Dual-luciferase Reporter Analysis Research Articles

Hsa_circ_0002473 inhibits GH3 cell proliferation and GH secretion as a competitive endogenous RNA for has-miR-4645-3p.

Growth hormone deficiency (GHD) diagnosis still lacks a gold standard or ideal diagnostic marker. Unlike other epigenetic mechanisms, non-coding RNAs regulate post-transcriptional levels. The information on non-coding RNAs in the field of GHD is limited. Therefore, this study aimed to explore the role of hsa_circ_0002473 as a competitive endogenous RNA for has-miR-4645-3p in attenuating the inhibitory effect of has-miR-4645-3p on SSTR2. In this study, we screened three significantly expressed circular RNAs (circRNAs) in five children with GHD, and selected the highest expressed hsa_circ_0002473 as the study object, and screened has-miR-4645-3p, which is the most likely to bind to hsa_circ_0002473, according to the microRNA (miRNA)-circRNA regulatory network, to study the role and mechanism of has-miR-4645-3p as a competitive endogenous RNA of has-miR-4645-3p on GH3 cells. Somatostatin receptor 2 (SSTR2) inhibits GH3 cell proliferation, and miRNA binding to SSTR2 inhibits the latter expression. Both bioinformatics and dual-luciferase reporter analyses showed targeting relationships between hsa_circ_0002473 and has-miR-4645-3p and between has-miR-4645-3p and SSTR2. We constructed the hsa_circ_0002473/has-miR-4645-3p axis and transfected it into GH3 cells and found that overexpression of hsa_circ_0002473 inhibited the proliferation and growth hormone (GH) secretion of GH3 cells, and that hsa-miR-4645-3p promoted the proliferation and GH secretion of GH3 cells by targeting SSTR2. Co-culture revealed that the inhibitory effect of hsa_circ_0002473 was reversed by has-miR-4645-3p. In conclusion, our findings suggest that hsa_circ_0002473 can act as a competitive endogenous RNA for has-miR-4645-3p to regulate GH3 cell proliferation and secretion by targeting SSTR2.

Circ_0001361/miR-490-5p/IGF2 Axis Regulates the Viability and Apoptosis of Neuroblastoma Cells.

Circular RNAs (circRNAs) are involved in the neuroblastoma (NB) development. Objectie: The study aimed to determine the biological behaviors of circ_0001361 and explore its underlying mechanism in NB. The circ_0001361, miR-490-5p, and IGF2 levels were measured using quantitative real-time polymerase chain reaction. Cellular processes were analyzed using MTT assay or fluorescence-activated cell sorting (FACS). Phosphorylated (p)-PI3K, p-AKT, Bax, and caspase-3 were tested by western blot. Dual-luciferase reporter analysis together with RNA pull-down analysis were utilized to evaluate the correlation of miR-490-5p and circ_0001361 or IGF2. The results in this study illustrated that an elevation of circ_0001361 levels was observed in NB. Depletion of circ_0001361 suppressed the viability but facilitated apoptosis of NB cells. Circ_0001361 sponged miR-490-5p, which targeted to regulate IGF2. Inhibition of miR-490-5p rescued the effect induced by circ_0001361 knockdown, while deletion of IGF2 rescued the effect induced by the miR-490-5p inhibitor. In summary, a loss of circ_0001361 inhibited NB progression via targeting the miR-490-5p/IGF2 axis, suggesting that circ_0001361 may be a novel therapeutical target of NB.

A novel hypoxic lncRNA, LOC110520012 sponges miR-206-y to regulate angiogenesis and liver cell proliferation in rainbow trout (Oncorhynchus mykiss) by targeting vegfaa

Long non-coding RNA (lncRNA) is a novel emerging type of competitive endogenous RNA (ceRNA) that performs key functions in multiple biological processes. However, little is known about the roles of lncRNA under hypoxia stress in fish. Here, vascular endothelial growth factor-Aa (vegfaa) was cloned in rainbow trout (Oncorhynchus mykiss), with the complete cDNA sequence of 2914 bp, encoding 218 amino acids. The molecular weight of the protein was approximately 25.33 kDa, and contained PDGF and VEGF_C domains. Time-course and spatial expression patterns revealed that LOC110520012 was a key regulator of rainbow trout in response to hypoxia stress, and LOC110520012, miR-206-y and vegfaa exhibited a ceRNA regulatory relationship in liver, gill, muscle and rainbow trout liver cells treated with acute hypoxia. Subsequently, the targeting relationship of LOC110520012 and vegfaa with miR-206-y was confirmed by dual-luciferase reporter analysis, and overexpression of LOC110520012 mediated the inhibition of miR-206-y expression in rainbow trout liver cells, while the opposite results were obtained after LOC110520012 silencing with siRNA. We also proved that vegfaa was a target of miR-206-y in vitro and in vivo, and the vegfaa expression and anti-proliferative effect on rainbow trout liver cells regulated by miR-206-y mimics could be reversed by LOC110520012. These results suggested that LOC110520012 can positively regulate vegfaa expression by sponging miR-206-y under hypoxia stress in rainbow trout, which facilitate in-depth understanding of the molecular mechanisms of fish adaptation and tolerance to hypoxia.

MaHsf24, a novel negative modulator, regulates cold tolerance in banana fruits by repressing the expression of HSPs and antioxidant enzyme genes.

Transcriptional regulation mechanisms underlying chilling injury (CI) development have been widely investigated in model plants and cold-sensitive fruits, such as banana (Musa acuminata). However, unlike the well-known NAC and WRKY transcription factors (TFs), the function and deciphering mechanism of heat shock factors (HSFs) involving in cold response are still fragmented. Here, we showed that hot water treatment (HWT) alleviated CI in harvested banana fruits accomplishing with reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activities. A cold-inducible but HWT-inhibited HSF, MaHsf24, was identified. Using DNA affinity purification sequencing (DAP-seq) combined with RNA-seq analyses, we found three heat shock protein (HSP) genes (MaHSP23.6, MaHSP70-1.1 and MaHSP70-1.2) and three antioxidant enzyme genes (MaAPX1, MaMDAR4 and MaGSTZ1) were the potential targets of MaHsf24. Subsequent electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and dual-luciferase reporter (DLR) analyses demonstrated that MaHsf24 repressed the transcription of these six targets via directly binding to their promoters. Moreover, stably overexpressing MaHsf24 in tomatoes increased cold sensitivity by suppressing the expressions of HSPs and antioxidant enzyme genes, while HWT could recover cold tolerance, maintaining higher levels of HSPs and antioxidant enzyme genes, and activities of antioxidant enzymes. In contrast, transiently silencing MaHsf24 by virus-induced gene silencing (VIGS) in banana peels conferred cold resistance with the upregulation of MaHSPs and antioxidant enzyme genes. Collectively, our findings support the negative role of MaHsf24 in cold tolerance, and unravel a novel regulatory network controlling bananas CI occurrence, concerning MaHsf24-exerted inhibition of MaHSPs and antioxidant enzyme genes.

Ciliary neurotrophic factor promotes the development of homocysteine-induced vascular endothelial injury through inflammation mediated by the JAK2/STAT3 signaling pathway

Elevated homocysteine (Hcy) levels have been recognized as significant risk factor for cardiovascular and cerebrovascular diseases, closely related to endothelial injury. While expression of Ciliary Neurotrophic Factor (CNTF) significantly increases during Hcy-induced vascular endothelial cell injury, the precise molecular pathways through which CNTF operates remain to be clarified. To induce vascular endothelial cell injury, human umbilical vein endothelial cells (HUVECs) were treated with Hcy. Cell viability and apoptosis in HUVECs were assessed using the CCK-8 assay and flow cytometry. Western blot analysis determined the expression levels of the JAK2-STAT3 pathway, inflammation-related factors (IL-1β, NLRP3, ICAM-1, VCAM-1), and apoptosis-related factors (cleaved Caspase-3 and Bax). Immunofluorescence staining and western blotting were employed to examine CD31 and α-SMA expression. Knockdown of CNTF was achieved using lentiviral interference, and its effects on inflammation and cell injury were evaluated. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter analysis were conducted to investigate the interaction between the MAFK and CNTF promoters. Our results indicated that Hcy induced high expression of CNTF and activated the JAK2-STAT3 signaling pathway, thereby upregulating factors associated with inflammation and cell apoptosis. Inhibiting CNTF alleviated Hcy-induced inflammation and cell injury. MAFK was identified as a transcription factor promoting CNTF transcription, and its overexpression exacerbated inflammation and cell injury in Hcy-treated HUVECs through the CNTF-JAK2-STAT3 axis, which could be reversed by knocking down CNTF. Activation of MAFK leads to CNTF upregulation, which activates the JAK2-STAT3 signaling pathway, regulating inflammation and inducing injury in Hcy-exposed vascular endothelial cells. Targeting CNTF or its upstream regulator MAFK may represent potential therapeutic strategies for mitigating endothelial dysfunction associated with hyperhomocysteinemia and cardiovascular diseases.

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.

Silencing AREG Enhances Sensitivity to Irradiation by Suppressing the PI3K/AKT Signaling Pathway in Colorectal Cancer Cells.

It has been established that Spalt-Like Transcription Factor 4 (SALL4) promotes Colorectal Cancer (CRC) cell proliferation. Furthermore, Amphiregulin (AREG) is crucially involved in cancer cell proliferation and therapeutic resistance regulation. In this regard, this study aimed to establish whether SALL4 affects the radiosensitization of CRC cells via AREG expression regulation. Transcriptome sequencing and the Human Transcription Factor Database (HumanTFDB) were used to identify the potential SALL4 targets. The dual-luciferase reporter analysis was used to confirm the SALL4-induced AREG activation. Western Blot (WB) and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays were used to examine the effect of X-ray irradiation on SALL4 and AREG expression. The AREG-KD (Knockdown) stable cell lines were created through lentiviral infection. Cell proliferation was tracked using Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU)-incorporation assays. Cell cycle and apoptosis were examined through flow cytometry. The cells were exposed to a controlled X-ray radiation dose (6 Gy) for imaging purposes. SALL4 could bound to the AREG promoter, enhancing AREG expression.Furthermore, irradiation upregulated SALL4 and AREG in CRC cells. Additionally, AREG knockdown in CRC cells led to reduced DNA replication efficiency, suppressed cell proliferation, increased DNA damage, and enhanced G1 phase arrest and apoptosis following irradiation. On the other hand, AREG overexpression reversed the inhibitory effects of SALL4 downregulation on AREG expression. In CRC cells, SALL4 downregulation suppressed AREG expression, regulating CRC cell radiosensitivity via the PI3K-AKT pathway, thus presenting a potential therapeutic pathway for CRC treatment using Radiotherapy (RT).

METTL3 Inhibition Suppresses Cell Growth and Survival in Colorectal Cancer via ASNS Downregulation.

Background: Colorectal cancer (CRC) presents a significant global health burden, with high rates of incidence and mortality, and an urgent need to improve prognosis. STM2457, a novel small molecule inhibitor specific for N6-methyladenosine (m6A) catalytic enzyme Methyltransferase-like 3 (METTL3) has implicated significant treatment potentials in a few of types of cancer. However, its impact and underlying mechanism are still unclear in CRC cells. Methods: We used CCK-8 and colony formation assay to observe cell growth, flow cytometry and TUNEL approaches to detect cell apoptosis under the treatment of STM2457 on CRC cells in vitro or in vivo. RNA-sequencing, qRT-PCR and western blotting were performed to explore downstream effectors of STM2457. Messenger RNA stability was evaluated by qRT-PCR after treatment with actinomycin D. The methylated RNA immunoprecipitation (MeRIP) qPCR, dual-luciferase reporter analyses and m6A dot blotting were carried out to measure the m6A modification. Associated gene expression pattern and clinical relevance in CRC clinical tissue samples were analyzed using online database. Results: STM2457 exhibited a strong influence on cell growth suppression and apoptosis of CRC cells in vitro and subcutaneous xenograft growth in vivo. Asparagine synthetase (ASNS) was markedly downregulated upon STM2457 treatment or METTL3 knockdown and exogenous overexpression of ASNS could rescue the biological defects induced by STM2457. Mechanistically, the downregulation of ASNS by STM2457 may be due to the decrease of m6A modification level in ASNS mRNA mediated by METTL3. Conclusions: Our findings suggest that STM2457 may serve as a potential therapeutic agent and ASNS may be a new promising therapeutic target for CRC.

CircUBE2D2 regulates HMGB1 through miR-885-5p to promote ovarian cancer malignancy

BackgroundThe newly discovered CircUBE2D2 has been shown to abnormally upregulate and promote cancer progression in a variety of cancers. The present study explored circUBE2D2 (hsa_circ_0005728) in Ovarian Cancer (OC) progression. MethodsCircUBE2D2, miR-885-5p, and HMGB1 were examined by RT-qPCR or WB. SKOV-3 cell functions (including cell viability, apoptosis, migration, and invasion) were validated using the CCK-8, flow cytometry, scratch assay, and transwell assay, respectively. The direct relationship between miR-885-5p and circUBE2D2 or HMGB1 was confirmed by a dual-luciferase reporter and RNA pull-down analysis. circUBE2D2′s role in vivo tumor xenograft experiment was further probed. ResultsOC tissue and cell lines had higher circUBE2D2 and HMGB1 and lower miR-885-5p. Mechanically, CircUBE2D2 shared a binding relation with miR-885-5p, while miR-885-5p can directly target HMGB1. Eliminating circUBE2D2 or miR-885-5p induction inhibited OC cell activities. However, these functions were relieved by down-regulating miR-885-5p or HMGB1 induction. Furthermore, circUBE2D2 knockout reduced tumor growth. ConclusionCircUBE2D2 regulates the expression of HMGB1 by acting as a sponge of ceRNA as miR-885-5p, thereby promoting the control of OC cell proliferation and migration and inhibiting cell apoptosis. Targeting CircUBE2D2 could serve as a new potential treatment strategy for OC.

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.