Notch1 mRNA Research Articles

MicroRNA‑455 inhibits cell proliferation and invasion of epithelial ovarian cancer by directly targeting Notch1.

Ovarian cancer (OC) is the fifth leading cause of cancer‑associated mortality among women in developed countries. Numerous microRNAs are aberrantly expressed in epithelial (E)OC, and are involved in EOC formation and progression. As such, microRNAs may be investigated as diagnostic and prognostic molecular biomarkers, and therapeutic targets for patients with EOC. For instance, microRNA‑455 (miR‑455) is abnormally expressed in various types of human cancer. However, details on the expression level, biological roles and underlying mechanism of miR‑455 in EOC remain unclear. In the present study, the expression of miR‑455 in EOC was detected, and its association with clinical characteristics was analysed. The functional roles and underlying mechanisms of miR‑455 in EOC were also investigated. The results revealed that miR‑455 expression in EOC tissues and cell lines was significantly downregulated. Furthermore, miR‑455 downregulation was correlated with tumor size, International Federation of Gynecology and Obstetrics stage and lymph node metastasis of patients with EOC. Restoration of miR‑455 expression efficiently inhibited cell proliferation and invasion invitro. Notch1 was identified as a direct target of miR‑455 in EOC. The mRNA and protein expression levels of Notch1 were higher in EOC tissues compared with in normal ovarian epithelial tissues. Spearman's correlation analysis indicated that miR‑455 expression was negatively correlated with the mRNA level of Notch1 in EOC tissues. Notch1 overexpression was able to restore the EOC cell proliferation and invasion abilities suppressed by miR‑455. The present study provided evidence that the dysregulation of the miR‑455/Notch1 signalling pathway may be essential for EOC occurrence and development. In addition, the results confirmed the tumor‑suppressive roles of miR‑455 in modulating EOC proliferation and invasion through regulation of Notch1 expression. Thus, miR‑455 may be a novel miRNA‑based therapeutic target to treat patients with EOC.

Role and importance of the expression of transcription factor FOXC2 in cervical cancer.

The aim of the present study was to investigate the relationship between the expression of transcription factor forkhead box C2 (FOXC2) and the clinical features of cervical cancer. A total of 66 patients with cervical cancer, 42 patients with cervical intraepithelial neoplasia (CIN) and 25 patients with cervical inflammation were enrolled. The positive expression rates and expression levels of mRNA of FOXC2, E-cadherin, N-cadherin, vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), Notch protein and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in cervical tissues were detected using immunohistochemistry and RT-PCR. The positive expression rates and expression levels of mRNA of FOXC2, N-cadherin, VEGF, SDF-1, Notch and LYVE-1 in cervical cancer were significantly higher than those in CIN, and those in the inflammatory tissues were the lowest, while the positive expression rate of E-cadherin in cervical cancer was lower than that in CIN, and that in the inflammatory tissues was the highest (P<0.05). The positive expression rates of FOXC2, N-cadherin, VEGF, SDF-1, Notch and LYVE-1 in patients with cervical cancer [human papillomavirus (HPV) positive, squamous cell carcinoma, Stages III–IV, maximal diameter ≥3.8 cm and low differentiation] were increased, and the positive expression rate of E-cadherin was decreased (P<0.05). Correlation analysis revealed that FOXC2 was positively correlated with the positive expression rates of N-cadherin, VEGF, SDF-1, Notch and LYVE-1, and negatively correlated with E-cadherin (P<0.05). In conclusion, the high expression of FOXC2 is correlated with the HPV infection, pathological pattern, clinical stage, tumor diameter and differentiation grade of cervical cancer, which may be involved in the epithelial-mesenchymal transition, vascular and matrix formation, Notch signaling pathway and lymphangiogenesis.

MTA-induced Notch activation enhances the proliferation of human dental pulp cells by inhibiting autophagic flux.

To determine the roles of autophagy and Notch signalling in mineral trioxide aggregate (MTA)-mediated proliferation and differentiation of human dental pulp cells (hDPCs). hDPCs were separated from human healthy molar teeth using collagenase I/dispase II digestion and then cultured in α-MEM medium with 15% foetal bovine serum. hDPCs were seeded in 96-well plates, and cell counting kit assays were carried out to test their viability. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate ALP, Runx2, Notch1, Hes1 and Hey1 mRNA levels. Notch1, hes1, LC3 and p62 protein levels were quantified by Western blotting. Colocalization of LC3 and Notch1 was measured by immunofluorescence. Two-tailed Student's t-tests were used for statistical analysis. Autophagic flux was significantly (P<0.05) inhibited by MTA extracts, causing Notch degradation arrest. This resulted in the promotion of cell proliferation and inhibition of differentiation during the logarithmic phase of cell growth. MTA extract promoted the proliferation of hDPCs in part by activating Notch signalling through inhibition of autophagic flux during the early stage and, thus, might potentially induce rapid restoration of injured pulps.

Mechanism of Chlorogenic Acid in Apoptotic Regulation through Notch1 Pathway in Non-small Cell Lung Carcinoma in Animal Level

背景与目的计算机断层扫描（computed tomography, CT）随访评估结节体积及密度变化是临床针对不定性肺结节采用的常用策略。在保证测量精度前提下降低CT剂量是需要考虑的问题。本研究旨在评估不同管电流及重建算法对肺体模结节容积定量及CT值测量的影响。方法应用64排螺旋CT，管电压120kV，7种管电流（10 mA、20 mA、50 mA、80 mA、100 mA、150 mA、350 mA）对肺结节体模进行扫描，采用滤波反投影（filtered back projection, FBP）、自适应迭代重建（ASIR: 30%, 50%, 80%）算法进行重建，获取28套CT图像。应用肺结节分析软件对3种直径（2.5 mm, 5 mm, 10 mm）、三种CT值（-100 HU, 60 HU, 100 HU）共9个球型结节测量容积及平均CT值数据。应用重复测量方差分析评估不同管电流及原始数据重建算法对容积及CT值测量的影响。结果直径为2.5 mm结节的容积测量相对误差（100.8%±28%）及三维CT值绝对误差（-756±80）HU最大；直径为5 mm及10 mm结节的容积相对误差小[(-0.9%±1.1%)vs(0.9%±1.4%)]，但CT值绝对误差大[(-243±26)HU vs(-129±7)HU]。针对直径为5 mm及10 mm结节使用重复测量方差分析结果显示，应用不同管电流及原始数据重建算法时容积测量相对误差没有显著性差异（F=5.60, P=0.10 vs F=11.13, P=0.08），三维CT值的绝对误差有显著影响（F=34.79, P < 0.001 vs F=156.14, P < 0.001）。结论不同管电流及重建算法对直径5mm及10 mm的结节容积定量影响很小，因此较低管电流及迭代重建算法可以应用在5 mm以上肺结节的CT随访中。结节分析软件提供的平均CT值与标准CT值在不同大小、密度结节中均具有较大误差，不能应用于临床。

Expression and association of VEGF-Notch pathways in infantile hemangiomas.

The vascular endothelial growth factor (VEGF) and Notch signaling pathways have been identified to be involved in the neovascularization, and angiogenesis of various tumor types. However, there is little data regarding their roles and association in infantile hemangiomas (IHs). In the present study, the significance and association of the VEGF-VEGF receptor (R)2, and δ like canonical Notch ligand 4 (Dll4)-Notch1 pathways in different clinical phases of IHs were investigated. Specimens in the proliferating phase (n=15) and involuting phase (n=12) were collected. Quantitative polymerase chain reaction was used to analyze the mRNA levels of VEGF, VEGFR2, Dll4 and Notch1. A further 61 paraffin-embedded IHs (26 in the proliferating group and 35 in the involuting group) specimens were collected to investigate the protein levels of Notch1 and Dll4 using immunohistochemistry, and then analyzed for the association between these factors and microvessel density (MVD). The relative expression levels of VEGF, VEGFR2 and Dll4 mRNA in the proliferating group were significantly higher compared with that in the involuting group. In addition, the relative levels of Notch1 mRNA were similar in the proliferating and involuting phases. Expression levels of VEGFR2 and Dll4 mRNA were positively correlated with Notch1 expression in the proliferating IHs. The relative expression of VEGFR mRNA was positively correlated with Dll4 in the proliferating and involuting groups. Immunohistochemical staining demonstrated that Notch1 and Dll4 protein were upregulated in the proliferating IHs compared with the control group. In the proliferating IHs, Notch1 and Dll4 protein expression levels were positively correlated with MVD, while in the involuting phase, only Dll4 was positively correlated with MVD. The expression levels of related factors in the VEGF and Notch pathways, and the associations among them suggest that they may be involved in the regulation of IH neovascularization and angiogenesis.

Antineoplastic effects of histone deacetylase inhibitors in neuroendocrine cancer cells are mediated through transcriptional regulation of Notch1 by activator protein 1.

Notch signaling is minimally active in neuroendocrine (NE) cancer cells. While histone deacetylase inhibitors (HDACi) suppress NE cancer growth by inducing Notch, the molecular mechanism underlying this interplay has not yet been defined. NE cancer cell lines BON, H727, and MZ‐CRC‐1 were treated with known HDACi Thailadepsin‐A (TDP‐A) and valproic acid (VPA), and Notch1 mRNA expression was measured with RT‐PCR. Truncated genomic fragments of the Notch1 promotor region fused with luciferase reporter were used to identify the potential transcription factor (TF) binding site. The key regulatory TF was identified with the electrophoretic mobility shift assay (EMSA). The effect of HDACi on Notch1 level was determined before and after silencing the TF. TDP‐A and VPA induced Notch1 mRNA in a dose‐dependent manner. A functional DNA motif at −80 to −52 from the Notch1 start codon responsible for the HDACi‐dependent Notch1 induction was identified. Mutation of this core sequence failed to induce luciferase activity despite HDACi treatment. EMSA showed the greatest gel shift with AP‐1 in nuclear extracts. Knockdown of AP‐1 significantly attenuated the effect of HDACi on Notch1 induction. Interestingly, AP‐1 transfection did not alter Notch1 level, suggesting that AP‐1 is necessary but insufficient for HDACi activation of Notch1. Therefore, AP‐1 is the TF that binds to a specific transcription‐binding site within the Notch1 promotor region to trigger Notch1 transcription. Elucidating the HDACi activation mechanism may lead to the development of novel therapeutic options against NE cancers and facilitate the identification of clinical responders and prevent adverse effects.

Overexpression of Pofut1 and activated Notch1 may be associated with poor prognosis in breast cancer

PurposeThe present study was to evaluate the prognostic value of protein expression of Pofut1 and Notch1 signaling in breast cancer. Material and methodsFormalin-fixed paraffin-embedded 314 breast specimens including 174 infiltrating ductal carcinoma(IDC), 50 ductal carcinoma in situ(DCIS) and 90 adjacent normal tissue(ANT) were immunohistochemically examined to evaluate the protein expression of Pofut1, activated Notch1(N1IC) and Slug on specimens. Survival analysis was performed by Kaplan-Meier method and Cox's proportional-hazards model. A online database was computationally used to further explore the prognostic role of Pofut1 and Notch1 mRNA expression by Kaplan-Meier Plotter. ResultsPofut1, Slug and N1IC expression were significantly increased in IDC compared to ANT(all p < 0.05). High expression of Pofut1, Slug and N1IC were associated with tumor aggressiveness including lymph node metastasis (LNM: p = 0.005 for Pofut1, p < 0.001 for N1IC, p = 0.017 for Slug), advanced stage(p = 0.039 for Pofut1, p = 0.025 for N1IC) and higher histological grade(p = 0.001 for N1IC). Additionally, high expression of Pofut1 was found to be significantly associated with high expressions of N1IC and Slug in IDC(r = 0.244, p = 0.001; r = 0.374, p < 0.001, respectively), similar correlation was also observed between high N1IC and Slug expression(r = 0.496, p < 0.001). Moreover, Kaplan-Meier and Cox's regression analysis indicated the significant prognostic value of elevated Pofut1, N1IC, Slug expressions, positive LNM and advanced tumor stage for the prediction of a shorter disease-free survival (DFS) and overall survival(OS). The web-based analysis also suggested a significant association of high Pofut1 and Notch1 mRNA expression with worse survival outcome. ConclusionOur findings suggested that overexpression of Pofut1 and activated Notch1 signaling may be associated with a poor prognosis in breast cancer.

Expression levels and mechanism of microRNA-34a and Notch1 signaling pathway in hippocampi of temporal lobe epilepsy rats

Objective To explore the expression levels of microRNA(miR)-34a in hippocampus of temporal lobe epilepsy rats and the effect of miR-34a on its target signaling pathway Notch1. Methods Rats were divided randomly into experiment group (n=40) and control group (n=40) by adopting random number table method.The status epilepticus model and the temporal lobe epilepsy model were induced by using lithium-pilocarpine for experiment group.The control group rats received an injection of an equal amount of 9 g/L saline as instead of pilocarpine.Racine grading was performed at 24 hours, day 3, day 7, day 15, and 1 month after modeling to evaluate the behavior.Real-time fluorescent quantitative polymerase chain reaction was used to test the mRNA expressions of miR-34a and Notch1.Western blot was performed to explore the protein expression of Notch1. Results The expression levels of miR-34a at post-status epilepticus in 24 hours, day 3, day 7, day 15 were 2.55±0.29, 2.11±0.17, 1.68±0.49 and 1.84±0.42, respectively, which showed statistically significant difference compared with the control group (1.00±0.00) (t=-1.55, -1.11, -0.68, -0.84, all P<0.05). The expression levels of Notch1 mRNA at post-status epilepticus in 24 hours, day 3, day 7, day 15, 1 month were 1.44±0.31, 1.27±0.13, 1.52±0.28, 0.91±0.33, and 0.80±0.09 respectively.There were significant differences at 24 hours, day 7 in Notch1 mRNA expression (t=-0.44, -0.52, all P<0.05) compared with the control group(1.00±0.00). The expression level of Notch1 mRNA on day 15 was significantly lower than 24 hours and day 7 (t=-0.54, -0.62, all P<0.05), and the expression in 1 month was significantly lower than in 24 hours, or day 3 and day 7 (t=-0.64, -0.46, -0.72, all P<0.05). The expression levels of Notch1 protein at post-status epilepticus 24 hours, day 3, day 7, day 15, 1 month were 0.78±0.09, 0.57±0.13, 0.55±0.16, 0.42±0.13, and 0.33±0.09, respectively.There was significantly up-regulated at 24 hours of Notch1 protein expression compared with control group (0.51±0.15)(t=-0.20, P<0.05); and the expression level at day 15 were significantly lower than 24 hours (t=-0.26, P<0.05), while the expression in 1 month was significantly lower than in 24 hours and on day 3 (t=-0.36, -0.24, all P<0.05). Conclusion miR-34a is significantly up-regulated in the post-status epilepticus rat hippocampus, and it may contribute to temporal lobe epilepsy by activating Notch1 signaling pathway. Key words: microRNA-34a; Temporal lobe epilepsy; Notch1

Human prostate luminal cell differentiation requires NOTCH3 induction by p38-MAPK and MYC

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.

Regulation of Notch Signaling by the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex in Drosophila melanogaster.

Notch signaling is an evolutionarily conserved pathway that is found to be involved in a number of cellular events throughout development. The deployment of the Notch signaling pathway in numerous cellular contexts is possible due to its regulation at multiple levels. In an effort to identify the novel components integrated into the molecular circuitry affecting Notch signaling, we carried out a protein-protein interaction screen based on the identification of cellular protein complexes using co-immunoprecipitation followed by mass-spectrometry. We identified Hrp48, a heterogeneous nuclear ribonucleoprotein in Drosophila, as a novel interacting partner of Deltex (Dx), a cytoplasmic modulator of Notch signaling. Immunocytochemical analysis revealed that Dx and Hrp48 colocalize in cytoplasmic vesicles. The dx mutant also showed strong genetic interactions with hrp48 mutant alleles. The coexpression of Dx and Hrp48 resulted in the depletion of cytoplasmic Notch in larval wing imaginal discs and downregulation of Notch targets cut and wingless Previously, it has been shown that Sex-lethal (Sxl), on binding with Notch mRNA, negatively regulates Notch signaling. The overexpression of Hrp48 was found to inhibit Sxl expression and consequently rescued Notch signaling activity. In the present study, we observed that Dx together with Hrp48 can regulate Notch signaling in an Sxl-independent manner. In addition, Dx and Hrp48 displayed a synergistic effect on caspase-mediated cell death. Our results suggest that Dx and Hrp48 together negatively regulate Notch signaling in Drosophila melanogaster.

Prognostic value of Notch receptors in postsurgical patients with hepatitis B virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and a major cause of cancer involved death worldwide. Prognosis remains poor because of high recurrence rates and lack of effective relapse prevention strategies. Notch pathway plays an important role in tumor progression and metastasis, and it is associated with the prognosis of cancer. A total of 465 hepatitis B virus (HBV)‐related HCC patients who underwent surgery were enrolled. Single nucleotide polymorphisms (SNP) of Notch pathway receptors were genotyped using Sanger DNA sequencing. Kaplan–Meier curves and the Cox proportional hazards regression model were adopted to analyze the association of polymorphisms and mRNA expression with clinical and pathological features, respectively. Four SNPs (rs1043996 in Notch3 and rs422951, rs520692, rs3830041 in Notch4) were significantly associated with overall survival (OS) (P = 0.023, P = 0.042, P = 0.028, and P = 0.001 respectively). Patients carrying the AA genotype in rs1043996 and TT/TC genotypes in rs422951 and rs520692 significantly decreased risks of death, compared to those carrying the AG/GG genotype in rs1043996 and CC genotype in rs422951 and rs520692, respectively. Patients carrying the TT genotype in rs3830041 showed poorer OS, compared with those carrying the TC/CC genotype. A haplotype block (rs422951 was in strong LD with rs520692, r 2 = 0.843) was identified in Notch4. Notch3 mRNA expression significantly increased in tumor tissue, compared with nontumor normal tissue (P < 0.0001). Moreover, higher expression of Notch3 was associated with poorer OS (HR = 2.11, 95% CI = 1.32–3.37, P = 0.002) and shorter recurrence time of HBV‐related HCC (HR = 1.96, 95% CI = 1.31–2.93, P = 0.001). Our findings collectively indicate that Notch receptors variants (rs1043996 in Notch3 and rs422951, rs520692, rs3830041 in Notch4) are independent predictive targets for OS in HBV‐related HCC patients. Notch3 expression is a potential prognostic biomarker of OS and recurrence‐free survival (RFS) prediction in HBV‐related HCC patients following surgical treatment.

Th17/Treg dysregulation in allergic asthmatic children is associated with elevated notch expression

ABSTRACTBackground: Notch signaling pathway is critically involved in the differentiation of T helper (Th) cells, key players in the pathogenesis of allergic diseases. Objective: The study is to explore whether Th17/Treg dysregulation in children with allergic asthma (AA) is associated with alteration of Notch expression. Methods: Thirty-five patients with AA and thirty-five healthy control children were selected. Flow cytometry was used to detect Th17 and Treg cells. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to measure the expression of Notch1 mRNA. The correlations among Notch1 mRNA expression, the percentage of Th17 cells, and Th17/Treg ratio were calculated. Results: Th17 and Treg cells were significantly increased and decreased, respectively, in children with AA than in healthy control (p < 0.001). mRNA level of Notch1 was elevated in children with AA comparing to healthy controls (p < 0.001). The mRNA expression of Notch1 was positively correlated with the percentage of Th17 cells (r = 0.775, p < 0.001) and Th17/Treg ratio (r = 0.698, p < 0.001). Conclusion: Children with AA showed dysregulation of Th17/Treg cells in peripheral blood. Such change is accompanied with overexpression of Notch1, indicating Th17/Treg dysregulation in children with AA is associated with elevated Notch expression.

Interferon regulatory factor 4 (IRF4) is overexpressed in human non‑small cell lung cancer (NSCLC) and activates the Notch signaling pathway.

The transcription factor, interferon regulatory factor 4 (IRF4), serves an essential role in the regulation of immune responses, and has been reported to act as a diagnostic and prognostic marker for various hematological malignancies. The present study aimed to investigate whether IRF4 could exert effects on human non-small cell lung cancer (NSCLC) and to explore the underlying mechanism. The mRNA and protein expression of IRF4 was detected in NSCLC tissues using reverse-transcription quantitative polymerase chain reaction and western blotting, respectively. In the in vitro experiment, IRF4 expression was knocked down or overexpressed using lentivirus in human lung adenocarcinoma A549 and lung squamous cell carcinoma LC-AI cell lines. Cell proliferation and colony number were analyzed using MTT and colony formation assays, respectively. The expression levels of IRF4 mRNA and protein were significantly higher in NSCLC tissues (n=54) compared with that in adjacent non-tumor tissues. Similarly, the expression levels of Notch1 and Notch2 mRNA were significantly higher in NSCLC tissues. Furthermore, the expression level of IRF4 mRNA was positively correlated with the levels of Notch1 and Notch2 mRNA in NSCLC tissues. Consequently, using NSCLC cell lines, it was demonstrated that the knockdown of IRF4 expression significantly reduced the cell proliferation rate and colony formation, whereas IRF4-overexpression significantly increased them. Notably, the IRF4 knockdown significantly decreased the expression levels of Notch1 and Notch2 mRNA, and phosphorylated protein kinase B (AKT), whereas IRF4 overexpression resulted in the opposite. The results of the present study indicate that IRF4 is overexpressed and serves as a tumor promoter in human NSCLC, at least partially, through activating the Notch-Akt signaling pathway.

Nox4 is involved in high glucose-induced apoptosis in renal tubular epithelial cells via Notch pathway.

It has previously been demonstrated that nicotinamide adenine dinucleotide phosphate‑oxidase (NADPH) oxidase4 (Nox4), is important in the pathogenesis of diabetic nephropathy (DN), however the exact mechanisms remain to be elucidated. The present study aimed to examine the effect of Nox4 on the alteration of the Notch pathway and cell apoptosis in the renal tubular epithelial cell line, HKC, under conditions of high glucose (HG; 30mmol/l glucose). Nox4 and the Notch pathway were inhibited by N‑acetylcysteine (NAC), diphenylene iodonium (DPI) or γ‑secretase inhibitor (DAPT). The protein levels of Nox4, Notch1, Notch intracellular domain1 (NICD1), phosphorylated (p) Ras‑related C3 botulinum toxin substrate1 (Rac1), Rac1, B‑cell lymphoma 2 apoptosis regulator (Bcl‑2), Bcl‑2 associated protein X apoptosis regulator (Bax) and cleaved caspase‑3 were determined by western blotting. The Nox4 and Notch1 mRNA levels were detected by reverse transcription‑quantitative polymerase chain reaction. Intracellular reactive oxygen species (ROS) levels were detected via chloromethyl‑2',7'‑dichlorodihydrofluorescein diacetate. Apoptotic cells were determined using an Annexin V/propidium iodide apoptosis detection kit. HG upregulated Nox4, Notch1, NICD1, p‑Rac1, Bax and cleaved caspase‑3 expression levels and downregulated Bcl‑2 expression in cultured HKC cells, compared with cells cultured in normal glucose levels. Inhibition of the Notch pathway via DAPT increased Bcl‑2 expression, decreased Bax and cleaved caspase‑3 levels and prevented HKC cell apoptosis. Inhibition of Nox4 by NAC and DPI inhibited the Notch signaling pathway and ROS generation, which prevented HKC cell apoptosis. These findings indicated that Nox4 potentially mediates HG‑induced HKC cell apoptosis via the Notch pathway, and may be involved in renal tubular epithelial cell injury in DN.

From the Cover: ImpairedProliferation and Differentiation of the Conducting Airway Epithelium Associated With Bronchiolitis Obliterans After Sulfur Mustard Inhalation Injury in Rats.

Adult male rats were exposed to a sublethal dose of SM inhalation (1.0-1.2 mg/kg) for 50 min. Histological sections of the bronchiolar epithelium were analyzed for changes using hematoxylin and eosin, trichrome, and immunofluorescent staining for acetylated tubulin (AT) and club cell secretory protein (CCSP). CCSP in bronchoalveolar lavage fluid was assessed using western blot. A bromodeoxyuridine (BRDU) assay was used to assess for epithelial proliferation, and real-time PCR measured changes in Notch mRNA expression. SM caused significant proximal bronchiolar epithelial injury with epithelial denudation, loss of acetylated tubulin and CCSP staining, and reduced bronchoalveolar lavage fluid CCSP levels. bromodeoxyuridine (BRDU) + staining of proximal bronchiolar epithelial cells was not increased, but staining was increased in the distal bronchiolar epithelium. One month after injury, the proximal bronchiolar epithelium was not fully repaired. Significant collagen deposition surrounded proximal bronchioles with luminal obstruction, consistent with bronchiolitis obliterans. These changes corresponded with a downregulation of Notch1, Notch3, and Hes1 mRNA expressions. This study demonstrates that SM exposure resulted in severe proximal airway epithelial injury, persistent morphological changes, impaired epithelial proliferation and, ultimately, bronchiolitis obliterans. These changes occurred at the same time that the Notch signaling genes were downregulated. Thus, the lung epithelium and the Notch signaling pathway may be worthy targets for the prevention of chronic airway remodeling after SM inhalation injury.

Clinical significance of S100A6 and Notch1 in multiple myeloma patients

目的探讨S100A6、Notch1在多发性骨髓瘤（MM）患者中的表达及其临床意义。方法以28例MM患者为研究对象，以20例白细胞、血小板略低但骨髓检查未见异常者为对照，采用real time PCR法检测骨髓单个核细胞S100A6、Notch1的表达；采用免疫组化染色法检测S100A6、Notch1蛋白在MM患者骨髓和髓外浸润组织活检病理切片中的表达；采用real time PCR法和Western blot法检测siRNA沉默骨髓瘤U266细胞S100A6基因后对Notch1 mRNA和蛋白水平的影响。并结合临床进行相关分析。结果①S100A6、Notch1 mRNA表达水平：初发MM患者组分别为2.19±1.25、2.98±0.64，均高于对照组（0.71±0.20、0.58±0.39）和稳定期患者组（0.85±0.26、0.72±0.40）（P值均<0.05）；伴髓外转移组（8例）分别为3.36±1.23、5.71±3.96，均高于无髓外转移组（20例）（1.40±0.25、1.16±1.00）。②S100A6与Notch1 mRNA表达呈正相关（r=0.505，P=0.007）。③MM患者骨髓和髓外浸润组织活检病理切片均可见浆细胞S100A6、Notch1阳性表达。④siRNA转染U266细胞48 h后S100A6基因表达沉默，Notch1 mRNA及蛋白水平明显下降。结论S100A6、Notch1表达与MM疾病发生、进展、髓外转移相关，二者具有显著相关性，可作为MM诊断及预后的指标。

Notch signaling pathway promotes the development of ovine ovarian follicular granulosa cells

The Notch signaling pathway regulates cell proliferation, differentiation and apoptosis involved in development of the organs and tissues such as nervous system, cartilage, lungs, kidneys and prostate as well as the ovarian follicles. This study aimed to investigate the mRNA expression and localization of NOTCH2, as the key factor in Notch signaling pathway. This was determined by PCR, real-time PCR and immunohistochemistry. Additionally, the effects of inhibiting Notch signaling pathway with different concentrations (5μM, 10μM and 20μM) of N-[N-(3, 5-Difuorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling pathway, on ovine granulosa cells was determined in vitro by detecting estradiol production using enzyme linked immunosorbent assay and expressions of the genes related to the cell cycle and apoptosis using real-time polymerase chain reaction (PCR). NOTCH2, the key member of Notch signaling pathway, was found in ovine follicles, and the expression of NOTCH2 mRNA was highest in the theca cells of the follicles in medium sizes (3–5mm in diameter) and granulosa cells of the follicles in large sizes (>5mm in diameter). Immunohistochemical results demonstrated that NOTCH2 protein was expressed in granulosa cells of preantral follicles, in both granulosa cells and theca cells of antral follicles. Compared with DAPT-treated groups, the control group had a higher number of granulosa cells (P<0.05) and a higher estradiol production (P<0.05). Compared with the control group, the mRNA abundances of HES1, MYC, BAX, BCL2 and CYP19A1 in DAPT-treated groups was lower (P<0.05), respectively; whereas, the expression of CCND2, CDKN1A and TP53 mRNA showed no remarkable difference compared with control group. Collectively, Notch signaling pathway could be involved in the ovine follicular development by regulating the growth and estradiol production of granulosa cells.

Prognostic roles of Notch receptor mRNA expression in human ovarian cancer.

Aberrant activation of Notch signaling pathway has been correlated with high grade ovarian carcinoma and carcinogenesis. However, the predictive and prognostic values of Notch signaling pathway in ovarian cancer patients remains unclear. We utilize “The Kaplan-Meier plotter” (KM plotter) background database to access the prognostic values including overall survival (OS), progression-free survival (PFS), as well as post-progression survival (PPS) of four Notch receptor mRNA expression in ovarian cancer patients. Notch1 mRNA high expression was not correlated with OS, PFS and PPS for all ovarian cancer patients, but significantly correlated with poor PFS in TP53 wild type and favorite PFS in TP53 mutation type ovarian cancer patients. Notch2 mRNA high expression was significantly correlated with poor PFS for all ovarian cancer patients, especially in grade II patients. Notch3 mRNA high expression was significantly correlated with favorite PFS for all ovarian cancer patients. Notch4 mRNA high expression was significantly correlated with favorite OS, but not PFS and PPS for all ovarian cancer patients. The results strongly support that there are distinct prognostic values of four Notch receptor mRNA expression in ovarian cancer patients.

Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

BackgroundInflammatory breast cancer (IBC), a particularly aggressive form of breast cancer, is characterized by cancer stem cell (CSC) phenotype. Due to a lack of targeted therapies, the identification of molecular markers of IBC is of major importance. The heparan sulfate proteoglycan Syndecan-1 acts as a coreceptor for growth factors and chemokines, modulating inflammation, tumor progression, and cancer stemness, thus it may emerge as a molecular marker for IBC.MethodsWe characterized expression of Syndecan-1 and the CSC marker CD44, Notch-1 & -3 and EGFR in carcinoma tissues of triple negative IBC (n = 13) and non-IBC (n = 17) patients using qPCR and immunohistochemistry. Impact of siRNA-mediated Syndecan-1 knockdown on the CSC phenotype of the human triple negative IBC cell line SUM-149 and HER-2-overexpressing non-IBC SKBR3 cells employing qPCR, flow cytometry, Western blotting, secretome profiling and Notch pharmacological inhibition experiments. Data were statistically analyzed using Student’s t-test/Mann-Whitney U-test or one-way ANOVA followed by Tukey’s multiple comparison tests.ResultsOur data indicate upregulation and a significant positive correlation of Syndecan-1 with CD44 protein, and Notch-1 & -3 and EGFR mRNA in IBC vs non-IBC. ALDH1 activity and the CD44(+)CD24(-/low) subset as readout of a CSC phenotype were reduced upon Syndecan-1 knockdown. Functionally, Syndecan-1 silencing significantly reduced 3D spheroid and colony formation. Intriguingly, qPCR results indicate downregulation of the IL-6, IL-8, CCL20, gp130 and EGFR mRNA upon Syndecan-1 suppression in both cell lines. Moreover, Syndecan-1 silencing significantly downregulated Notch-1, -3, -4 and Hey-1 in SUM-149 cells, and downregulated only Notch-3 and Gli-1 mRNA in SKBR3 cells. Secretome profiling unveiled reduced IL-6, IL-8, GRO-alpha and GRO a/b/g cytokines in conditioned media of Syndecan-1 knockdown SUM-149 cells compared to controls. The constitutively activated STAT3 and NFκB, and expression of gp130, Notch-1 & -2, and EGFR proteins were suppressed upon Syndecan-1 ablation. Mechanistically, gamma-secretase inhibition experiments suggested that Syndecan-1 may regulate the expression of IL-6, IL-8, gp130, Hey-1, EGFR and p-Akt via Notch signaling.ConclusionsSyndecan-1 acts as a novel tissue biomarker and a modulator of CSC phenotype of triple negative IBC via the IL-6/STAT3, Notch and EGFR signaling pathways, thus emerging as a promising therapeutic target for IBC.

Notch Signaling Participates in TGF-β-Induced SOST Expression Under Intermittent Compressive Stress.

Notch signaling is regulated by mechanical stimuli in various cell types. It has previously been reported that intermittent compressive stimuli enhanced sclerostin (SOST) expression in human periodontal ligament cells (hPDLs) by regulating transforming growth factor-β (TGF-β) expression. The aim of the present study was to determine the involvement of Notch signaling in the TGF-β-induced SOST expression in hPDLs. Cells were treated with intermittent compressive stress in a computer-controlled apparatus for 24 h. The mRNA and protein expression of the cells were determined by real-time polymerase chain reaction and Western blot analysis, respectively. In some experiments, the target signaling pathway was impeded by the addition of a TGF-β receptor kinase inhibitor (SB431542) or a γ-secretase inhibitor (DAPT). The results demonstrated that hPDLs under intermittent compressive stress exhibited significantly higher NOTCH2, NOTCH3, HES1, and HEY1 mRNA expression compared with control, indicating that mechanical stress induced Notch signaling. DAPT pretreatment markedly reduced the intermittent stress-induced SOST expression. The expression of NOTCH2, NOTCH3, HES1, and HEY1 mRNA under compressive stress was significantly reduced after pretreatment with SB431542, coinciding with a reduction in SOST expression. Recombinant human TGF-β1 enhanced SOST, Notch receptor, and target gene expression in hPDLs. Further, DAPT treatment attenuated rhTGF-β1-induced SOST expression. In summary, intermittent compressive stress regulates Notch receptor and target gene expression via the TGF-β signaling pathway. In addition, Notch signaling participates in TGF-β-induced SOST expression in hPDLs. J. Cell. Physiol. 232: 2221-2230, 2017. © 2016 Wiley Periodicals, Inc.