Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1 / 中国医学科学院学报

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

Regulation of Notch1 and Foxp1 by MiR-34a in psoriasis vulgaris / Regulación de Notch1 y Foxp1 por MiR-34ª en psoriasis vulgaris

SUMMARY: This study is to investigate the regulation of Notch1 and Foxp1 by miR-34a in the development of psoriasis vulgaris. RT-PCR was used to compare the levels of miR-34a in the skin lesions of 20 patients with psoriasis vulgaris and 20 normal skin tissues. Immunohistochemistry was used to detect the expression of Notch1 and Foxp1 in 51 patients with psoriasis vulgaris, which were further compared with that in 29 normal control tissues. In addition, in HaCaT cells, we used miR-34a mimics and inhibitors to overexpress and inhibit miR-34a, respectively, and detected the mRNA and protein levels of miR-34a, Notch1, and Foxp1. The level of miR-34a in the skin lesions of patients with psoriasis vulgaris was significantly higher than that in normal skin tissues (t=2.192, P<0.05). The positive rate of Notch1 in the skin lesions of patients with psoriasis vulgaris was 76.47 %, which was significantly higher than that in normal skin tissues (13.79 %) (t=29.215, P<0.01). The positive rate of FOXP1 in the psoriasis vulgaris group was 92.16 %, which was also significantly higher than that in the normal skin group (65.52 %) (t=9.087, P<0.01). In addition, overexpression of miR-34a significantly promoted the expression of Notch1 and Foxp1. However, inhibition of miR-34a significantly reduced Notch1 and Foxp1 levels. miR- 34a is highly expressed in the skin tissues of patients with psoriasis vulgaris, and may participate in the development of psoriasis vulgaris by regulating Notch1 and Foxp1.

RESUMEN: El objetivo de este estudio fue investigar la regulación de Notch1 y Foxp1 por miR-34a en el desarrollo de la psoriasis vulgar. Se utilizó RT-PCR con el fin de comparar los niveles de miR-34a en las lesiones cutáneas de 20 pacientes con psoriasis vulgar y 20 tejidos de piel normales. Se utilizó inmunohistoquímica para detectar la expresión de Notch1 y Foxp1 en 51 pacientes con psoriasis vulgar, que se compararon además con la de 29 tejidos normales control. Además, en las células HaCaT, usamos miméticos e inhibidores de miR-34a para sobreexpresar e inhibir miR-34a, respectivamente, y detectamos los niveles de ARNm y proteína de miR-34a, Notch1 y Foxp1. El nivel de miR- 34a en las lesiones cutáneas de pacientes con psoriasis vulgar fue significativamente mayor que en los tejidos normales de la piel (t=2,192, P<0,05). La tasa de positividad de Notch1 en las lesiones cutáneas de pacientes con psoriasis vulgar fue del 76,47 %, que fue significativamente mayor que la de los tejidos normales de la piel (13,79 %) (t=29,215, P<0,01). La tasa positiva de FOXP1 en el grupo de psoriasis vulgar fue del 92,16 %, que también fue significativamente mayor que la del grupo de piel normal (65,52 %) (t=9,087, P<0,01). Además, la sobreexpresión de miR-34a promovió significativamente la expresión de Notch1 y Foxp1. Sin embargo, la inhibición de miR-34a redujo de manera importante los niveles de Notch1 y Foxp1. miR-34a se expresa en gran medida en los tejidos de la piel en pacientes con psoriasis vulgar y puede participar en el desarrollo de la psoriasis vulgar mediante la regulación de Notch1 y Foxp1.

Chidamide inhibits the NOTCH1-MYC signaling axis in T-cell acute lymphoblastic leukemia / 医学前沿

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most dangerous hematological malignancies, with high tumor heterogeneity and poor prognosis. More than 60% of T-ALL patients carry NOTCH1 gene mutations, leading to abnormal expression of downstream target genes and aberrant activation of various signaling pathways. We found that chidamide, an HDAC inhibitor, exerts an antitumor effect on T-ALL cell lines and primary cells including an anti-NOTCH1 activity. In particular, chidamide inhibits the NOTCH1-MYC signaling axis by down-regulating the level of the intracellular form of NOTCH1 (NICD1) as well as MYC, partly through their ubiquitination and degradation by the proteasome pathway. We also report here the preliminary results of our clinical trial supporting that a treatment by chidamide reduces minimal residual disease (MRD) in patients and is well tolerated. Our results highlight the effectiveness and safety of chidamide in the treatment of T-ALL patients, including those with NOTCH1 mutations and open the way to a new therapeutic strategy for these patients.

Valproic acid withdrawal ameliorates impairments of hippocampal-spatial working memory and neurogenesis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Valproic acid (VPA), an agent that is used to treat epileptic seizures, can cause spatial memory impairment in adults and children. This effect is thought to be due to the ability of VPA to inhibit neurogenesis in the hippocampus, which is required for learning. We have previously used an animal model to show that VPA significantly impairs hippocampal-spatial working memory and inhibits neuronal generation in the sub-granular zone of the dentate gyrus. As there are patient reports of improvements in memory after discontinuing VPA treatment, the present study investigated the recovery of both spatial memory and hippocampal neurogenesis at two time points after withdrawal of VPA. Male Wistar rats were given intraperitoneal injections of 0.9% normal saline or VPA (300 mg/kg) twice a day for 10 d. At 1, 30, or 45 d after the drug treatment, the novel object location (NOL) test was used to examine spatial memory; hippocampal cell division was counted using Ki67 immunohistochemistry, and levels of brain-derived neurotrophic factor (BDNF) and Notch1 were measured using western immunoblotting. Spatial working memory was impaired 1 and 30 d after the final administration, but was restored to control levels by 45 d. Cell proliferation had increased to control levels at 30 and 45 d. Both markers of neurogenesis (BDNF and Notch1 levels) had returned to control levels at 45 d. These results demonstrate that memory recovery occurs over a period of six weeks after discontinuing VPA treatment and is preceded by a return of hippocampal neurogenesis to control levels.

HtrA1 suppresses the growth of pancreatic cancer cells by modulating Notch-1 expression

Pancreatic cancer is well known to be the most deadly malignancy with the worst survival rate of all cancers. High temperature requirement factor A1 (HtrA1) plays an important role in cancer cell proliferation, migration, apoptosis, and differentiation. This study aimed to explore the function of HtrA1 in pancreatic cancer cell growth and its underlying mechanism. We found that the expression of HtrA1 was lower in pancreatic cancer tissue compared to the adjacent normal tissue. Consistently, HtrA1 levels were also decreased in two human pancreatic cancer cell lines, PANC-1 and BXPC-3. Moreover, enforced expression of HtrA1 inhibited cell viability and colony formation of PANC-1 and BXPC-3 cells. Overexpression of HtrA1 promoted apoptosis and suppressed migratory ability of tumor cells. On the contrary, siRNA-mediated knockdown of HtrA1 promoted the growth potential of pancreatic cancer cells. In addition, we found that up-regulation of HtrA1 reduced the expression of Notch-1 in pancreatic cancer cells. On the contrary, knockdown of HtrA1 increased the expression levels of Notch-1. Furthermore, overexpression of Notch-1 abolished the anti-proliferative effect of HtrA1 on pancreatic cancer cells. Taken together, our findings demonstrated that HtrA1 could inhibit pancreatic cancer cell growth via regulating Notch-1 expression, which implied that HtrA1 might be developed as a novel molecular target for pancreatic cancer therapy.

Co-expression patterns of Notch1, Snail, and p53 in grade III hepatocellular carcinoma with postoperative recurrence: a preliminary study / 대한간학회지

BACKGROUND/AIMS: We aimed to determine the association between the co-expression patterns of Notch1, Snail, and p53 proteins (NSP) and the postoperative prognosis of hepatocellular carcinoma (HCC). METHODS: The immunoblot data for molecular expression (147 HCC/corresponding non-HCC tissues and 15 dysplastic nodules) and the sequencing data for p53 mutations (110 HCCs) were obtained from our previous study. Data analyses were restricted to cases with HCC differentiation grade III (n=47), due to its high p53 mutation rate. RESULTS: Nineteen of the 47 patients (40.4%) -comprising 12 in the liver and 7 in distant organs-had relapsed at 1-2 years after surgery. There was no relationship between p53 mutation and postoperative recurrence in the grade III HCCs. Seven (87.5%) of the eight relapsed cases with Notch1, Snail, and p53 (wild) co-expression experienced recurrence only within the liver, and all tumors were smaller than 5 cm in diameter. Extrahepatic relapse occurred mostly in HCC patients with tumors larger than 5 cm in diameter, without any deviation in the NSP pattern. CONCLUSIONS: The results of this preliminary study suggest that the co-expression of Notch1, Snail, and p53 (wild) is not inferior to the patterns with p53 mutation as an indicator of postoperative recurrence of grade III HCC.

The mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells into myocardial cells induced by 5-azacytidine.

Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.

Isolation of stem cells from adult rat kidneys

The kidney has an inherent ability for recovery and regeneration following acute damage. However, there has been much contention as to the source of regenerating renal cells. The aim of this study was to isolate and characterize these cells. Normal rat kidneys were minced and cells were isolated with collagenase I and were cultured in an expansion medium. Adherent cells were isolated and expanded for more than 120 days in vitro. These cells had the potential of trans-lineage differentiation into neural cells, adipocytes and osteocytes. These cells also expressed Nucleostemin, Cyclin D1, Notch1 and Survivin which are commonly expressed in stem cells. The results of the current work show that the adult kidney contains a population of multipotent stem cells.

Sequential expressions of Notch1, Jagged2 and Math1 in molar tooth germ of mouse

The Notch signaling pathway is an evolutionary conserved mechanism that plays an important role in cell-cell communication and cell fate in a wide range of tissues. The mammalian family of Notch receptors consists of 4 members: Notch1/2/3/4. The Notch ligand family consists of 5 members: Delta1/3/4 and Jagged1/2. Math1 encodes a murine Basic helix-loop-helix (bHLH) transcription factor that acts as positive regulator of cell differentiation. Recently, links between Notch and Math1 pathways were demonstrated in various tissues. Expression of Notch1, Jagged2 and Math1 were analyzed in the mouse molar tooth germ during embryonic stage (E) 13 and E15 and during postnatal stage (PN) 1, PN3, PN5, PN10 and PN14 by using in situ hybridization. Positive Notch1 expression was found at the tooth bud during embryonic stages, but its expression was absent from the basal cells in contact with the dental mesenchyme. Jagged2 and Math1 were strongly expressed in differentiated ameloblasts and odontoblasts and Math1 strong expression was even maintained until PN14 stage. Math1 showed the strongest expression. Our results suggest that the Notch1 signaling pathway through Jagged2 could be importantly related to Math1, directing the process of odontogenesis toward cell differentiation.

Expression of conserved signalling pathway genes during spontaneous vascular differentiation of R1 embryonic stem cells and in Py-4-1 endothelial cells.

Embryonic stem (ES) cells are an invaluable model for identifying subtle phenotypes as well as severe outcomes of perturbing gene function that may otherwise result in lethality. However,though ES cells of different origins are regarded as equally pluripotent,their in vitro differentiation potential varies, suggesting that their response to developmental signals is different. The R1 cell line is widely used for gene manipulation due to its good growth characteristics and highly efficient germline transmission. Hence, we analysed the expression of Notch, Wnt and Sonic Hedgehog (Shh) pathway genes during differentiation of R1 cells into early vascular lineages. Notch-, Wnt- and Shh-mediated signalling is important during embryonic development. Regulation of gene expression through these signalling molecules is a frequently used theme, resulting in context-dependent outcomes during development. Perturbing these pathways can result in severe and possibly lethal developmental phenotypes often due to primary cardiovascular defects. We report that during early spontaneous differentiation of R1 cells, Notch-1 and the Wnt target Brachyury are active whereas the Shh receptor is not detected. This expression pattern is similar to that seen in a mouse endothelial cell line. This temporal study of expression of genes representative of all three pathways in ES cell differentiation will aid in further analysis of cell signalling during vascular development.