Direct Target Research Articles

Isoflurane preconditioning attenuates OGD/R-induced cardiomyocyte cytotoxicity by regulating the miR-210/BNIP3 axis.

Isoflurane, a commonly used inhaled anesthetic, has been found to have a cardioprotective effect. However, the precise mechanisms have not been fully elucidated. Here, we found that isoflurane preconditioning enhanced OGD/R-induced upregulation of miR-210, a hypoxia-responsive miRNA, in AC16 human myocardial cells. To further test the roles of miR-210 in regulating the effects of isoflurane preconditioning on OGD/R-induced cardiomyocyte injury, AC16 cells were transfected with anti-miR-210 or control anti-miRNA. Results showed that isoflurane preconditioning attenuated OGD/R-induced cardiomyocyte cytotoxicity (as assessed by cell viability, LDH and CK-MB levels), which could be reversed by anti-miR-210. Isoflurane preconditioning also prevented OGD/R-induced increase in apoptotic rate, caspase-3 and caspase-9 activities, and Bax level and decrease in Bcl-2 expression level, while anti-miR-210 blocked these effects. We also found that anti-miR-210 prevented the inhibitory effects of isoflurane preconditioning on OGD/R-induced decrease in adenosine triphosphate content; mitochondrial volume; citrate synthase activity; complex I, II, and IV activities; and p-DRP1 and MFN2 expression. Besides, the expression of BNIP3, a reported direct target of miR-210, was significantly decreased under hypoxia condition and could be regulated by isoflurane preconditioning. In addition, BNIP3 knockdown attenuated the effects of miR-210 silencing on the cytoprotection of isoflurane preconditioning. These findings suggested that isoflurane preconditioning exerted protective effects against OGD/R-induced cardiac cytotoxicity by regulating the miR-210/BNIP3 axis.

Trifolirhizin targets PTK6 to induce autophagy and exerts antitumor effects in nasopharyngeal carcinoma.

Trifolirhizin, a natural flavonoid glycoside, has been proved to exert antitumor activities in various human malignant tumors. PTK6 was identified as a direct target of trifolirhizin based on public database SuperPred (https://prediction.charite.de/). Overexpressed PTK6 in a variety of tumors is closely associated with the malignant development of tumors. Herein, this present research was formulated to elaborate the effects of trifolirhizin on the biological behaviors of nasopharyngeal carcinoma (NPC) cells and to probe into the intrinsic mechanisms. The current study firstly elucidated the tumor-inhibiting functions of trifolirhizin in NPC malignant progression from the perspective of targeting inhibition of PTK6. In this work, CCK-8 for cell viability, EdU staining for cell proliferation, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for cell apoptosis and immunofluorescence staining for LC3 expression were performed. Besides, levels of proliferation-related, apoptosis-related and autophagy-related proteins were detected by western blot analysis. Moreover, molecular docking of trifolirhizin with PTK6 was conducted to seek the compound-protein binding potential. It was demonstrated that trifolirhizin treatment inhibited the proliferation and promoted the apoptosis of NPC cells as well as strengthened autophagy in NPC cells. Furthermore, it was verified that trifolirhizin targeted PTK6 and negatively regulated PTK6 expression. The suppressive effects of trifolirhizin on the malignant behaviors of NPC cells and the enhancing effect of trifolirhizin on autophagy in NPC cells were partly abolished upon upregulation of PTK6. To conclude, findings suggested that trifolirhizin may downregulate PTK6 expression to induce autophagy and exert the antitumor activities in NPC.

MiRNA-driven sensitization of breast cancer cells to Doxorubicin treatment following exposure to low dose of Zinc Oxide nanoparticles.

The impact of Engineered nanomaterials (ENMs) (i.e., Zinc Oxide nanoparticles (ZnO NPs)) on human health has been investigated at high and unrealistic exposure levels, overlooking the potential indirect harm of subtoxic and long exposures. Therefore, this study aimed to investigate the impacts of subtoxic concentrations of zinc oxide (ZnO NPs) on breast cancer cells' response to Doxorubicin. Zinc oxide nanoparticles caused a concentration-dependent reduction of cell viability in multiple breast cancer cell lines. A subtoxic concentration of 1.56µg/mL (i.e., no observed adverse effect level) was used in subsequent mechanistic studies. Molecularly, miRNA profiling revealed significant downregulation of 13 oncogenic miRNAs (OncomiRs) in cells exposed to the sub-toxic dose of ZnO NPs followed by doxorubicin treatment. Our comprehensive bioinformatic analysis has identified 617 target genes enriched in ten pathways, mainly regulating gene expression and transcription, cell cycle, and apoptotic cell death. Several tumor suppressor genes emerged as validated direct targets of the 13 OncomiRs, including TFDP2, YWHAG, SMAD2, SMAD4, CDKN1A, CDKN1B, BCL2L11, and TGIF2. This study insinuates the importance of miRNAs in regulating the responsiveness of cancer cells to chemotherapy. Our findings further indicate that being exposed to environmental ENMs, even at levels below toxicity, might still modulate cancer cells' response to chemotherapy, which highlights the need to reestablish endpoints of ENM exposure and toxicity in cancer patients receiving chemotherapeutics.

MiRNA-519e promotes cardiomyocyte cell division accompanied by pro-angiogenic and anti-apoptotic effects

Abstract Background MicroRNAs have the therapeutic potential for heart repair after myocardial infarction(MI). Using a functional high-throughput screening approach, we identified microRNA-519e(miR-519e) as an inducer of cell-cycling in human-derived iPSC-cardiomyocytes(hiPSC-­CM). Purpose This study examines miR-519e for cell division in hiPSC-­CM, additional beneficial functions in heart-related non-CMs, and direct target identification and validation in vitro and in vivo. Methods MiR-519e-mimics or miR-scrambled(miR-scr) were transfected into hiPSC-­CM, human aortic endothelial cells(HAEC) and human cardiac fibroblasts(cFB). Proliferative markers (EdU(5-Ethynyl-2'- deoxyuridine), H3P(Phospho-Histone H3) and AURKB(Aurora B-kinase)) and apoptotic response using MTT assay after exposure to doxorubicin were assessed in hiPSC­-CMs. Angiogenic capacity of HAEC was evaluated by tube formation assay and fibrotic response of cFBs using EdU and α-SMA(smooth muscle actin) staining. In vivo, a mouse MI model with ligation of the left anterior descending followed by intramyocardial injection of miR-519e or miR-scr was used. Direct target identification of miR-519e was assessed using in silico analysis combined with RNA-Seq of miR-519e-mimic treated hiPSC-CMs and validated using Ago2-RNA immunoprecipitation(RIP) followed by qPCR. Results After transfection of miR-519e-mimics in hiPSC-­CM, a significant proliferative response indicative of cell division was observed in immunofluorescence staining and on protein level as compared with miR-scr treated hiPSC-CMs (EdU, H3P and AURKB, p<0.01). Apoptosis was blunted in miR-519e-treated hiPSC-CMs (p<0.05). MiR-519e-treatment of HAEC resulted in more meshes (p<0.01) and longer tube length (p<0.05), indicating enhanced pro-angiogenic capacity. Importantly, EdU-uptake and α-SMA were similar in miR-519e-treated cFB as compared to miR-scr, suggesting neutral effects of miR-519e on myofibroblast transition. In mice undergoing MI, injection of miR-519e-mimics significantly downregulated CDKN1A/P21(Cyclin dependent kinase inhibitor 1A) (p<0.05 vs. miR-scr), a direct target of miR-519e and negative master regulator of cell-cycling. For further novel target identification of miR-519e, we performed in silico analysis combined with RNA-Seq data from miR-519e-treated hiPSC-CM and validation using Ago2-RIP followed by qPCR. Herein, we identified PTEN(Phosphatase and tensin homolog) and TGFBR2(Transforming growth factor β receptor 2) that are involved in cardiomyocyte proliferation and hypoxic response, as direct targets of miR-519e. Conclusion Collectively, miR-519e is a potent inducer of cell division in human cardiomyocytes, with anti-apoptotic and pro-angiogenic capacity in the absence of pro-fibrotic effects. We show that miR-519e treatment downregulates key negative cell-cycle regulators in vivo and we identified novel direct targets of miR-519e that are involved in regulatory pathways for heart regeneration.

GULP1 as a Downstream Effector of the Estrogen Receptor-β Modulates Cisplatin Sensitivity in Bladder Cancer.

Precise molecular mechanisms underlying resistance to cisplatin-based chemotherapy remain unclear, while the activity of estrogen receptor-β (ERβ) has been suggested to be associated with chemosensitivity in urothelial cancer. We aimed to determine if GULP1, an adapter protein known to facilitate phagocytosis, could represent a downstream effector of ERβ and thereby modulate cisplatin sensitivity in bladder cancer. GULP1 expression and cisplatin cytotoxicity were compared in bladder cancer lines. Immunohistochemistry was used to determine the expression of GULP1 and ERβ in two sets of tissue microarray (TMA) consisting of transurethral resection specimens. The levels of GULP1 expression were considerably higher in ERβ-knockdown sublines than in the respective control ERβ-positive sublines. Estradiol treatment reduced GULP1 expression in ERα-negative/ERβ-positive lines, which was restored by the anti-estrogen tamoxifen. Chromatin immunoprecipitation assay revealed the binding of ERβ to the GULP1 promoter in bladder cancer cells. Moreover, GULP1 knockdown sublines were significantly more resistant to cisplatin treatment, but not to other chemotherapeutic agents, including gemcitabine, methotrexate, vinblastine, and doxorubicin. In the first set of TMA (n=129), the expression of ERβ and GULP1 was inversely correlated (p=0.023), and ERβ(-)/GULP1(+) in 51 muscle-invasive tumors was associated with significantly lower risk of disease progression and cancer-specific mortality. Similarly, in the second set (n=43), patients with ERβ(-)/GULP1(+) muscle-invasive disease were significantly (p=0.021) more likely to be responders to cisplatin-based neoadjuvant chemotherapy before radical cystectomy. ERβ activation was found to reduce the expression of GULP1 as a direct downstream target in bladder cancer cells, resulting in the induction of cisplatin resistance.

Serum exosomal long non-coding RNA H19 as a theragnostic target for atrial fibrillation

Abstract Background/Introduction Exosomes are membrane vesicles of 30- to 150-nm secreted by most cell types that can mediate intercellular communication by transmitting various forms of RNAs. Long non-coding RNAs (lncRNAs, ≥ 200 nucleotides length) have been reported to play important roles in the pathophysiological processes that promote the development and progression of many diseases. Interestingly, recent studies have shown that exosomal lncRNAs exhibit different expression profiles in various diseases and are being extensively studied in the medical field as an ideal target for the diagnosis and treatment. However, there is still little known about their role in atrial fibrillation (AF). Purpose The aim of this study was to identify a novel theragnostic target exosomal lncRNA for AF. Methods First, serum exosomes from controls and patients with persistent AF were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Next, serum exosomal lncRNA expression profiles were explored using RNA-sequencing analysis. In addition, the expression levels of candidate exosomal lncRNAs were confirmed using qRT-PCR (n = 50 per group). Finally, AF-related pathophysiological mechanisms of exosomal lncRNA were investigated in angiotensin II (Ang II)-treated human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-atrial CMs). Results After RNA-sequencing analysis, we identified 27 differentially expressed lncRNAs (i.e., 4 upregulated and 23 downregulated lncRNAs with a |fold change| ≥ 2 and p < 0.05) in serum exosomes from patients with persistent AF compared with the controls. In fact, qRT-PCR reveled that exosomal lncRNA H19 was consistently downregulated in the serum of patients with persistent AF compared with the controls (p < 0.01). In addition, exosomal lncRNA H19 exhibited significant diagnostic validity for AF. Notably, we confirmed that exosomal lncRNA H19 was involved in AF-related pathophysiological mechanisms. In Ang II-treated iPSC-atrial CMs, inhibition of lncRNA H19 significantly aggravated Ang II-induced increases in levels of hypertrophic markers (ANP, BNP and β-MHC), cell surface area and inflammation (p < 0.05). Mechanistically, we found that loss of lncRNA H19 weakens the inhibition of its direct target microRNAs, miR-141-3p and miR-200a-3p, leading to downregulation of their target PTEN, thereby promoting the atrial remodeling and the consequent AF. Conclusion In conclusion, serum-derived exosomal lncRNA H19 could serve as a potential target for the diagnosis and treatment of AF.

MiR-144/451 attenuates lipopolysaccharide-induced lung inflammation by downregulating Rac1 and STAT-3 in macrophages.

MicroRNAs have been shown to play a critical role in lung inflammatory diseases. Here, we report that knocking out miR-144/451 in mice exacerbates lipopolysaccharide (LPS)-induced lung inflammation. The lung inflammation in mice was induced by intratracheal instillation of LPS. Loss-of-function experiments demonstrated that miR-144/451 gene knockout (KO) increased LPS-induced lung inflammation and oxidant stress compared with wild-type (WT) mice, as manifested by increased total bronchoalveolar lavage fluid cells and neutrophil counts, elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid, enhanced myeloperoxidase activity, and reduced catalase and glutathione peroxidase activity in lung tissues. We also found that LPS significantly decreased miR-451 expression in lung tissues and macrophages; while miR-451 overexpression in LPS-induced RAW264.7 cells remarkably reduced TNF-α and IL-6 levels as well as reactive oxygen species (ROS) production, suggesting a feedback loop might exist in inflammatory cells. Rac1 mRNA and protein levels were downregulated in miR-451-overexpressed RAW264.7 cells. Ex vivo stimulation experiments, performed using alveolar macrophages isolated from miR-144/451 KO mice, confirmed that Rac1 inhibitor alleviated levels of TNF-α and ROS in response to LPS stimulation compared with WT controls. Luciferase reporter assay demonstrated that STAT-3 is a direct target of miR-451. STAT-3 protein levels were elevated in miR-144/451 KO macrophages. LPS treatment also resulted in higher phosphorylation levels of STAT-3 in macrophages from KO mice than in WT cells. Our study identified miR-144/451 as an anti-inflammatory factor in LPS-induced lung inflammation that acts by downregulating Rac1 and STAT-3.

METTL3 drives heart failure by regulating Spp1 and Fos m6A modification in myocardial infarction

Abstract Background While m6A modification has been reported in myocardial infarction (MI), the detailed mechanism by which METTL3 regulates the progression of the disease has not yet been elucidated, and it remains unclear why m6A modification increases after MI. Purpose To investigate the role and mechanism of METTL3 in regulating its targets through m6A methylation in MI. The research results will not only add new content to the basic mechanism of myocardial protection but also provide new ideas and new targets for the prevention and treatment of MI. Methods Through MeRIP-seq and extensive bioinformatics analysis, the target genes SPP1 and FOS with the most significant m6A modification and differential expression in MI were screened. We successfully constructed heart specific Mettl3 knockout mice (Mettl3CKO) to verify that METTL3 promotes the deterioration of cardiac function after MI. We performed complementary molecular methods to assess protein quantity and interactions to identify mechanisms regulating this response. We manipulated select molecular pathways using both genetic and pharmacological methods to validate these mechanisms. Results Here, we showed that METTL3 exerted methyltransferase activity-dependent functions in gene regulation in MI, and a significant transcription factor HuR assisted the function of METTL3. and demonstrated that METTL3 was critical for the promotion of heart failure after MI. More specifically, METTL3 directly interacted with HuR through its nuclear localization domain in the cell nucleus under normoxia condition. When hypoxia developed, METTL3 separated from HuR and deposited m6A into 5’UTR of Spp1 and Fos mRNA to maintain their stability. In contrast, HuR bound to the ARE domain of 3’UTR of Spp1 and Fos mRNA to take them to the cytosol, maintaining their stability. Moreover, HIF-1α directly interacted with the HRE domain of Mettl3 to promote its transcription, and HuR bound to the ARE domain of 3’UTR of Mettl3 mRNA to maintain its stability to promote following translation. Conclusions Collectively, our studies revealed previously unappreciated functions of METTL3 with the help of HuR, and a direct target of HIF-1α under normoxia condition, which together contribute to its essential function in MI, suggesting therapeutic potential for targeting the METTL3/HuR/Spp1(Fos) mRNA axis.Figure1

MiR-124-3p inhibits cell stemness in glioblastoma via targeting EPHA2 through ALKBH5-mediated m6A modification.

Glioblastoma (GBM) is the most aggressive form of glioma, characterized by high mortality and poor prognosis. Dysregulation of microRNAs (miRNAs) plays a critical role in the progression and metastasis of GBM. This study aimed to investigate the role and molecular mechanism of miR-124-3p in GBM. Levels of miR-124-3p, EPHA2, and ALKBH5 were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasion, and stemness were assessed using the Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and sphere formation assays, respectively. Bioinformatics prediction, dual-luciferase reporter assays, and RNA pull-down experiments were employed to validate the target of miR-124-3p. RNA binding protein immunoprecipitation (RIP) and methylated RNA immunoprecipitation (Me-RIP) were utilized to evaluate the regulation of miR-124-3p maturation by ALKBH5. The results indicated that overexpression of miR-124-3p inhibited the proliferation, migration, invasion, and stemness of GBM cells. EPHA2 was identified as a direct downstream target of miR-124-3p, and its overexpression reversed the inhibitory effects of miR-124-3p on cellular functions. Furthermore, miR-124-3p targeted EPHA2 to inactivate the Wnt/β-catenin pathway. Additionally, ALKBH5 negatively regulated miR-124-3p by impeding its processing. In conclusion, knockdown of ALKBH5 promoted the processing of pri-miR-124-3p, increasing mature miR-124-3p levels, which inhibited the malignant behaviors of GBM cells by targeting EPHA2. These findings highlight the importance of the ALKBH5/miR-124-3p/EPHA2 axis in GBM.

Long Non-coding RNA FOXD2-AS1 Silencing Inhibits Malignant Behaviors of Ovarian Cancer Cells Via miR-324-3p/SOX4 Signaling Axis.

It is urgent to develop new therapeutic strategies for ovarian cancer (OC). Long-noncoding RNAs (lncRNAs) have participated in multiple biological processes including tumor recurrence and progression. This study aimed to determine the effects and potential regulatory mechanism of lncRNA FOXD2-AS1 in OC progression. Levels of lncRNA FOXD2-AS1 and miR-324-3p in OC tissues and cell lines were analyzed using quantitative real-time PCR (qRT-PCR). The direct target between FOXD2-AS1 or miR-324-3p was determined using bioinformatics tools and further verified by dual-luciferase reporter assay. Cell viability, apoptosis, migration, along invasion were assessed by MTT, flow cytometry, as well as Transwell assays, respectively. In addition, the levels of miR-324-3p, PCNA, MMP9, Bax, Bcl-2, and SOX4 in OC cells were evaluated using qRT-PCR and western blot assays. We observed that lncRNA FOXD2-AS1 was up-regulated while miR-324-3p was down-regulated in OC tissues and cell lines, especially in SKOV3 cells. Moreover, miR-324-3p was a direct target of lncRNA FOXD2-AS1. Meanwhile, SOX4 interacted with miR-324-3p and was negatively regulated by miR-324-3p in SKOV3 cells. Function assays confirmed that lncRNA FOXD2-AS1 silenced depressed cell proliferation, migration, and invasion while accelerating apoptosis. These functions of lncRNA FOXD2-AS1 were attenuated by miR-324-3p inhibition. Our research demonstrated that FOXD2-AS1 silencing restrained cell growth and metastasis of OC via regulating miR-324-3p/SOX4 axis, indicating that lncRNA FOXD2-AS1 could be a novel potential therapeutic target for OC.

Decreased number of satellite cells-derived myonuclei in both fast- and slow-twitch muscles in HeyL-KO mice during voluntary running exercise

BackgroundSkeletal muscles possess unique abilities known as adaptation or plasticity. When exposed to external stimuli, such as mechanical loading, both myofiber size and myonuclear number increase. Muscle stem cells, also known as muscle satellite cells (MuSCs), play vital roles in these changes. HeyL, a direct target of Notch signaling, is crucial for efficient muscle hypertrophy because it ensures MuSC proliferation in surgically overloaded muscles by inhibiting the premature differentiation. However, it remains unclear whether HeyL is essential for MuSC expansion in physiologically exercised muscles. Additionally, the influence of myofiber type on the requirement for HeyL in MuSCs within exercised muscles remains unclear.MethodsWe used a voluntary wheel running model and HeyL-knockout mice to investigate the impact of HeyL deficiency on MuSC-derived myonuclei, MuSC behavior, muscle weight, myofiber size, and myofiber type in the running mice.ResultsThe number of new MuSC-derived myonuclei was significantly lower in both slow-twitch soleus and fast-twitch plantaris muscles from exercised HeyL-knockout mice than in control mice. However, expect for the frequency of Type IIb myofiber in plantaris muscle, exercised HeyL-knockout mice exhibited similar responses to control mice regarding myofiber size and type.ConclusionsHeyL expression is crucial for MuSC expansion during physiological exercise in both slow and fast muscles. The frequency of Type IIb myofiber in plantaris muscle of HeyL-knockout mice was not significantly reduced compared to that of control mice. However, the absence of HeyL did not affect the increased size and frequency of Type IIa myofiber in plantaris muscles. In this model, no detectable changes in myofiber size or type were observed in the soleus muscles of either control or HeyL-knockout mice. These findings imply that the requirement for MuSCs in the wheel-running model is difficult to observe due to the relatively low degree of hypertrophy compared to surgically overloaded models.

A derivative of tanshinone IIA and salviadione, 15a, inhibits inflammation and alleviates DSS-induced colitis in mice by direct binding and inhibition of RIPK2.

Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions primarily affecting the gastrointestinal tract. Previous studies established the role of the NF-κB signaling pathway in the development of IBDs, suggesting that anti-inflammatory therapies might offer a viable treatment strategy. Tanshinone IIA and salviadione, both derived from Salviae Miltiorrhizae Radix et Rhizoma, possess anti-inflammatory and anti-oxidative activities. A series of new compounds were synthesized by hybridizing salviadione with tanshinone. Among these compounds, 15a showed beneficial effects in LPS-induced acute lung injury and diabetes-induced renal injury mouse models. The current study explored the therapeutic efficacy of 15a using both acute and chronic colitis models and elucidated the underlying mechanisms. DSS-induced colitis models were established in mice, where acute colitis was treated with compound 15a (5 or 10 mg·kg-1·d-1) for 8 days, while chronic colitis mice received compound 15a (5 or 10 mg·kg-1·d-1, i.g.) during 2.5% DSS administration. The 15a treatment significantly alleviated DSS-induced pathological and inflammatory damages in both acute and chronic colitis mouse models. In mouse intestinal epithelial cell line MODE-K, pretreatment with compound 15a (5 or 10 μM) significantly suppressed LPS + L18-MDP-induced inflammatory responses. The receptor-interacting serine/threonine kinase 2 (RIPK2) was identified as a direct binding target of compound 15a using microarrays and recombinant human proteins. Moreover, 15a could directly bind to and inhibit the phosphorylation of RIPK2, leading to the suppression of the NF-κB and MAPK signaling pathways. Furthermore, LEU153 and VAL32 were identified within the KD domain of RIPK2 as critical amino residues for the binding of 15a. Briefly, the current findings demonstrate that compound 15a holds promise as a therapeutic agent for managing acute and chronic colitis.

Loss of miR-634 contributes to the formation FOXA1-positive triple negative breast cancer subtype

BackgroundTriple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, lacks targeted therapies, posing a substantial challenge for treatment. Therefore, investigating its pathogenesis is a crucial research focus. FOXA1 and miR-634 are involved in tumorigenesis. However, the molecular mechanisms underlying the aberrant upregulation of FOXA1 expression in TNBC remain unclear. Therefore, we explore the role of miR-634 in the FOXA1-positive TNBC subtype.MethodsQuantitative reverse transcription polymerase chain reaction was used to detect miR-634 expression in breast cancer tissues and cell lines. Aberrantly activated signaling pathways and related genes in TNBC were analyzed using The Cancer Genome Atlas. The potential target of miR-634 was predicted by TargetScan, the TNBC cell proliferation rate was detected using an MTT assay, the in vitro metastatic capacity was determined by transwell assay and the cell cycle distribution was tested using flow cytometry. Western blotting was performed to measure the expression of proteins involved in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway.ResultsThe expression of miR-634 was significantly down-regulated in both TNBC tissues and cells, compared with adjacent non-cancerous tissues and MCF10A, respectively. Ectopic expression of miR-634 inhibits breast cancer cell proliferation and in vitro metastasis. The TCGA-based expression profile analysis of TNBC revealed that aberrantly activated PI3K/AKT signaling may contribute to its malignant phenotype. FOXA1, the top hit of aberrantly upregulated genes in TNBC, was a direct target of miR-643. Moreover, forced expression of miR-643 drastically suppressed FOXA1 expression by the inactivation PI3K/AKT signaling.ConclusionMiR-634 suppresses FOXA1 to inhibit the proliferation and metastasis of TNBC cells by inactivating the PI3K/AKT signaling pathway.

PD-L1 Acts Independently of PD-1 as a Marker of Pathologic Fibroblasts in Laryngotracheal Stenosis.

Laryngotracheal stenosis (LTS) describes fibrotic airway obstruction that is life-threatening without treatment. Targeted therapies are needed as an adjunct to surgical management. We have previously observed the upregulation of immune checkpoint programmed cell death (PD)-1 and its ligand, PD-L1, in patients with LTS. This study aims to determine whether PD-1 and PD-L1 play a role in the pathophysiology of LTS. Basic science. Laboratory. Fibroblasts derived from the subglottic scar of 5 iSGS patients were cultured ex vivo with transforming growth factor β (TGFβ), PD-L1 agonist (PD-1), and PD-L1 blockade (anti-PD-L1). PD-L1, TGFβ receptor II (TGFβRII), and Collagen-1 expression were quantified by flow cytometry. A validated chemomechanical injury model of subglottic stenosis was applied in PD-1 knockout and wild-type (WT) mice, and subglottic thickening was assessed by histologic analysis. TGFβ significantly increased the expression of PD-L1 and Collagen-1 in human airway scar fibroblasts (P < .05). PD-1 knockout mice demonstrated no significant difference in subglottic airway fibrosis compared to WT mice. Ex vivo PD-L1 modulation had no impact on fibroblast Collagen-1 expression. PD-L1 high-intensity fibroblasts expressed greater Collagen-1 and TGFβRII compared to PD-L1 low-intensity fibroblasts. PD-1 knockout does not protect mice from the development of laryngotracheal fibrosis. However, its ligand, PD-L1 is highly expressed on pathologic fibroblasts unique to scar, characterized by high Collagen-1 and TGFβRII expression. PD-L1 is also upregulated in conjunction with Collagen-1 by TGFβ stimulation. PD-L1 may act independently of PD-1 to sensitize fibroblasts to TGFβ, suggesting direct targeting of PD-L1 may have therapeutic potential in LTS.

MALAT1/miR-582-5p/GALNT1/MUC1 axis modulates progression of AML leukemia stem cells by regulating JAK2/STAT3 pathway.

Acute myeloid leukemia (AML) is characterized by uncontrolled clonal expansion and differentiation block of immature myeloid cells. Some studies have shown that leukemia stem cells (LSC) are thought to be responsible for the initiation and development of leukemia. Moreover, abnormal O-glycosylation is a key modification in the process of cancer malignancy. In this study, GALNT1 expression was significantly upregulated in LSCs, while knockdown of GALNT1 inhibited cell viability and promoted apoptosis. Importantly, GALNT1 was the direct target of miR-582-5P, and MALAT1 directly interacted with miR-582-5P. In addition, Our investigation corroborated that MALAT1 functioned as an endogenous sponge of miR-582-5P to regulate mucin1 (MUC1) expression, catalyzed by GALNT1, which modulated the activity of JAK2/STAT3 pathway. MALAT1 and MUC1 were targets of transcription factor STAT3 and were regulated by STAT3. In general, these new findings indicated that MALAT1/miR-582-5P/GALNT1 axis is involved in the progression of LSCs, illuminating the possible mechanism mediated by O-glycosylated MUC1 via JAK2/STAT3 pathway.

Predicting and Characterizing piRNAs and their functions Using an Integrated Machine Learning Approach

One class of short non-coding RNA molecule that is well recognized is called PIWI-interacting RNA (piRNA). PiRNAs are involved in the creation of novel medications as well as the identification of different kinds of tumors. Additionally, it is associated with stopping transposes, managing gene transcription, and maintaining genomic integrity. The important role that piRNAs play in biological processes has led to a growing body of research in bioinformatics on the discovery of piRNAs and their functionality. In this research, a powerful model is proposed to improve PiRNA prediction and functionality. The suggested model uses four classifiers (Logistic Regression, SVC, Random Forest, and Gradient Boosting Classifier) for classification. Moreover, TNC and DNC are used to acquire features. There are two layers involved in developing the suggested model. A sequence's potential to be piRNA is predicted in the first layer, and its potential to direct target mRNA deadenylation is predicted in the second. In the first layer, the model's accuracy is 98.59%, and in the second layer, it is 94.55%. One class of short non-coding RNA molecule that is well recognized is called PIWI-interacting RNA (piRNA). PiRNAs are involved in the creation of novel medications as well as the identification of different kinds of tumors. Additionally, it is associated with stopping transposes, managing gene transcription, and maintaining genomic integrity. The important role that piRNAs play in biological processes has led to a growing body of research in bioinformatics on the discovery of piRNAs and their functionality. In this research, a powerful model is proposed to improve PiRNA prediction and functionality. The suggested model uses four classifiers (Logistic Regression, SVC, Random Forest, and Gradient Boosting Classifier) for classification. Moreover, TNC and DNC are used to acquire features. There are two layers involved in developing the suggested model. A sequence's potential to be piRNA is predicted in the first layer, and its potential to direct target mRNA deadenylation is predicted in the second. In the first layer, the model's accuracy is 98.59%, and in the second layer, it is 94.55%. One class of short non-coding RNA molecule that is well recognized is called PIWI-interacting RNA (piRNA). PiRNAs are involved in the creation of novel medications as well as the identification of different kinds of tumors. Additionally, it is associated with stopping transposes, managing gene transcription, and maintaining genomic integrity. The important role that piRNAs play in biological processes has led to a growing body of research in bioinformatics on the discovery of piRNAs and their functionality. In this research, a powerful model is proposed to improve PiRNA prediction and functionality. The suggested model uses four classifiers (Logistic Regression, SVC, Random Forest, and Gradient Boosting Classifier) for classification. Moreover, TNC and DNC are used to acquire features. There are two layers involved in developing the suggested model. A sequence's potential to be piRNA is predicted in the first layer, and its potential to direct target mRNA deadenylation is predicted in the second. In the first layer, the model's accuracy is 98.59%, and in the second layer, it is 94.55%.

6-Gingerol Inhibits De Novo Lipogenesis by Targeting Stearoyl-CoA Desaturase to Alleviate Fructose-Induced Hepatic Steatosis

Metabolic-associated fatty liver disease (MAFLD), also known as non-alcoholic fatty liver disease (NAFLD), is a worldwide liver disease without definitive or widely used therapeutic drugs in clinical practice. In this study, we confirm that 6-gingerol (6-G), an active ingredient of ginger (Zingiber officinale Roscoe) in traditional Chinese medicine (TCM), can alleviate fructose-induced hepatic steatosis. It was found that 6-G significantly decreased hyperlipidemia caused by high-fructose diets (HFD) in rats, and reversed the increase in hepatic de novo lipogenesis (DNL) and triglyceride (TG) levels induced by HFD, both in vivo and in vitro. Mechanistically, chemical proteomics and cellular thermal shift assay (CETSA)–proteomics approaches revealed that stearoyl-CoA desaturase (SCD) is a direct binding target of 6-G, which was confirmed by further CETSA assay and molecular docking. Meanwhile, it was found that 6-G could not alter SCD expression (in either mRNA or protein levels), but inhibited SCD activity (decreasing the desaturation levels of fatty acids) in HFD-fed rats. Furthermore, SCD deficiency mimicked the ability of 6-G to reduce lipid accumulation in HF-induced HepG2 cells, and impaired the improvement in hepatic steatosis brought about by 6-G treatment in HFD supplemented with oleic acid diet-induced SCD1 knockout mice. Taken together, our present study demonstrated that 6-G inhibits DNL by targeting SCD to alleviate fructose diet-induced hepatic steatosis.

Deciphering the Landscape of GATA-Mediated Transcriptional Regulation in Gastric Cancer.

Gastric cancer (GC) is an asymptomatic malignancy in early stages, with an invasive and cost-ineffective diagnostic toolbox that contributes to severe global mortality rates on an annual basis. Ectopic expression of the lineage survival transcription factors (LS-TFs) GATA4 and 6 promotes stomach oncogenesis. However, LS-TFs also govern important physiological roles, hindering their direct therapeutic targeting. Therefore, their downstream target genes are particularly interesting for developing cancer-specific molecular biomarkers or therapeutic agents. In this work, we couple inducible knockdown systems with chromatin immunoprecipitation and RNA-seq to thoroughly detect and characterize direct targets of GATA-mediated transcriptional regulation in gastric cancer cells. Our experimental and computational strategy provides evidence that both factors regulate the expression of several coding and non-coding RNAs that in turn mediate for their cancer-promoting phenotypes, including but not limited to cell cycle, apoptosis, ferroptosis, and oxidative stress response. Finally, the diagnostic and prognostic potential of four metagene signatures consisting of selected GATA4/6 target transcripts is evaluated in a multi-cancer panel of ~7000 biopsies from nineteen tumor types, revealing elevated specificity for gastrointestinal tumors. In conclusion, our integrated strategy uncovers the landscape of GATA-mediated coding and non-coding transcriptional regulation, providing insights regarding their molecular and clinical function in gastric cancer.

Inhibition of long non-coding RNA NEAT1 suppressed the epithelial mesenchymal transition through the miR-204-5p/Six1 axis in asthma.

Asthma, a prevalent chronic respiratory condition, is characterized by airway remodeling. Long non-coding RNA (lncRNA) NEAT1 has been demonstrated to participate in airway fibrosis. Furthermore, the miR-204-5p/Six1 axis significantly influences epithelial mesenchymal transition (EMT). However, the function of NEAT1/miR-204-5p/Six1 in asthmatic EMT remains unclear. This study intends to elucidate the function of NEAT1/miR-204-5p/Six1 axis in asthmatic EMT. TGF-β1 was used to induce the EMT model in BEAS-2B cells. Immunofluorescence and western blot were executed to verify the establishment of the EMT model. NEAT1, miR-204-5p, and Six1 expression levels were evaluated using RT-qPCR. The role of NEAT1 in EMT in vitro was explored by CCK8 assays and flow cytometry. The luciferase reporter assay was performed to validate the interaction between NEAT1 and miR-204-5p/Six1. NEAT1 expression was increased during EMT. Functional experiments showed that the knockdown of NEAT1 suppressed cell proliferation and promoted cell apoptosis in vitro. Furthermore, inhibition of NEAT1 decreased the expression of N-cadherin, vimentin, and α-SMA and increased the expression of E-cadherin. Mechanistically, NEAT1 was identified as a sponge for miR-204-5p, and Six1 was found to be a direct target of miR-204-5p. Down-regulation of NEAT1 reduced the Six1 expression via targeting miR-204-5p to inhibit the process of EMT in asthma. This study may provide new insight to reveal the underlying mechanisms of asthma.

STOP1 dominates Arabidopsis tolerance to ammonium over NRT1.1/NPF6.3/CHL1

ABSTRACT The Arabidopsis nitrate transceptor NITRATE TRANSPORTER 1.1 (NRT1.1/NPF6.3/CHL1) plays significant roles even in the absence of nitrate. The loss-of-function of NRT1.1 alleviates growth suppression and chlorosis under toxic levels of ammonium as the sole nitrogen source. Recently, we reported that acidic stress-inducible genes are downregulated by NRT1.1 deficiency under ammonium toxicity conditions, implying that NRT1.1 may exacerbate ammonium-dependent acidic stress. The transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) enhances Arabidopsis tolerance to ammonium and acidic stresses. Furthermore, STOP1 directly activates NRT1.1 transcription. These previous findings prompted us to analyze the association between NRT1.1 and STOP1 on ammonium tolerance. Here, we show that ammonium-inducible STOP1 plays a dominant role over NRT1.1 in ammonium tolerance and expression of direct target genes of STOP1. We present a novel scheme in which NRT1.1 and STOP1 jointly regulate common target(s) associated with ammonium tolerance.