Notch1 Axis Research Articles

MiR-137 inhibits cervical cancer progression via down-modulating Notch1 and inhibiting the PI3K/AKT/mTOR signaling pathway

BackgroundCervical cancer (CC) is frequently diagnosed, and accounts for a large proportion of cancer-related mortality worldwide. Therefore, it is an urgent need to explore the mechanisms of CC progression and seek new therapeutic targets. We know that microRNA-137 (miR-137) is involved in CC progression; however, it’s mechanism in CC has not yet been clarified.MethodsQuantitative real-time PCR (qRT-PCR) was applied to detect the expressions of miR-137 and Notch homolog 1 messenger RNA (Notch1 mRNA) in CC cells. Cell Counting Kit-8 (CCK-8) and Transwell assay were utilized to monitor the proliferation, invasion, and migration of CC cells. The binding relationship of miR-137 with Notch1 was detected by dual luciferase reporter gene assay. Western blot was used to determine the expressions of Notch1, p-AKT/AKT, and p-mTOR/mTOR signaling pathways.ResultsThe results showed that miR-137 was down-regulated and Notch1 was up-regulated in CC cells. MiR-137 was reversely correlated with Notch1, and Notch1 partially reversed the promotion of miR-137 silencing on malignant phenotype of CC cells. Furthermore, in vitro experiments confirmed that miR-137 partially impeded CC progression via regulation of the Notch1 and PI3K/AKT/mTOR axis.ConclusionsMiR-137 targets Notch1 and regulates CC through the PI3K/Akt/mTOR pathway. The miR-137/Notch1 axis may be a promising target for CC patients, and these findings can provide a reference for the clinical treatment of CC.

Cr(VI) promotes tight joint and oxidative damage by activating the Nrf2/ROS/Notch1 axis

This study aimed to investigate whether Cr(VI) induced tight joint and oxidative damage in the small intestine, as mediated by the nuclear factor erythroid 2-related factor 2 (Nrf2)/reactive oxygen species (ROS)/Notch1 axis crosstalk. Thirty-two ICR mice were obtained and subjected to Cr(VI) via intragastric administration daily for 5 days. Western blot (WB) analysis, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) staining, and immunofluorescence (IF) staining were applied to detect small intestinal damage, Nrf2, Notch1, and respective downstream targets in this research. Results showed that Cr(VI) led to the tight joint and oxidative damage in the small intestine of mice. Nrf2 was stimulated, and Notch1 (Notch intracellular domain, NICD1) was activated to translocate into the nucleus and activate an antioxidant action. These findings were validated by WB analysis and IF staining. ROS levels increased as the Cr(VI) concentration increased. The colocalization analysis of Nrf2 and NICD1 implied that a crosstalk between Nrf2 and Notch1 existed. Therefore, this study indicated that the Nrf2/ROS/Notch1 axis crosstalk could aggravate the tight joint and oxidative damage in the small intestine after Cr(VI) treatment.

Linc00641 promotes the progression of gastric carcinoma by modulating the miR-429/Notch-1 axis.

Linc00641 plays different roles in various types of human cancers. However, the function of linc00641 and its underlying mechanism of action in gastric cancer have not been fully elucidated. Therefore, the aim of our current study was to explore the molecular mechanism of linc00641 in gastric cancer. MTT assays, flow cytometry, wound healing assays, and Transwell invasion assays were utilized to measure cell viability, apoptosis, migration and invasion, respectively. Western blotting and RT-PCR assays were carried out to explore the mechanism of linc00641 in gastric cancer cells. We found that silencing linc00641 suppressed the viability and stimulated the apoptosis of gastric cancer cells, while linc00641 overexpression had the opposite effects. Moreover, linc00641 sponged the expression of miR-429 and subsequently upregulated Notch-1 expression in gastric cancer cells. We concluded that linc00641 promoted the malignant progression of gastric cancer by modulating the miR-429/Notch-1 axis.

A Procarcinogenic Colon Microbe Promotes Breast Tumorigenesis and Metastatic Progression and Concomitantly Activates Notch and β-Catenin Axes.

The existence of distinct breast microbiota has been recently established, but their biological impact in breast cancer remains elusive. Focusing on the shift in microbial community composition in diseased breast compared with normal breast, we identified the presence of Bacteroides fragilis in cancerous breast. Mammary gland as well as gut colonization with enterotoxigenic Bacteroides fragilis (ETBF), which secretes B. fragilis toxin (BFT), rapidly induces epithelial hyperplasia in the mammary gland. Breast cancer cells exposed to BFT exhibit "BFT memory" from the initial exposure. Intriguingly, gut or breast duct colonization with ETBF strongly induces growth and metastatic progression of tumor cells implanted in mammary ducts, in contrast to nontoxigenic Bacteroides fragilis. This work sheds light on the oncogenic impact of a procarcinogenic colon bacterium ETBF on breast cancer progression, implicates the β-catenin and Notch1 axis as its functional mediators, and proposes the concept of "BFT memory" that can have far-reaching biological implications after initial exposure to ETBF. SIGNIFICANCE: B. fragilis is an inhabitant of breast tissue, and gut or mammary duct colonization with ETBF triggers epithelial hyperplasia and augments breast cancer growth and metastasis. Short-term exposure to BFT elicits a "BFT memory" with long-term implications, functionally mediated by the β-catenin and Notch1 pathways.This article is highlighted in the In This Issue feature, p. 995.

A Novel PAK1-Notch1 Axis Regulates Crypt Homeostasis in Intestinal Inflammation.

Background & Aimsp21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/β-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice.MethodsIL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT.ResultsWhen compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein–coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased β-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation.ConclusionsPAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1–Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

Long noncoding RNA HCG18 inhibits the differentiation of human bone marrow-derived mesenchymal stem cells in osteoporosis by targeting miR-30a-5p/NOTCH1 axis

BackgroundRecent studies have demonstrated that long non-coding RNAs (LncRNAs) can influence bone cell differentiation and formation. However, it is unclear whether lncRNA HCG18 is involved in osteoporosis (OP). This study was conducted to investigate the regulation of HCG18 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsBMSCs were isolated and cultured from mouse pathological models and osteoporosis patients. RT-qPCR was performed to detect the expression of HCG18 and miR-30a-5p in BMSCs. The interaction between HCG18 and miR-30a-5p was analyzed by dual luciferase assay and RNA pulldown assay. The interaction between miR-30a-5p and NOTCH1 3′-UTR was analyzed by dual luciferase assay. RT-qPCR and Western blotting were used to detect the expression of osteogenic genes Runx2, OCN and OPN. Hindlimb-unloaded (HU) mice model was established, and HCG18 was knocked down on bone-formation surfaces by using lentivirus mediated shRNA transfection.ResultsThe expression of HCG18 was increased in BMSCs of OP patients, while the expression of miR-30a-5p was decreased. The expression of HCG18 and miR-30a-5p was negatively correlated in BMSCs. During the differentiation from BMSCs to osteoblasts, the expression of HCG18 was significantly downregulated, and the expression of miR-30a-5p was significantly upregulated. Overexpression of HCG18 was able to reverse the osteogenic-induced upregulation of miR-30a-5p expression, and knockdown of HCG18 further promoted the expression of miR-30a-5p. In addition, miR-30a-5p partially abolished the effect of HCG18 on osteogenic differentiation of BMSCs. NOTCH1 was a target protein of miR-30a-5p, and upregulation of NOTCH1 reversed the effect of miR-30a-5p on osteogenic differentiation of BMSCs. Furthermore, this study found that lentivirus mediated HCG18 knockdown on the bone-formation surfaces of hindlimb-unloaded (HU) mice partially alleviated unloading-induced bone lossConclusionsHCG18 inhibited osteogenic differentiation of BMSCs induced by OP via the miR-30a-5p/NOTCH1 axis. HCG18 can be identified as a regulator of osteogenic differentiation of BMSCs.

Xiaoai Jiedu Recipe suppresses hepatocellular carcinogenesis through the miR-200b-3p /Notch1 axis.

PurposeXiaoai Jiedu recipe (XJR), a formula long used by Chinese National Medical Professor Zhou Zhongying, has potent antitumor properties, but the molecular mechanism is still unclear. The aim of the study was to investigate the antitumor mechanism of XJR on hepatocellular carcinoma (HCC) by focusing on miRNA.MethodsThree concentrations of XJR (low, middle, and high) were used to treat tumor xenograft mice models. Microarray technology was used to identify the differential expressed genes after XJR treatment, and bioinformatic tools and luciferase reporter assay to predict the potential pathways. HepG2 cells were transfected with inhibitor of miR-200b-3p to detect the effect of miR-200b-3p and Notch1 on tumor growth.ResultsXJR effectively exerted anti-HCC effect both in vitro and in vivo. MiRNA chip analysis results showed that the expression of 75 miRNAs was upregulated and 158 miRNAs was downregulated in blood from XJR-treated mice. Further validation by using real-time polymerase chain reaction (RT-PCR) assay showed that the expression of five miRNAs (miR-453, miR-200b-3p, miR-135a-1-3p, miR-1960, miR-378a-5p, and miR-466f) was consistent with the results of miRNA chip analysis. Among them, miR-200b-3p was selected as candidate for further research. Results of the MTT, migration, and wound healing assays showed that down-expression of miR-200b-3p abrogated the effect of XJR on cell growth and metastasis. Luciferase reporter assay confirmed that Notch1 was the direct target of miR-200b-3p. XJR significantly decreased Notch1 expression in HepG2 cells, whereas miR-200B-3p inhibitor abrogated the XJR-induced decrease in Notch1 expression.ConclusionThis study indicated that XJR could effectively inhibit HCC and might exert its antitumor effect through the miR‐200b-3p/Notch1 axis. These findings provided new avenues for the use of XJR for prevention and treatment of HCC.

LncRNA Xist Contributes to Endogenous Neurological Repair After Chronic Compressive Spinal Cord Injury by Promoting Angiogenesis Through the miR-32-5p/Notch-1 Axis.

Endogenous repair after chronic compressive spinal cord injury (CCSCI) is of great clinical interest. Ischemia-hypoxia-induced angiogenesis has been proposed to play an important role during this repair process. Emerging evidence indicates that long non-coding RNAs (lncRNAs) are involved in the pathophysiological processes of various diseases. Here, we identified a lncRNA (Xist; X-inactive specific transcript) with upregulated expression in cervical spine lesions during endogenous neurological repair in CCSCI rats. Therapeutically, the introduction of Xist to rats increased neurological function in vivo as assayed using the Basso, Beattie, and Bresnahan (BBB) score and inclined plane test (IPT). We found that the introduction of Xist enhanced endogenous neurological repair by promoting angiogenesis and microvessel density after CCSCI, while depletion of Xist inhibited angiogenesis and cell sprouting and migration. Mechanistically, Xist promoted angiogenesis by sponging miR-32-5p and modulating Notch-1 expression both in vitro and in vivo. These findings suggest a role of the Xist/miR-32-5p/Notch-1 axis in endogenous repair and provide a potential molecular target for the treatment of ischemia-related central nervous system (CNS) diseases.

LINC00612/miR-31-5p/Notch1 Axis Regulates Apoptosis, Inflammation, and Oxidative Stress in Human Pulmonary Microvascular Endothelial Cells Induced by Cigarette Smoke Extract.

BackgroundLong non-coding RNAs (lncRNAs) have been reported as key regulators in chronic obstructive pulmonary disease (COPD). However, the precise role of LINC00612 remains unclear.MethodsThe real-time quantitative polymerase chain reaction (RT-qPCR) was used to quantify the expression levels of LINC00612, miR-31-5p, and Notch homolog 1 (Notch1) in lung tissues and cells. Under a cigarette smoke extract (CSE) stimulation condition, the apoptosis was analyzed by flow cytometry assay. Caspase-3 activity was examined with a caspase-3 activity assay kit; besides, inflammation and oxidative stress were assessed by measuring interleukin-6, tumor necrosis factor-α, glutathione/oxidized glutathione, reactive oxygen species, malondialdehyde, and superoxide dismutase activity. The interaction relationship between miR-31-5p and LINC00612 or Notch1 was predicted by bioinformatics databases, while dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays were performed to confirm prediction. Eventually, the related protein expression was estimated with western blot assay.ResultsLINC00612 was downregulated in COPD tissues when compared with controls. Consistently, CSE inhibited LINC00612 expression in HPMECs with a dose/time-dependent method. Gain-of-function experiments indicated that the upregulation of LINC00612 could repress cell apoptosis, inflammation, and oxidative stress in HPMECs induced by CSE. In addition, miR-31-5p was negatively regulated by LINC00612 in HPMECs treated with CSE. The overexpression of miR-31-5p could abolish LINC00612-induced effects on HPMECs exposed to CSE. Importantly, LINC00612 could weaken CSE-induced cell apoptosis, inflammation, and oxidative stress in HPMECs by regulating the miR-31-5p/Notch1 signaling pathway.ConclusionCurrent findings suggest that CSE-mediated cell apoptosis, inflammation, and oxidative stress in HPMECs were abolished by upregulation of LINC00612. Furthermore, the LINC00612/miR-31-5p/Notch1 axis may represent a novel regulator of apoptosis, inflammation, and oxidative stress of HPMECs, which may be a potential therapeutic target for COPD in the future.

Long non-coding RNA RP11-59H7.3 promotes cell proliferation and invasion metastasis in colorectal cancer by miR-139-5p/NOTCH1 axis.

Increasing evidence suggests long non-coding RNAs (lncRNAs) are distinctively expressed in several cancers. However, the functions of these lncRNAs in cancer development remain unknown. In the current study, we report high expression of a novel lncRNA, RP11-59H7.3, and its association with prognosis in colorectal cancer (CRC) patients. Functional analyses of this lncRNA revealed its role in promoting proliferation and progression of the cell cycle, as well as enhancement of cell migration and invasion. Furthermore, our results revealed that knockdown of RP11-59H7.3 promoted cell apoptosis, with luciferase reporter assays showing that it directly binds to miR-139-5p. Knockdown of this lncRNA significantly reduced expression of NOTCH1, a direct target of miR-139-5p. Additionally, we show that suppression NOTCH1 by miR-139-5p could be partially rescued by overexpressing RP11-59H7.3. Analysis of the relationship between RP11-59H7.3 and miR-139-5p, in CRC tissues, showed a negative correlation while a positive association was observed between the RP11-59H7.3 expression and levels of NOTCH1. Taken together, these results demonstrated that the RP11-59H7.3/miR-139-5p/NOTCH1 axis functions as a key regulator in CRC metastasis. RP11-59H7.3 represents a potential biomarker for CRC diagnosis and could be an important target for development of novel therapies to manage the disease.

<p>Toosendanin Suppresses Glioma Progression Property and Induces Apoptosis by Regulating miR-608/Notch Axis</p>

BackgroundGlioma is one the most common and aggressive primary tumors of adult central nervous system worldwide, which tends to develop dysplasia and metastasis. Recently, toosendanin (TSN) has shown pharmacological effects in several cancers. However, little is known about the underlying mechanism of the effect of TSN on glioma and its relationship between miRNA in glioma.MethodsCell proliferation, cell cycle, cell apoptosis and cell migration were analyzed by CCK-8 cell viability, flow cytometry, wound scratch healing, transwell and Western blotting assays, respectively, in vitro. The regulation relationships between TSN and miR-608 or between miR-608 and Notch1 (Notch2) were examined using qRT-PCR, dual-luciferase and Western blotting assays. The functional effects of TSN through regulating miR-608 and Notch1 (Notch2) were further examined using a xenograft tumor mouse model in vivo.ResultsAfter TSN concentration was increased from 50 nM, 100 nM to 150 nM, cell proliferation and cell cycle were gradually reduced, and the cell apoptosis rate was increased in U-138MG or U-251MG cells. Wound-healing and transwell assays results showed that cell migration was significantly inhibited in TSN treatment cells (TSN treatment, 50 nM) compared to control cells. Mechanistic studies revealed that TSN up-regulated the expression of microRNA-608 (miR-608), while down-regulated the expression of miR-608’s target, Notch1 and Notch2. Over-expression of Notch1 and Notch2 partly attenuated TSN-induced tumor suppressive function. Moreover, in vivo experiments revealed that TSN treatment led to a significant inhibition of tumor growth, suggesting that it might be a promising drug for the treatment of glioma.ConclusionIn the present study, a novel established functional manner of TSN/miR-608/Notch1 (Notch2) axis was systematically indicated, which might provide prospective intervention ways for glioma therapy.

MicroRNA-34c-3p target inhibiting NOTCH1 suppresses chemosensitivity and metastasis of non-small cell lung cancer.

BackgroundChemotherapy is the standard treatment for non-small cell lung cancer (NSCLC). However, chemoresistance frequently occurs, making the treatment of NSCLC more difficult.MethodWe combined clinical and experimental studies to establish the role of microRNA (miR)-34c in NSCLC metastasis and chemoresistance.ResultsMiR-34c expression was significantly decreased in patients with NSCLC who showed a poor chemoresponse and metastasis. Overexpression of miR-34c sensitized NSCLC cells to paclitaxel and cisplatin both in vitro and in vivo. Furthermore, we found that NOTCH1 was target of miR-34c in NSCLC cells and played a key role in the effects of miR-34c on NSCLC.ConclusionNSCLC metastasis and chemoresistance are suppressed though the miR-34c/NOTCH1 axis. MiR-34c has important implications in the development of therapeutic strategies for metastasis and chemoresistance in NSCLC.

Long Noncoding RNA DSCAM-AS1 Facilitates Colorectal Cancer Cell Proliferation and Migration via miR-137/Notch1 Axis.

Growing evidences demonstrate that long noncoding RNAs (lncRNAs) participate in various cancers including colorectal cancer (CRC). In the current study, we found that the expression of DSCAM-AS1 in CRC tissues and cell lines was significantly upregulated, and was positively correlated with metastasis status and advanced stage of CRC. In addition, Kaplan-Meier assays also indicated that the expression of DSCAM-AS1 was correlated with poor prognosis in patients with CRC. Silence of DSCAM-AS1 inhibited proliferation and migration of CRC cells. Subcellular fractionation and FISH analyses suggested that DSCAM-AS1 was majorly distributed in cytoplasm of HT29 and LOVO cells. Thus, DSCAM-AS1 might act as a competing endogenous RNA (ceRNA). Subsequently, RT-qPCR results displayed that the expression of miR-137 in CRC tissues was relatively lower than that in the neighboring normal tissues. The interaction between miR-137 and DSCAM-AS1 was demonstrated by luciferase reporter assay. Functionally, miR-137 reversed the pro-proliferation and -metastasis effect of DSCAM-AS1 on CRC cells. Collectively, DSCAM-AS1 promotes CRC progression via sponging miR-137. MiR-137 can suppress the expression of Notch-1, a novel signaling regulating cell proliferation and EMT, by working on the 3'UTR of Notch-1. At last, Notch-1 overexpression or miR-137 inhibition could restore the DSCAM-AS1 silencing-mediated repressive function on cell proliferation and migration. The above data suggested that, DSCAM-AS1 may contribute to CRC cell proliferation and migration by targeting miR-137/Notch-1 axis.

Role of microRNA-449a in the progress of inflammatory bowel disease in children

This study aimed to investigate the expression and role of microRNA-449a (miR-449a) in inflammatory bowel disease (IBD) in children, and further to explore the underlying mechanism. To study the role of miR-449a in IBD development, an in vitro inflammatory model of IBD was conducted by stimulating the normal intestinal epithelial cells Caco-2 with 2% dextran sulfate sodium (DSS). The results showed that the miR-449a level was significantly higher in children with IBD than that in children without IBD. Notch1 was a direct target gene of miR-449a. The increased levels of TNF-α, IL-1β and IL-6, reduced cell viability and enhanced cell apoptosis in Caco-2 cells caused by DSS administration were significantly inhibited by miR-449a inhibition. Besides, DSS significantly decreased the level of Notch1, Hes-1, Hey-1 and Bcl-2, and increased Bax expression, and these changes were prevented by miR-449a inhibitor. Importantly, all the effects of miR-449a inhibitor on Caco-2 cells were eliminated by Notch1 gene silencing. In conclusion, the data of our study indicated that miR-449a was up-regulated in IBD in children, and miR-449a inhibitor could inhibit inflammatory response and cell apoptosis by targeting Notch1 in IBD in vitro. The miR-449a/Notch1 axis might be a novel target for IBD treatment.

JNK1 Induces Notch1 Expression to Regulate Genes Governing Photoreceptor Production.

c-Jun N-terminal kinases (JNKs) regulate cell proliferation and differentiation via phosphorylating such transcription factors as c-Jun. The function of JNKs in retinogenesis remains to be elucidated. Here, we report that knocking out Jnk1, but not Jnk2, increased the number of photoreceptors, thus enhancing the electroretinogram (ERG) responses. Intriguingly, Notch1, a well-established negative regulator of photoreceptor genesis, was significantly attenuated in Jnk1 knockout (KO) mice compared to wild-type mice. Mechanistically, light specifically activated JNK1 to phosphorylate c-Jun, which in turn induced Notch1 transcription. The identified JNK1–c-Jun–Notch1 axis strongly inhibited photoreceptor-related transcriptional factor expression and ultimately impaired photoreceptor opsin expression. Our study uncovered an essential function of JNK1 in retinogenesis, revealing JNK1 as a potential candidate for targeting ophthalmic diseases.

Low-Dose IFNγ Induces Tumor Cell Stemness in Tumor Microenvironment of Non-Small Cell Lung Cancer.

IFNγ is conventionally recognized as an inflammatory cytokine that plays a central role in antitumor immunity. Although it has been used clinically to treat a variety of malignancies, low levels of IFNγ in the tumor microenvironment (TME) increase the risk of tumor metastasis during immunotherapy. Accumulating evidence suggests that IFNγ can induce cancer progression, yet the mechanisms underlying the controversial role of IFNγ in tumor development remain unclear. Here, we reveal a dose-dependent effect of IFNγ in inducing tumor stemness to accelerate cancer progression in patients with a variety of cancer types. Low levels of IFNγ endowed cancer stem-like properties via the intercellular adhesion molecule-1 (ICAM1)-PI3K-Akt-Notch1 axis, whereas high levels of IFNγ activated the JAK1-STAT1-caspase pathway to induce apoptosis in non-small cell lung cancer (NSCLC). Inhibition of ICAM1 abrogated the stem-like properties of NSCLC cells induced by the low dose of IFNγ both in vitro and in vivo. This study unveils the role of low levels of IFNγ in conferring tumor stemness and elucidates the distinct signaling pathways activated by IFNγ in a dose-dependent manner, thus providing new insights into cancer treatment, particularly for patients with low expression of IFNγ in the TME. SIGNIFICANCE: These findings reveal the dose-dependent effect of IFNγ in inducing tumor stemness and elucidate the distinct molecular mechanisms activated by IFNγ in a dose-dependent manner.

Paeonol reverses promoting effect of the HOTAIR/miR-124/Notch1 axis on renal interstitial fibrosis in a rat model.

Renal interstitial fibrosis (RIF) is a common manifestation of inflammatory and noninflammatory renal diseases, which correlates to renal excretory dysfunction. Recently, the long noncoding RNAs (lncRNAs) have been demonstrated to be involved in the development of various renal diseases. Here, we aim to determine whether paeonol (PAE) affects RIF with involvement of the lncRNA HOX transcript antisense intergenic RNA (HOTAIR)/microRNA-124 (miR-124)/Notch1 axis. RIF rat models were established by performing unilateral ureteral occlusion (UUO), in which interactions between HOTAIR, Notch1, and miR-124 were determined. To identify the roles of PAE and HOTAIR in RIF, rats were injected with HOTAIR or PAE. Subsequently, to further investigate the underlying mechanism of PAE in RIF, epithelial to mesenchymal transition (EMT)- and migration-related genes in NRK-49F cells were measured. Next, rats were further treated with IMR-1 (inhibitor of the Notch1/Jagged1 signaling pathway) to determine how PAE influences the Notch1/Jagged1 signaling pathway. HOTAIR interacted with miR-124, and miR-124 directly targeted Notch1, and HOTAIR was observed to be upregulated in RIF rats. PAE was found to decrease HOTAIR and Notch1 expression but to increase the miR-124 expression in RIF rats. PAE inhibited EMT and migration of NRK-49F cells facilitated by HOTAIR. HOTAIR activated the Notch1/Jagged1 signaling pathway by downregulating miR-124, while PAE reversed these effects of HOTAIR on the Notch1/Jagged1 signaling pathway. Overall, our study demonstrates the contributory effect of lncRNA HOTAIR on RIF by activating the Notch1/Jagged1 signaling pathway via inhibition of miR-124, whereas administration of PAE can alleviate the effects of HOTAIR on RIF.

Distribution and Clinical Features of NOTCH1 Signaling Activating Alterations in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

IntroductionNOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations.MethodsA total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS).ResultsIn total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern.Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors.Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations.ConclusionsWe observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

Protective role of microRNA-374 against myocardial ischemia-reperfusion injury in mice following thoracic epidural anesthesia by downregulating dystrobrevin alpha-mediated Notch1 axis.

Ischemia-reperfusion (I/R) injury often leads to myocardial apoptosis and necrosis. Studies have demonstrated the role microRNAs (miRs) played in myocardial I/R injury. Thus, we established a myocardial I/R injury model and a thoracic epidural anesthesia (TEA) model in mice to explore whether microRNA-374 (miR-374) affects myocardial I/R injury. We collected myocardial tissues to evaluate whether TEA exerts a protection effect on myocardial tissues. In addition, the levels of miR-374, dystrobrevin alpha (DTNA), and the statue of the Notch1 axis were detected. Subsequently, cardiomyocytes extracted from TEA mice were treated to regulate their levels of miR-374 and DTNA. After that, cell viability, cell cycle distribution, and apoptosis of cardiomyocytes were assessed. This was followed by the detection of the myocardial infarction area. The mice models of myocardial I/R injury were associated with poorly expressed miR-374 and highly expressed DTNA. TEA was found to protect myocardial tissues against myocardial I/R injury by elevating miR-374 and reducing DTNA. Dual-luciferase reporter assay validated that DTNA was the target gene of miR-374. Cardiomyocytes with overexpressed miR-374 were shown to have downregulated DTNA levels and blocked Notch1 axis. Overexpressed miR-374 was also found to promote the viability and inhibit the apoptosis of cardiomyocytes, as well as to increase the number of cells arrested in the S phase. In accordance with this, the myocardial infarction area was decreased with the upregulated miR-347 and downregulated DTNA. Collectively, these results demonstrated that, by inhibiting the activity of DTNA-mediated Notch1 axis, miR-374 could protect against myocardial I/R injury in mice after TEA.

MicroRNA-744 Inhibits Cellular Proliferation and Invasion of Colorectal Cancer by Directly Targeting Oncogene Notch1.

Accumulated studies have strongly implicated aberrantly expressed microRNAs (miRNAs) in carcinogenesis and cancer progression of various cancers, including colorectal cancer (CRC). Hence, a comprehensive study of miRNAs and their association with CRC may be a promising therapeutic method for patients with this malignancy. MicroRNA-744 (miR-744) is abnormally expressed in several types of human cancer. Thus far, little is known about the expression, biological roles, and exact mechanisms of miR-744 in CRC. Thus, the present study measured the expression level of miR-744 and investigated its roles and associated molecular mechanisms in CRC. This study demonstrated that miR-744 expression was significantly underexpressed in CRC tissues and cell lines. Low miR-744 expression was positively associated with lymphatic metastasis and TNM stage. Functional experiments revealed that miR-744 overexpression obviously inhibited the proliferation and invasion of CRC cells. Furthermore, Notch1 was identified as a direct target of miR-744 in CRC. Moreover, the inhibition of Notch1 phenocopied the inhibitory effects of miR-744 overexpression on CRC cells. Restored Notch1 expression markedly rescued the tumor-suppressive effects of miR-744 overexpression on CRC cells. Overall, miR-744 exhibits an essential role in CRC progression, and the miR-744/Notch1 axis may provide novel insights into future treatments for patients with CRC.