Luciferase Reporter Assay Research Articles

Atomoxetine suppresses radioresistance in glioblastoma via circATIC/miR-520d-5p/Notch2-Hey1 axis

BackgroundResistance acquired after radiotherapy is directly related to the failure of various cancer treatments, including GBM. Because the mechanism for overcoming radioresistance has not yet been clearly identified, the development of diagnostic and therapeutic markers to treat radioresistance is necessary. Since increased expression of stemness- and EMT-related markers are reported to be closely correlated with radioresistance, research is underway to develop new drugs targeting these factors.MethodsTo develop an anticancer drug that overcomes radioresistance, a library of drugs already approved by the FDA was used. After treating radioresistant GBM cells with each drug, the expression of stemness- and EMT-related markers was confirmed by qRT-PCR, and as a result, Atomoxetine (ATX) was selected. It was confirmed that radioresistance-induced cell migratory, invasive, sphere formation abilities, and tumor growth using a xenograft mouse model were suppressed upon ATX treatment. Using a miRNA prediction tool, we discovered miR-520d-5p, which targets Notch2 and Hey1, key factors in radioresistance, and discovered circATIC targeting this miRNA, revealing its relationship with ATX. We demonstrated the expression regulation mechanism and signaling mechanism between circATIC, miR-520d-5p, Notch2, and Hey1 factors using a luciferase reporter assay. In addition, the results at the cellular level were clinically verified by confirming the correlation between radiation, miR-520d-5p, and circATIC using patient plasma by qRT-PCR.ResultsATX showed potential as a treatment for radioresistance by suppressing the malignant phenotype by regulating the circATIC/miR-520d-5p/Notch2-Hey1 signaling mechanism in vitro and in vivo using radioresistant GBM cells.ConclusionsThis study revealed that ATX suppresses radioresistance through the circATIC/miR-520d-5p/Notch2-Hey1 signaling pathway. These results showed the potential of ATX as a new drug that can overcome radioresistance, a major challenge in cancer treatment, and the signaling factors identified in this mechanism suggest the possibility of use as potential targets for the diagnosis and treatment of radioresistance.

The role of lncRNA TSIX in osteoarthritis pathogenesis: mechanistic insights and clinical biomarker potential

BackgroundThis study seeks to elucidate the expressions of lncRNA TSIX in Osteoarthritis (OA) and to explore its mechanisms in regulating OA progression.MethodsRT-qPCR was employed to analyze the expression of TSIX in OA patients classified by Kellgren-Lawrence (K-L) grades. Receiver operator characteristic (ROC) was conducted to evaluate the diagnostic value of TSIX. Correlation between TSIX levels and clinical scores such as Lysholm and visual analogue scale (VAS) score was evaluated using Pearson method. IL-1β-induced SW1353 cells served as an in vitro model. The cell function were assessed by flow cytometry and cell counting kit-8 (CCK-8) assay. The relationship between TSIX and miR-320a was verified by luciferase reporting system, while bioinformatics approaches were utilized to predict the downstream target genes of miR-320a.ResultsThe findings revealed that TSIX level in OA patients was elevated compared to that of the control group, with a notable progressive increase in TSIX expression correlated with higher K-L grades. In OA patients, the Lysholm score showed a negative correlation with TSIX expression, while the VAS score displayed a positive correlation with TSIX levels. Cell studies demonstrated that inhibition of TSIX enhanced cell viability and mitigated IL-1β-induced apoptosis by targeting miR-320a, in addition to promoting Aggrecan and Collagen II secretion. Luciferase reporter assay further validated the targeting interaction among TSIX, miR-320a, and PTEN.ConclusionsThis study demonstrated an increased expression of TSIX in OA patients. It suggests that TSIX may play a role in chondrocyte dysfunction during OA by modulating the miR-320a/PTEN axis.

STUB1 suppresses paclitaxel resistance in ovarian cancer through mediating HOXB3 ubiquitination to inhibit PARK7 expression

Paclitaxel (PTX) is a first-line drug for ovarian cancer (OC) treatment. However, the regulatory mechanism of STUB1 on ferroptosis and PTX resistance in OC remains unclear. Genes and proteins levels were evaluated by RT-qPCR, western blot and IHC. Cell viability and proliferation were measured by CCK-8 and clone formation. The changes of mitochondrial morphology were observed under a transmission electron microscope (TEM). Reactive oxygen species (ROS), iron, malondialdehyde (MDA) and glutathione (GSH) were measured using suitable kits. The interactions among STUB1, HOXB3 and PARK7 were validated using Co-IP, and dual luciferase reporter assay. Our study found that STUB1 was decreased and PARK7 was increased in tumor tissue, especially from chemotherapy resistant ovarian cancer tissue and resistant OC cells. STUB1 overexpression or PARK7 silencing suppressed cell growth and promoted ferroptosis in PTX-resistant OC cells, which was reversed by HOXB3 overexpression. Mechanistically, STUB1 mediated ubiquitination of HOXB3 to inhibit HOXB3 expression, and HOXB3 promoted the transcription of PARK7 by binding to the promoter region of PARK7. Furthermore, STUB1 overexpression or PARK7 silencing suppressed tumor formation in nude mice. In short, STUB1 promoted ferroptosis through regulating HOXB3/PARK7 axis, thereby suppressing chemotherapy resistance in OC.

MiR-525-5p modulates cell proliferation, cell cycle, and apoptosis in Burkitt's lymphoma by targeting MyD88 and regulating the NF-κB signaling pathway.

MiR-525-5p functions as an oncomiRNA or tumor suppressor, and has been reported in various cancer types, including laryngeal squamous cell carcinoma, glioma, breast cancer, and cervical cancer. However, the biological functions and precise mechanisms of miR-525-5p remain unclarified in Burkitt's lymphoma (BL). This study aimed to explore the roles of miR-525-5p in BL, with the goal of ascertaining its regulatory effects on the nuclear factor-kappaB (NF-κB) signaling pathway by targeting Myeloid differentiation factor 88 (MyD88). The expression levels of miR-525-5p and MyD88 were measured by quantitative real-time PCR and immunohistochemical staining, respectively. The effects of miR-525-5p overexpression on BL cell proliferation, colony-forming, and migration were evaluated by cell counting kit-8, soft agar colony-forming, and transwell assays, while cell cycle and cell apoptosis were analyzed by flow cytometry. Possible interactions between miR-525-5p and MyD88 was examined via luciferase reporter assay. The expression of MyD88 and NF-κB signaling pathway-related proteins, including p65, p-p65, IκBa, and p-ΙκBa was determined by western blotting. BL cells overexpressing miR-525-5p were treated with phorbol 12-myristate 13-acetate (PMA), and Hoechst 33258 staining and Calcein AM/EthD-I staining were used to analyze the changes in chemotherapy sensitivity of BL cells to doxorubicin (DOX). Compared with reactive lymphoid hyperplasia, miR-525-5p was dramatically downregulated in BL tissues, while the rate of MyD88 protein positivity was significantly increased. Upregulation of miR-525-5p suppressed cell proliferation, colony-forming, and migration, induced cell cycle arrest and apoptosis, and enhanced the chemosensitivity to DOX in BL cells. MiR-525-5p targeted MyD88 to inhibit the activation of NF-κB signaling pathway. PMA treatment reactivated the NF-κB pathway and reversed apoptosis mediated by miR-525-5p overexpression. These findings revealed that miR-525-5p acts as a tumor suppressor, targeting MyD88 to modulate proliferation, cell cycle progression, and apoptosis in BL cells by regulation of NF-κB signaling pathway.

METTL14 attenuates cancer stemness by suppressing ATF5/WDR74/β-catenin axis in gastric cancer.

Stemness is a key factor contributing to treatment failure in gastric cancer (GC). Methyltransferase-like 14 (METTL14) has been linked to various cancers, though its specific role in regulating stemness in GC remains undefined. In this study, we assessed METTL14 expression levels in GC tissues using public datasets and clinical specimens and investigated its impact on cell proliferation, metastasis, and stemness both invitro and invivo. Through m6A RNA immunoprecipitation (MeRIP) and luciferase reporter assays, we identified downstream targets of METTL14. Rescue assays were performed to examine whether METTL14 overexpression could reverse stemness in GC. We also explored the underlying mechanisms using chromatin immunoprecipitation (ChIP) and western blot analysis, focusing on the role of ATF5 and the upstream regulation of METTL14. Our findings show that lower METTL14 expression is associated with poorer overall survival in GC patients. Functionally, METTL14 knockdown enhanced stemness traits in GC cells. Mechanistically, METTL14 facilitated m6A modification, promoting the degradation of ATF5 mRNA. Overexpression of ATF5 reversed the stemness inhibition caused by METTL14 overexpression by increasing WDR74 transcription and enhancing β-catenin nuclear translocation. Furthermore, histone H3 lactylation at Lys18 was found to upregulate METTL14 expression. In conclusion, METTL14 knockdown promotes stemness in GC by mediating m6A modification of ATF5 mRNA, which activates the WDR74/β-catenin axis, making METTL14 a potential therapeutic target for gastric cancer treatment.

YAP regulates HER3 signaling-driven adaptive resistance to RET inhibitors in RET-aberrant cancer.

Rearranged during transfection (RET) aberrations represent a targetable oncogene in several tumor types, with RET inhibitors displaying marked efficacy. However, some patients with RET-aberrant cancer are insensitive to RET tyrosine kinase inhibitors (TKIs). Recently, drug-tolerant mechanisms have attracted attention as targets for initial therapies to overcome drug resistance. The underlying mechanisms of drug-tolerant cell emergence treated with RET-TKIs derived from RET-aberrant cancer cells remain unknown. This study investigated the role of YAP-mediated HER3 signaling in the underlying mechanisms of adaptive resistance to RET-TKIs in RET-aberrant cancer cells. Four RET-aberrant cancer cell lines were used to assess sensitivity to the RET-TKIs selpercatinib and pralsetinib and to elucidate molecular mechanisms underlying adaptive resistance using RNA sequencing, phospho-RTK antibody arrays, chromatin immunoprecipitation assay, and luciferase reporter assays. Clinical specimens from patients with RET-fusion-positive lung cancer were analyzed for pre-treatment YAP expression and correlated with treatment outcomes. In high YAP-expressing RET-aberrant cancer cells, YAP-mediated HER3 signaling activation maintained cell survival and induced the emergence of cells tolerant to the RET-TKIs selpercatinib and pralsetinib. The pan-ErBB inhibitor afatinib and YAP/TEAD inhibitors verteporfin and K-975 sensitized YAP-expressing RET-aberrant cancer cells to the RET-TKIs selpercatinib and pralsetinib. Pre-treatment YAP expression in clinical specimens obtained from patients with RET-fusion-positive lung cancer was associated with poor RET-TKI treatment outcomes. The YAP-HER3 axis is crucial for the survival and adaptive resistance of high YAP-expressing RET-aberrant cancer cells treated with RET-TKIs. Combining YAP/HER3 inhibition with RET-TKIs represents a highly potent strategy for initial treatment.

FoxO1 Deficiency in M-MDSCs Exacerbates B Cell Dysfunction in Systemic Lupus Erythematosus.

Myeloid-derived suppressor cells (MDSCs) contribute to the pathogenesis of systemic lupus erythematosus (SLE), in part due to promoting the survival of plasma cells. Forkhead box protein O1 (FoxO1) expression in monocytic MDSCs (M-MDSCs) exhibits a negative correlation with the SLE Disease Activity Index (SLEDAI) score. This study aimed to investigate the hypothesis that M-MDSCs specific FoxO1 deficiency enhances aberrant B cell function in aggressive SLE. We used GEO datasets and clinical cohorts to verify the FoxO1 expression and circulating M-MDSCs clinical significance. Using Cre-LoxP technology, we generated myeloid FoxO1 deficiency mice (mFoxO1-/-) to establish murine lupus-prone models. The transcriptional stage was assessed by integrating ChIP-seq with transcriptomic analysis, luciferase reporter assay and ChIP-qPCR. Methylated RNA immunoprecipitation sequencing, RNA sequencing and CRISPR-dCas9 were used to identify m6A modification. In vitro B cell co-culture experiments, Capmatinib intragastric administration, m6A-modulated MDSCs adoptive transfer, and SLE patient sample validation were performed to determine the role of FoxO1 on M-MDSCs dysregulation during B cell autoreacted with SLE. We present evidence that low FoxO1 is predominantly expressed in M-MDSCs in both SLE patients and lupus mice, and mice with myeloid FoxO1 deficiency (mFoxO1-/-) are more prone to B cell dysfunction. Mechanically, FoxO1 inhibits Met transcription by binding to the promoter region. M-MDSCs FoxO1 deficiency blocks the Met/COX2/PGE2 secretion pathway, promoting B cell proliferation and hyperactivation. Met antagonist Capmatinib effectively mitigates lupus exacerbation. Furthermore, ALKBH5 targeting catalyzes m6A modification on FoxO1 mRNA in CDS and 3'-UTR regions. Upregulation of FoxO1 mediated by ALKBH5 overexpression in M-MDSCs improves lupus progression. Finally, these correlations were confirmed in untreated SLE patients. Our findings indicate that effective inhibition of B cells mediated by ALKBH5/FoxO1/Met axis in M-MDSCs could offer a novel therapeutic approach to manage SLE.

Compound #41 Targets Acute Myelogenous Leukemia by Inhibiting the Wnt/β-catenin Signaling Pathway.

Aberrant activation of the Wnt/β-catenin signaling pathway contributes to the pathogenesis of acute myelogenous leukemia (AML). Thus, targeting this pathway offers a promising therapeutic strategy against AML. Here, we synthesized a novel dipeptide-type inhibitor of the Wnt/β-catenin signaling pathway, compound #41, and explored its anti-tumor effects on AML cells. We evaluated the inhibitory effect of compound #41 on T cell factor (TCF)/β-catenin transcriptional activity using a luciferase (Luc) reporter assay. The anti-tumor effects were assessed on KG1a and MV4;11 human AML cells using RT-qPCR, western blotting, and WST-8, cell cycle, and apoptosis assays. Differentially expressed genes were analyzed by RNA-sequencing (RNA-seq). Additionally, we investigated the in vivo effects of compound #41 using KG1a-Luc/GFP cells in an orthotopic mouse model. The Luc reporter assay showed that compound #41 decreased the TCF/β-catenin transcriptional activity. Compound #41 blocked the cell cycle progression, inhibited cell proliferation, and induced apoptosis in AML cells. Treatment with compound #41 down-regulated the expression of β-catenin, Survivin, and β-catenin-specific target genes, as demonstrated by RNA-seq. In vivo analysis showed that compound #41 blocked the expansion of KG1a-Luc/GFP cells in the bone marrow and prolonged the overall survival of KG1a-Luc/GFP-transplanted mice. Compound #41 suppressed the Wnt/β-catenin signaling pathway by reducing CTNNB1 levels and induced apoptosis in AML cells. Furthermore, compound #41 inhibited the proliferation of KG1a-Luc/GFP cells in the bone marrow and extended the overall survival of mice. Thus, compound #41 is an attractive Wnt/β-catenin signaling pathway inhibitor of AML.

Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoids in lipopolysaccharide-treated HEI-OC1 cells through modulation of the FOXP3/Nrf2 signaling pathway.

Glucocorticoids (GCs) are commonly used to treat sudden sensorineural hearing loss (SSNHL), although some patients are resistant to this therapeutic approach. Clinical studies have demonstrated the efficacy of tanshinone IIA (TA) in combination with GC for managing various human ailments. However, it remains unclear whether TA can mitigate GC resistance in SSNHL. Our aim is to elucidate the role of NRF2-induced transcriptional regulation of HDAC2 in influencing GC resistance and investigate the involvement of TA-related molecular pathways in GC resistance. Here, HEI-OC1 cells are treated with lipopolysaccharide (LPS) to establish an in vitro model for SSNHL. The cells are subsequently treated with dexamethasone (DXE) or DXE+TA. RT-qPCR and western blot analysis are used to measure the mRNA and protein levels of Forkhead box P3 (FOXP3), nuclear factor erythroid 2-related factor 2 (NRF2), and histone deacetylase 2 (HDAC2). Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays are carried out to assess cell proliferation. Flow cytometry analysis is performed to evaluate apoptosis. Mechanistic studies involve chromatin immunoprecipitation (ChIP), luciferase reporter, and DNA pull-down assays. Our results show that treatment with TA+DEX significantly increases proliferation and suppresses apoptosis in LPS-treated HEI-treated OC1 cells. TA upregulates HDAC2 expression by activating NRF2-mediated transcription of HDAC2, with the NRF2-HDAC2 binding site located at bases 419-429 (ATGACACTCCA) in the promoter sequence of HDAC2. Furthermore, TA upregulates FOXP3 expression to activate NRF2 transcription, with the predicted FOXP3-binding site located at bases 864-870 (GCAAACA) in the promoter sequence of NRF2. In summary, these findings suggest that TA enhances the therapeutic effects of GC on the proliferation and apoptosis of HEI OC1 cells by increasing FOXP3/Nrf2 expression. These results indicate that TA may be promising for ameliorating GC resistance in patients with SSNHL.

Ras-related Protein in Brain 4A (Rab4A) is Downregulated by miR-496 to inhibit the Progression of Gastric Cancer.

Ras-related protein in brain 4A (Rab4A), as a member of the Rab family, is involved in the intracellular circulation of membrane receptors or endocytic substances and regulates the progression of multiple tumors. From our results, the knockdown of Rab4A inhibited the proliferation, migration and invasion in AGS cells. Importantly, the surface expression of epidermal growth factor receptor (EGFR) declined significantly in Rab4A knockdown cells. The downstream pathway of EGFR was also inhibited after the transfection of Rab4A-specific siRNA, including AKT and β-catenin pathways. In addition, miR-496 down-regulated the expression of Rab4A in AGS cells. The result of the luciferase reporter assay showed that miR-496 could bind to the 3'UTR of Rab4A. In conclusion, the expression of Rab4A is inhibited by miR-496, and the knockdown of Rab4A inhibits the proliferation, migration and invasion through down-regulating the surface expression of EGFR. Rab4A is a potential target in the treatment of gastric cancer.

A 65 kilobase deletion of the upstream TYR gene region in a family with oculocutaneous albinism type 1

Oculocutaneous albinism type 1 is caused by variants in the TYR (tyrosinase) gene. We describe a family with two affected sibs who inherited the pathogenic missense TYR variant c.1146C > A;p.(Asn382Lys) from their mother and a deletion encompassing 65 kilobase pairs of the upstream region of the gene between hg38 coordinates chr11:89110944 and chr11:89175770, from their father. The deletion likely arose by non-homologous recombination since the regions including the two deletion breakpoints share no sequence homology. The deletion contains a single enhancer element that is homologous to a 5′ Tyr core regulatory element in the mouse. A luciferase reporter assay showed that this element had a positive regulatory activity. This represents to our knowledge the first deletion solely restricted to non-coding upstream sequences of the TYR gene. It is assumed that the deletion down-regulates expression of the TYR gene and is therefore pathogenic, allowing to establish the diagnosis of OCA 1 in the patients. This study underscores the need to extend the search for pathogenic variants to regulatory regions either by whole genome sequencing or by targeted next generation sequencing of a panel including entire genes (exons, introns, flanking sequences) in order to improve the diagnostic rate in patients with albinism.

Hsa_Circ_0105596/FTO Inhibits Progression of Parkinson's disease by Sponging miR-187-3p and Regulating eEF2

Backgroud:Parkinson's disease (PD) characterized by inflammation and protein erroneous deposition, whose pathological mechanisms have not been elucidated. NcRNA plays important role in PD, especially when circRNA sponges miRNA, which leads to the breakdown of downstream regulation. The aim of this study is to investigate the dynamic changes between upregulated circRNA and downregulated miRNA during the pathogenesis of PD and their impact on downstream miRNA targets. MethodWe conducted bioinformatics on sequencing data of substantia nigra (SN) and striatum, and intersected differentially expressed genes (Degs) to determine core role of circFTO-miR-187-3p-EEF2 axes in the progression of PD. Firstly, therapeutic effect of knock-down circFTO in PD and its impact on EEF2 were determined in mouse, using immunohistochemistry, HE, Nissl, TUNEL staining and western blot (WB). Targeted binding relationship between circFTO, miR-187-3p, and EEF2 was determined through RNA binding protein immunoprecipitation assays (RIP) and dual luciferase reporter assay. The significance of gene in apoptosis was confirmed through flow cytometry, lentiviral transduction, quantitative real-time PCR, and WB. ResultCircFTO is upregulated and miR-187-3p is downregulated in SN of PD. EEF2 was the core of neural repair related modules in both SN and striatum using Weighted Correlation Network Analysis (WGCNA) and protein-protein interaction (PPI). The binding regulation relationship between circFTO-miR-187-3p-EEF2 was determined through structural analysis, RIP, and dual luciferase reporter assay. After knocking-down circFTO, animals showed alleviated symptoms, decreased levels of oxidative stress and EEF2. Upregulation of miR-187-3p or si-circFTO in SH-SY5Y cells can reduce cell apoptosis, EEF2, and oxidative stress. Moreover, individual interference with EEF2 can partially counteract the induction of 6-OHDA on apoptosis. ConclusionExcessive circFTO sponging miR-187-3p leads to the inability of miR-187-3p to effectively regulate expression of EEF2, resulting in the progression of PD. Moreover, interference with circFTO can effectively reduce brain inflammation and oxidative stress.

Lnc NEAT1 facilitates the progression of melanoma by targeting the miR-152-3p/CDK6 axis: An observational study.

Long noncoding (Lnc) RNAs are novel regulators in melanoma. Lnc nuclear enriched autosomal transcript 1 (NEAT1) was reportedly upregulated in melanoma; however, the functional roles and mechanisms of Lnc NEAT1 need further investigation. Therefore, we used quantitative real-time PCR to determine the mRNA levels of Lnc NEAT1, miR-152-3p, and cyclin-dependent protein kinase 6 (CDK6). The protein level of CDK6 was determined by Western blot. Cell counting kit 8 and colony formation assays were used to assess cell proliferation. Cell migration was measured by wound healing and Transwell assays. Direct binding of the indicated molecules was verified by an RNA-binding protein immunoprecipitation assay and a dual luciferase reporter assay. The results revealed that Lnc NEAT1 and CDK6 were elevated, while miR-152-3p was downregulated in melanoma. Furthermore, Lnc NEAT1 was positively correlated with CDK6 expression and negatively correlated with miR-152-3p level. Furthermore, Lnc NEAT1 facilitated proliferation, migration, and invasion of melanoma cells. The underlying mechanism is that Lnc NEAT1 serves as a sponge for miR-152-3p to suppress the inhibitory effect of miR-152-3p on CDK6. Furthermore, the miR-152-3p/ CDK6 axis was implicated in the progression of melanoma accelerated by Lnc NEAT1. Taken together, Lnc NEAT1 may promote melanoma development by serving as an endogenous sponge of miR-152-3p, increasing CDK6 expression, and identifying a new target for the treatment of melanoma.

DEAD/H-box helicase 11 is transcriptionally activated by Yin Yang-1 and accelerates oral squamous cell carcinoma progression.

Oral squamous cell carcinoma (OSCC) is the most common oral malignancy. DEAD/H-box helicase 11 (DDX11), a DNA helicase, has been implicated in the progression of several cancers. Yet, the precise function of DDX11 in OSCC is poorly understood. The DDX11 expression in OSCC cells and normal oral keratinocytes was evaluated in the Gene Expression Omnibus database (GSE146483 and GSE31853). SCC-4 and CAL-27 cells expressing doxycycline-inducible DDX11 or DDX11 shRNA were generated by lentiviral infection. The role of DDX11 in OSCC cells was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, colony formation assay, flow cytometry assay, TUNEL staining, and western blot. The effects of DDX11 on tumor growth were explored in a xenograft nude mouse model. The relationship between DDX11 and transcription factor Yin Yang-1 (YY1) was researched using the dual luciferase report assay and chromatin immunoprecipitation assay. DDX11 expression was significantly upregulated in OSCC cells. Knockdown of DDX11 inhibited cell proliferation, induced cell cycle arrest, and suppressed PI3K-AKT pathway, while DDX11 overexpression showed opposite effects. The number of apoptotic cells was increased in DDX11 silenced cells. DDX11 upregulation or knockdown accelerated or suppressed tumor growth in vivo, respectively. Moreover, the YY1 bound and activated the DDX11 promoter, resulting in increasing DDX11 expression. Forced expression DDX11 reversed the anticancer effects of YY1 silencing on OSCC cells. DDX11 has tumor-promoting function in OSCC and is transcriptionally regulated by YY1, indicating that DDX11 may serve as a potential target for the OSCC treatment.

Fructus Arctii Mitigates Depressive Disorder via the Let-7e-Modulated Toll-Like Receptor (TLR) Signaling Pathway.

Depressive disorder is a common and serious public health challenge globally. Fructus arctii is a traditional medicinal plant ingredient with diverse pharmacological effects. This study aimed to investigate the therapeutic potential of Fructus arctii in alleviating depressive-like behaviors. We established a chronic unpredictable mild stress (CUMS)-induced depression mouse model to assess the antidepressant effects of Fructus arctii. BV2 cells treated with lipopolysaccharide (LPS) were used to mimic neuronal damage. Behavioral tests, including the sucrose preference test, tail-suspension test, and forced swim test, were conducted to evaluate the impact of Fructus arctii on depressive-like behaviors. Let-7e expression was detected by RT-qPCR, and TLR4 signaling pathway activation was evaluated by western blot analysis, which also assessed the inflammatory response by measuring levels of IL-6, IL-1β, MCP-1, TNF-α, and iNOS. Immunohistological analysis was conducted to detect the expression of microglia markers. Luciferase reporter assays verified the interaction between let-7e and TLR4. Fructus arctii administration effectively alleviated depressive-like behaviors induced by CUMS in mice, as evidenced by improved sucrose preference and reduced immobility time in behavioral tests. Mechanistically, Fructus arctii reversed the CUMS-induced downregulation of let-7e and upregulation of TLR4 and MyD88 protein levels in mice hippocampus tissues. In addition, Fructus arctii suppressed microglial activation and reduced the levels of inflammatory factors by upregulating let-7e. Let-7e was verified to bind to TLR4, thereby negatively regulating its expression. TLR4 overexpression reversed the suppressive effect of let-7e upregulation on inflammatory reactions and microglial activation. Furthermore, intracerebroventricular injection of let-7e agomiR alleviated depressive-like behavior and inhibited microglial activation in vivo. In summary, Fructus arctii mitigates depression by regulating the let-7e/TLR4/MyD88 pathway, offering new insights into potential depression therapies.

STK33 as the functional substrate of miR-454-3p for suppression and apoptosis in neuroblastoma

miR-454-3p has been reported to be a tumor suppressive micro-RNA (miRNA) in multiple cancer types. We identified the kinase STK33 mRNA, which is a high-risk factor for survival in neuroblastoma (NB) patients, as being a substrate of miR-454-3p in NB. Even though STK33 is an attractive target for several cancers, development of inhibitors of STK33 has been challenging. For the various cell lines tested, we demonstrated reduced growth and viability with the miR-454-3p mimic. From among the candidate NB-associated miRNAs, miR-454-3p mimic and its antagonist had the most profound effect on STK33 mRNA and protein level changes. Under various conditions of growth and external stress for the cells, the RNA levels for miR-454-3p and STK33 also negatively correlated. Luciferase reporter assays demonstrated STK33 as a substrate for miR-454-3p, and recombinant versions of STK33 resistant to miR-454-3p significantly blunted the suppressive effect of the miR-454-3p and established STK33 as the major functional substrate of miR-454-3p. Overexpression of miR-454-3p or knockdown of STK33 mRNA promoted autophagy, and at the same time, increased the apoptotic markers in the tested NB cells, indicating a mechanism for the suppressive effect of the agents. Given the difficult-to-drug targets such as STK33 and the recent successes in RNA delivery methods for cancer treatment, it is thought that targeting cancer cells with a suppressive miRNA such as miR-454-3p for STK33-dependent cancer types may be an alternative means of NB therapy.

Design, Synthesis, and Hypoxia-Inducible Factor-1α Inhibitory Activity Evaluation of Panaxadiol Derivatives Containing a Thiazole Moiety.

The hypoxia-inducible factor-1α (HIF-1α) pathway has been implicated in tumor angiogenesis, growth, and metastasis. Therefore, the inhibition of this pathway is an important therapeutic target for cancer. Thiazole derivatives have been reported to have diverse biological activities, especially in terms of anti-tumor. Consequently, we hypothesized that the introduction of a thiazole functional group in PD was likely to improve the biological potency. Here, three series of PD derivatives containing a thiazole moiety were synthesized, including (a) sulfonyl-containing thiazole derivatives (5 a-l), (b) urea-containing thiazole derivatives (7 a-i), and (c) thiourea-containing thiazole derivatives (9 a-i), and evaluated for HIF-1α inhibitory activity using a Hep3B cell-based luciferase reporter assay. The results showed that about 1/3 of the target compounds showed moderate or strong HIF-1α inhibitory activity, among which compounds 5 d and 7 b showed the strongest inhibitory activity with IC50 values of 17.37 and 6.42 μM, respectively, and did not show any significant cytotoxicity. Western blot assay results indicated that these two compounds exhibited more potent inhibition, compared with panaxadiol, of the expression of HIF-1α protein in Hep3B cells at a concentration of 50 μM. Molecular docking experiments were also performed to investigate the structure-activity relationship. Compounds 5 d and 7 b can be used as leads for further study and development of novel antitumor drugs.

Transport of miR-766–3p to A549 cells by plasma-derived exosomes and its effect on intracellular survival of Mycobacterium tuberculosis by regulating NRAMP1 expression in A549 cells

Exosomal microRNAs (miRNAs) in circulation were recognized as potential biomarkers for the diagnosis of multiple diseases. However, its potential as a diagnostic hallmark for tuberculosis (TB) has yet to be explored. Here, we comprehensively analyze miRNA profiles in exosomes derived from the plasma of active TB patients and healthy persons to evaluate its efficacy in TB diagnosis. Small-RNA transcriptomic profiling analysis identified a total of 14 differentially expressed miRNAs (DEmiRNAs), among which the diagnostic potential of exosomal miR-766–3p, miR-376c-3p, miR-1283, and miR-125a-5p was evident from their respective areas under the ROC curve, which were 0.8963, 0.8313, 0.8097, and 0.8050, respectively. The bioinformatics analysis and Luciferase reporter assays confirmed that the 3′-untranslated region of natural resistance-associated macrophage protein 1 (NRAMP1) mRNA was targeted by miR-766–3p. The exosomes could be internalized by the A549 cells in co-culturing experiments. Furthermore, both increased miR-766–3p and decreased NRAMP1 expression were observed in Mtb-infected A549 cells. MiR-766–3p overexpression reduced the NRAMP1 levels, but increased intracellular Mtb, suggesting that miR-766–3p may facilitate Mtb survival by targeting NRAMP1. Moreover, miR-766–3p-transfected cells exhibited increased apoptosis and reduced proliferation following Mtb infection. Taken together, circulating exosomal miR-766–3p, miR-1283, miR-125a-5p, and miR-376c-3p may serve as candidate hallmarks for TB diagnosis where the presence of miR-766–3p seems associated with the vulnerability to Mtb infection in humans and could be a new molecular target for therapeutic intervention of TB.

MiR-128-3p suppresses tumor growth and enhances chemosensitivity in tongue squamous cell carcinoma through MAP2K7 targeting.

MicroRNAs (miRNAs), which are key players in cancer cell resistance to chemotherapy, notably target genes associated with drug resistance. While miRNA-128-3p is recognized for its involvement in various cancers, its specific role in tumorigenesis and cisplatin (CIS) resistance in tongue cancer remains unclear. Therefore, in the present study, we endeavoured to elucidate the significance of miR-128-3p in tongue squamous cell carcinoma (TSCC), shedding light on its intricate functions and underlying mechanisms. We quantified the expression of miR-128-3p and its target genes using qRT-PCR, followed by a series of functional assays in vitro, such as proliferation and migration assays, flow cytometry analysis, and western blotting to unravel the mechanisms underlying the functions of miR-128-3p. Additionally, we validated the ability of miR-128-3p to target MAP2K7 genes through luciferase reporter assays. We observed that increased expression of miR-128-3p significantly inhibited TSCC cell migration, proliferation, and epithelial-mesenchymal transition (EMT), possibly by regulating MAP2K7 in the JNK/MAP kinase pathway through miRNA target binding. Furthermore, we showed that increased miR-128-3p levels enhanced the sensitivity of TSCC cells to CIS through the JNK/c-Jun cascade. We observed that miR-128-3p reduces the expression of c-Jun and ABC transporter genes by targeting MAP2K7, affecting JNK1/2. This inhibition possibly decreases drug efflux and thus enhances the TSCC sensitivity to CIS treatment. Our findings demonstrate oncosuppressive behaviour of miR-128-3p, which also potentially enhances the sensitivity of TSCC cells to CIS by suppressing MAP2K7 and JNK1/2, leading to evasion of apoptosis.

DNA Hypomethylation Activates the RpMYB2-Centred Gene Network to Enhance Regeneration of Adventitious Roots.

Plants, being immobile, are exposed to environmental adversities such as wind, snow and animals that damage their structure, making regeneration essential for their survival. The adventitious roots (ARs) primarily emerge from a detached explant to uptake nutrients; therefore, the molecular network involved in their regeneration needs to be explored. DNA methylation, a key epigenetic mark, influences molecular pathways, and recent studies suggested its role in regeneration. In our research, the application of 5-azacytidine (5-azaC), an inhibitor of DNA methylation, caused the earlier initiation and development of root primordia and consequently enhanced the AR regeneration rate in Robinia psuedoacacia L (black locust). The whole-genome bisulfite sequencing (WGBS) revealed a decrease in global methylation and an increase in hypomethylated cytosine sites and regions across all contexts including CHH, CHG and mergedCG caused transcriptional variations in 5-azaC-treated sample. The yeast two-hybrid (Y2H) assay revealed a RpMYB2-centred network of transcriptionally activated transcription factors (TFs) including RpWRKY23, RpGATA23, RpSPL16 and other genes like RpSDP, RpSS1, RpBEN1, RpGULL05 and RpCUV with nuclear localization suggesting their potential co-localization. Additionally, yeast one-hybrid (Y1H) assay showed the interaction of RpMYB2 interactors, RpGATA23 and RpWRKY23, with promoters of RpSK6 and RpCDC48, and luciferase reporting assay (LRA) validated their binding with RpSK6. Our results revealed that hypomethylation-mediated transcriptomic modifications activated the RpMYB2-centred gene network to enhance AR regeneration in black locust hypocotyl cuttings. These findings pave the way for genetic modification to improve plant regeneration ability and increase wood production while withstanding environmental damage.