and enhanced cell apoptosis. Mechanistic study data corroborated that circSLC7A11 targeted miR-877-5p, miR-877-5p targeted LASP1. LASP1 was negatively interrelated to miR-877-5p and was positively interrelated to circSLC7A11 in LSCC tissues. Also, circSLC7A11 knockdown reduced the LASP1 levels, and miR-877-5p inhibitor co-transfection reversed this reduction. Rescue assays further demonstrated that circSLC7A11 accelerated LSCC through miR-877-5p/LASP1. ConclusionCircSLC7A11 exerted oncogenic functions in LSCC by miR-877-5p/LASP1, hinting that circSLC7A11 was a novel biomarker for LSCC.

Silencing LncRNA SNHG16 suppresses the diabetic inflammatory response by targeting the miR-212-3p/NF-κB signaling pathway

BackgroundLong noncoding RNAs (LncRNAs) have been identified to play an important role in diabetes. The aim of the present study was to determine the expression and function of small nucleolar RNA host gene 16 (SNHG16) in diabetic inflammation.MethodsFor the in vitro experiments, quantitative real-time PCR (qRT-PCR), Western blotting and immunofluorescence were used to detect LncRNA SNHG16 expression in the high-glucose state. The potential microRNA sponge target of LncRNA SNHG16, miR-212-3p, was detected by dual-luciferase reporter analysis and qRT-PCR. For the in vivo experiments, glucose changes in mice were detected after si-SNHG16 treatment, and SNHG16 and inflammatory factor expression in kidney tissues were detected by qRT-PCR and immunohistochemistry.ResultsLncRNA SNHG16 was upregulated in diabetic patients, HG-induced THP-1 cells, and diabetic mice. Silencing SNHG16 inhibited the diabetic inflammatory response and the development of diabetic nephropathy. miR-212-3p was found to be directly dependent on LncRNA SNHG16. miR-212-3p could inhibitor P65 phosphorylation in THP-1 cells. The miR-212-3p inhibitor reversed the action of si-SNHG16 in THP-1 cells and induced an inflammatory response in THP-1 cells. LncRNA SNHG16 was also found to be higher in the peripheral blood of diabetic patients than in the normal person. The area under the ROC curve is 0.813.ConclusionThese data suggested that silencing LncRNA SNHG16 suppresses diabetic inflammatory responses by competitively binding miR-212-3p to regulate NF-κB. LncRNA SNHG16 can be used as a novel biomarker for patients with type 2 diabetes.

Circular RNA COL1A2 Mediates High Glucose-Induced Oxidative Stress and Pyroptosis by Regulating MiR-424-5p/SGK1 in Diabetic Nephropathy

Diabetic nephropathy (DN) represents a major diabetes-related complication, which could undermine renal function. CircCOL1A2 has been previously reported to show abnormal expression during DN. However, its functional role in the progression of DN, as well as the potential molecular mechanisms, remains unclear. The present work examined the expression of circCOL1A2 in the plasma of DN patients, and employed high glucose (HG)-challenged HK-2 cells as the in vitro cell model of hyperglycemia (HG)-induced DN. CircCOL1A2 was silenced using siRNA in HK-2 cells to clarify the functional engagement of circCOL1A2 in HG-induced DN. We examined the roles of circCOL1A2 in regulating oxidative stress by measuring reactive oxygen species (ROS), lipid peroxidation, and superoxide dismutase (SOD) levels. Besides, the effects of circCOL1A2 silencing on pyroptosis were investigated by RT-qPCR, western blot (WB), and ELISA assays. StarBase (version 2.0) was used to identify the downstream effector of circCOL1A2, and their interactions were further verified through dual-luciferase reporter analysis, RNA pull-down assays, and RNA immunoprecipitation (RIP) assay. CircCOL1A2 was highly expressed in DN patients and HG-induced HK-2 cells. Knocking down circCOL1A2 alleviated oxidative stress and pyroptosis upon HG treatment. In addition, we demonstrated that circCOL1A2 knockdown could promote miR-424-5p expression while inhibiting Serum/Glucocorticoid Regulated Kinase 1 (SGK1) level. Furthermore, miR-424-5p inhibitor or SGK1 overexpression impaired the effects of circCOL1A2 knockdown on HG-induced oxidative stress and pyroptosis. Hence, our results demonstrated that the circCOL1A2 mediates HG-exposed pyroptosis and oxidative stress through modulating miR-424-5p/SGK1 axis in diabetic nephropathy, indicating that silencing circCOL1A2 is a potential intervention strategy for DN management.

MicroRNA-27a inhibits porcine type I muscle fibre gene expression by directly targeting peroxisome proliferator-activated receptor-γ coactivator-1α.

MicroRNAs are one of the key determinants of muscle fibre development and phenotype in mammals. The preliminary experiment implied that microRNA-27a (miR-27a) might involve in regulation of muscle fibre type composition of pigs. Thereby, the present study aimed to confirm the regulatory effect of miR-27a on porcine type I muscle fibre-encoding gene (myosin heavy chain gene 7, MYH7) expression and its related mechanism. We firstly observed opposite expression patterns between miR-27a and MYH7 as well as between miR-27a and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) during differentiation of porcine skeletal muscle satellite cells. Through the subsequent transfection analysis in porcine myotubes, we found that miR-27a suppressed the expression of MYH7 and PGC-1α. Besides, miR-27a induced inhibition of PGC-1α downstream targets, namely myocyte enhancer factor-2C (MEF2C) along with mitochondrial biogenesis and oxidative metabolism-related factors such as nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), cytochrome c (Cytc)and cytochrome oxidase IV (COX Ⅳ) and succinodehydrogenase (SDH). Dual-luciferase reporter analysis revealed that miR-27a could bind to the predicted target site in the 3'-untranslated regions of PGC-1α mRNA, confirming a direct targeting of PGC-1α by miR-27a. Moreover, PGC-1α silencing abolished the promotive effects of miR-27a inhibitor on MYH7, PGC-1α and its downstream targets (MEF2C, NRF-1, mtTFA, COX Ⅳ, Cytcand SDH) in porcine myotubes. Collectively, miR-27a inhibits porcine MYH7 expression by negatively regulating PGC-1α and PGC-1α-controlled MEF2C expression as well as mitochondrial biogenesis and oxidative metabolism. Our findings may provide a molecular target for genetic or nutritional control of muscle fibre phenotype of pigs, probably having an important implication for regulating pork quality.

Exosomal microRNA-146a-5p from Adipose-Derived Mesenchymal Stem Cells: Preventing H2O2-Induced Oxidative Stress Injury in Human Keratinocyte Cell Line by Modulating SHC SH2 Domain-Binding Protein 1

Exosomes secreted by adipose-derived mesenchymal stem cell (ADMSC) may function as novel candidates for wound healing. Here, we isolated the exosomes from ADMSCs and authorized by electron microscope (TEM), nanoparticle tracking analysis, as well as western blotting assay. The effects of ADMSC-derived exosomes on the growth and migration of human keratinocyte cell line (HaCaT) treated with H2O2 were studied. Flow cytometry was applied for measuring cell apoptosis. Migration was evaluated by wound healing and Transwell assays. Reverse transcription-quantitative polymerase chain reaction examined miRNAs and SHC SH2 domain-binding protein 1 (SHCBP1) expression. The integration between miR-146a-5p and SHCBP1 was estimated by dual-luciferase reporter analysis. The results revealed that ADMSC-derived exosomes promoted H2O2-treated HaCaT cell growth, migration, and invasion. miR-146a-5p-silenced ADMSC suppressed the regulation of the exosomes on the biological behaviors of HaCaT cells. SHCBP1 was determined and verified to be a target of miR-146a-5p. Moreover, SHCBP1 inhibition abrogated the miR-146a-5p-mediated cellular processes. In conclusion, ADMSC-derived exosomes carrying miR-146a-5p could protect HaCaT cells from injury by negatively regulating SHCBP1 expression.

Overexpressed RBPMS-AS1 increased cell radiosensitivity by sponging miR-19a-3p in lung cancer cell lines (A549 and SK-MES-1) via regulating PTEN/AKT axis

Purpose This paper intended to study RBPMS-AS1 in lung cancer (LC) radiosensitivity. Materials and Methods LC cells were transfected with RBPMS-AS1 overexpression plasmid and miR-19a-3p mimic and treated with radiation. PTEN, AKT, p-AKT, RBPMS-AS1, and miR-19a-3p expressions were detected via Western blot and qRT-PCR. The localization of RBPMS-AS1 in cells was determined through fluorescence in situ hybridization assay. The targeting relationships of RBPMS-AS1 and miR-19a-3p/miR-19a-3p and PTEN were determined through RIP and dual luciferase reporter analysis. Cell survival, viability, and apoptosis were assessed through colony formation, CCK-8, and flow-cytometry assays. Results RBPMS-AS1 was downregulated in LC and mainly distributed in cytoplasm. RBPMS-AS1 targeted miR-19a-3p in LC cells. Radiation suppressed LC cell survival, viability, and induced apoptosis, as overexpressed RBPMS-AS1 performed the similar effects and enhanced those effects induced by radiation. MiR-19a-3p mimic reversed the effect of overexpressed RBPMS-AS1 on enhancing radiation-induced LC cell apoptosis. MiR-19a-3p targeted PTEN and miR-19a-3p mimic reversed the effect of overexpressed RBPMS-AS1 on PTEN and phosphorylation of AKT in LC cells. Conclusion Overexpressed RBPMS-AS1 sponged miR-19a-3p to increase cell radiosensitivity in LC via regulating PTEN/AKT axis.

Jun-mediated lncRNA-IMS promotes the meiosis of chicken spermatogonial stem cells via gga-miR-31-5p/stra8.

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs)are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis(lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells.