Knockdown Of Hes1 Research Articles

Notch signaling regulates limb regeneration through Hes1 and HeyL in the Chinese mitten crab

Tissue regeneration is an efficient strategy developed by animals to compensate for damaged tissues, involving various types of progenitor cells. Deciphering the signal network that modulates the activity of these progenitors during regeneration is crucial for understanding the differences in regenerative capacities across species. In this study, we evaluated the expression profile and phenotypic function of Notch signaling during limb regeneration in arthropod Chinese mitten crabs. The expression of key components of the Notch signaling pathway was upregulated at 7-day post-autotomy (7 DPA), and declined later at 18-day post-autotomy (18 DPA). To assess the role of Notch, we injected dsRNA targeting the Notch gene into the automized area and evaluated the regeneration efficiency. Our results indicated that blocking Notch signaling led to regenerative defects, manifested by delays in the wound closure and blastema emergence processes. Furthermore, the expression of Notch target genes, Hes1 and HeyL, was significantly reduced following Notch knockdown by dsRNA. Knockdown of Hes1 specifically impaired the proliferation and expression of neural progenitor cell markers, without affecting myogenic cells. In contrast, blockage of HeyL inhibited the proliferation and expression of markers in both activated neurogenic and myogenic progenitor cells, while up-regulating markers of quiescent neural progenitor cells. These findings suggest that Notch signaling plays an important role in limb regeneration of E. sinensis by activating downstream effectors Hes1 and HeyL, regulating neurogenesis and myogenesis through distinct mechanisms.

HES1 promotes aerobic glycolysis and cancer progression of colorectal cancer via IGF2BP2-mediated GLUT1 m6A modification.

Aerobic glycolysis has been shown to play a key role in tumor cell proliferation and metastasis. However, how it is directly regulated is largely unknown. Here, we found that HES1 expression was significantly higher in CRC tissues than that in adjacent normal tissues. Moreover, high HES1 expression is associated with poor survival in CRC patients. HES1 knockdown markedly inhibited cell growth and metastasis both in vitro and in vivo. Additionally, silencing of HES1 suppressed aerobic glycolysis of CRC cells. Mechanistic studies revealed that HES1 knockdown decreased the expression of GLUT1, a key gene of aerobic glycolysis, in CRC cells. GLUT1 overexpression abolished the effects of HES1 knockdown on cell aerobic glycolysis, proliferation, migration and invasion. ChIP-PCR and dual-luciferase reporter gene assay showed that HES1 directly bound the promoter of IGF2BP2 and promoted IGF2BP2 expression. Furthermore, our data indicated that IGF2BP2 recognized and bound the m6A site in the GLUT1 mRNA and enhanced its stability. Taken together, our findings suggest that HES1 has a significant promotion effect on CRC aerobic glycolysis and progression by enhancing the stability of m6A-modified GLUT1 mRNA in an IGF2BP2-dependent manner, which may become a viable therapeutic target for the treatment of CRC in humans. The mechanism of HES1 regulating glycolysis in CRC.

Notch Signaling Plays a Dual Role in Regulating the Neuron-to-Oligodendrocyte Switch in the Developing Dorsal Forebrain.

Neural progenitor cells in the developing dorsal forebrain generate excitatory neurons followed by oligodendrocytes (OLs) and astrocytes. However, the specific mechanisms that regulate the timing of this neuron-glia switch are not fully understood. In this study, we show that the proper balance of Notch signaling in dorsal forebrain progenitors is required to generate oligodendrocytes during late stages of embryonic development. Using ex vivo and in utero approaches in mouse embryos of both sexes, we found that Notch inhibition reduced the number of oligodendrocyte lineage cells in the dorsal pallium. However, Notch overactivation also prevented oligodendrogenesis and maintained a progenitor state. These results point toward a dual role for Notch signaling in both promoting and inhibiting oligodendrogenesis, which must be fine-tuned to generate oligodendrocyte lineage cells at the right time and in the right numbers. We further identified the canonical Notch downstream factors HES1 and HES5 as negative regulators in this process. CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated knockdown of Hes1 and Hes5 caused increased expression of the pro-oligodendrocyte factor ASCL1 and led to precocious oligodendrogenesis. Conversely, combining Notch overactivation with ASCL1 overexpression robustly promoted oligodendrogenesis, indicating a separate mechanism of Notch that operates synergistically with ASCL1 to specify an oligodendrocyte fate. We propose a model in which Notch signaling works together with ASCL1 to specify progenitors toward the oligodendrocyte lineage but also maintains a progenitor state through Hes-dependent repression of Ascl1 so that oligodendrocytes are not made too early, thus contributing to the precise timing of the neuron-glia switch.SIGNIFICANCE STATEMENT Neural progenitors make oligodendrocytes after neurogenesis starts to wind down, but the mechanisms that control the timing of this switch are poorly understood. In this study, we identify Notch signaling as a critical pathway that regulates the balance between progenitor maintenance and oligodendrogenesis. Notch signaling is required for the oligodendrocyte fate, but elevated Notch signaling prevents oligodendrogenesis and maintains a progenitor state. We provide evidence that these opposing functions are controlled by different mechanisms. Before the switch, Notch signaling through Hes factors represses oligodendrogenesis. Later, Notch signaling through an unknown mechanism promotes oligodendrogenesis synergistically with the transcription factor ASCL1. Our study underscores the complexity of Notch and reveals its importance in regulating the timing and numbers of oligodendrocyte production.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

REST, RCOR1 and RCOR2 expression is reduced in osteoarthritic chondrocytes and contributes to increasing MMP13 and ADAMTS5 expression through upregulating HES1

Expression of key transcriptional regulators is altered in chondrocytes in osteoarthritis (OA). This contributes to an increase in production of cartilage-catabolizing enzymes such as MMP13 and ADAMTS5. RCOR1 and RCOR2, binding partners for the transcriptional repressor REST, have previously been found to be downregulated in OA chondrocytes although their function in chondrocytes is unclear. HES1 is a known REST/RCOR1 target gene and HES1 has been shown to promote MMP13 and ADAMTS5 expression in murine OA chondrocytes. The purpose of this study was to determine whether reduced REST/RCOR levels leads to increased HES1 expression in human OA chondrocytes and whether HES1 also promotes ADAMTS5 and MMP13 expression in these cells.Chondrocytes were isolated from osteoarthritic and adjacent macroscopically normal cartilage obtained from patients undergoing total knee arthroplasty. RNA and protein levels of REST, RCOR1 and RCOR2 were lower, but levels of HES1 higher, in chondrocytes isolated from osteoarthritic compared to macroscopically normal cartilage. Over-expression of either REST, RCOR1 or RCOR2 resulted in reduced HES1 levels in OA chondrocytes whereas knockdown of REST, RCOR1 or RCOR2 led to increased HES1 expression in chondrocytes from macroscopically normal cartilage. In OA chondrocytes, ADAMTS5 and MMP13 expression were reduced following HES1 knockdown, but further enhanced following HES1 over-expression. Levels of phosphorylated CaMKII were higher in chondrocytes from OA cartilage consistent with previous findings that HES1 only promotes gene transcription in the presence of active CaMKII.These findings identify the REST/RCOR/HES1 pathway as a contributing factor leading to increased ADAMTS5 and MMP13 expression in OA chondrocytes.

Concentration-dependent effects of leptin on osteoarthritis-associated changes in phenotype of human chondrocytes

ABSTRACT Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.

Abstract P6-14-05: Regulation of cellular identity and spatial organization during collective breast cancer invasion

Abstract An early step in breast cancer progression is invasion of tumor cells into surrounding tissues. In many breast cancers, particularly ductal carcinomas, this invasion is accomplished by tumor cells migrating as a cohesive group. This often involves cells that take on heterogeneous roles as either leader or follower cells. While studies in common mouse and human breast cancer models have established that leader cells express high levels of keratin-14 (K14) and other basal epithelial markers, the molecular mechanisms regulating K14+ leader cell identity remain obscure. Here we performed time-sampled single cell RNA-sequencing in 3D type I collagen-embedded tumor organoids isolated from the MMTV-PyMT luminal B model of breast cancer. 11 distinct cellular transcriptional states were identified and correlated with K14 expression and invasive strand formation. Having identified the leader cell state we next asked what transcription factors were enriched, reasoning that transcription factors could be master regulators of leader cell fate. 30 different shRNAs targeting 10 genes were systematically evaluated for their effects on collective invasion. From this screen, suppression of Hes1, the downstream target of Notch signaling, yielded a marked switch from collective to single cell invasion. Disseminating single tumor cells maintained high expression of K14 in Hes1 knockdown organoids which was phenocopied by gamma-secretase inhibition in a human TNBC PDX model. Because K14+ tumor cells highly express the Notch ligand Jag1, these results support a model in which Notch signaling, specifically through activation of Hes1, dictates leader cell identity and spatial organization during collective invasion. Studies are ongoing investigating the impact of Hes1 dynamics on leader cell adhesion, hybrid EMT state, and preference for single versus collective metastasis. Because Notch suppression induces leader cell dissemination, we propose that Notch targeted therapy should be combined with therapies eradicating leader cells. Citation Format: Andrea E. Doak, Kevin J. Cheung. Regulation of cellular identity and spatial organization during collective breast cancer invasion. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-05.

Notch signaling inhibitor and anti-PD-L1 antibody combination therapies decelerate tumor progression in pancreatic cancer

Abstract Objective: Pancreatic cancer (PC) is a highly lethal malignancy with an immunosuppressive environment. Yet, current immune checkpoint inhibitor monotherapies have shown limited efficacy in PC, prompting the need for combination therapies. Herein, we hypothesized that combinations of Notch signaling inhibitor and anti-ligand programmed death-ligand 1 (PD-L1) antibody immunotherapy would show synergistic efficacy. Methods: The baseline expression of PD-L1 and HES1 was measured in PC cell lines, single-cell RNA-seq data of PC (GSA: CRA001160), and cBioPortal databases. In an in vitro study, MIA PaCa2 and SW1990 were used to explore the mechanism between Notch signaling and PD-L1. To study the effects in vivo, a subcutaneous tumor model was established using Pan02 cells treated with either anti-PD-L1 monoclonal antibody and/or Notch inhibitor DAPT. The study performed involving human samples was approved by the Ethics Committee of Peking Union Medical College Hospital (approval No. S-K460, approval date: April 23, 2018). Animal studies were approved by the Animal Research Ethics Committee of Peking Union Medical College Hospital (approval No. XHDW-2019-049, approval date: November 28, 2019). Results: The Notch signaling inhibitor upregulated PD-L1 expression in PC tumor cells both in vitro and in vivo. Notch effector HES1 knockdown produced PD-L1 upregulation in both MIA PaCa2 and SW1990 cells. Combined DAPT and anti-PD-L1 antibody treatment of Pan02 subcutaneous tumor model resulted in significantly reduced tumor weights compared to that with monotherapy, as well as significantly reduced Ki67 than that in the monotherapy group and control group. Flow cytometry analysis revealed significantly increased CD8+ T cell infiltration in tumors of the combination group compared with those of the monotherapy group. Conclusion: Notch signaling blockade might enhance the antitumor effect of anti-PD-L1 therapy in PC.

MiR-449a Repression Leads to Enhanced NOTCH Signaling in TMPRSS2:ERG Fusion Positive Prostate Cancer Cells.

Simple SummaryThe reason for the frequent presence of the TMPRSS2:ERG (T2E) gene fusion in prostate cancer (PCa) is not fully understood. We were interested in investigating epigenomic alterations associated with the T2E gene fusion in PCa cells. Making use of publicly available genome-wide data and in vitro analyses using an LNCaP cell line model with inducible T2E expression, we uncovered a molecular network driving NOTCH signaling in T2E-positive PCa. We show that ERG directly activates NOTCH1 and HES1 expression by interacting with their promoter regions. Furthermore, NOTCH is activated by downregulation of miR-449a in ERG overexpressing cells. Experimental NOTCH pathway inhibition as well as HES1 knockdown reduced oncogenic capacities in vitro, suggesting that the NOTCH pathway triggers oncogenic processes in TMPRSS2:ERG-positive PCa. HES1 and ERG expression are correlated in tissue samples from PCa patients. Our data suggest a novel epigenomic network driving NOTCH signaling in T2E+ PCa cells.About 50% of prostate cancer (PCa) tumors are TMPRSS2:ERG (T2E) fusion-positive (T2E+), but the role of T2E in PCa progression is not fully understood. We were interested in investigating epigenomic alterations associated with T2E+ PCa. Using different sequencing cohorts, we found several transcripts of the miR-449 cluster to be repressed in T2E+ PCa. This repression correlated strongly with enhanced expression of NOTCH and several of its target genes in TCGA and ICGC PCa RNA-seq data. We corroborated these findings using a cellular model with inducible T2E expression. Overexpression of miR-449a in vitro led to silencing of genes associated with NOTCH signaling (NOTCH1, HES1) and HDAC1. Interestingly, HDAC1 overexpression led to the repression of HES6, a negative regulator of the transcription factor HES1, the primary effector of NOTCH signaling, and promoted cell proliferation by repressing the cell cycle inhibitor p21. Inhibition of NOTCH as well as knockdown of HES1 reduced the oncogenic properties of PCa cell lines. Using tissue microarray analysis encompassing 533 human PCa cores, ERG-positive areas exhibited significantly increased HES1 expression. Taken together, our data suggest that an epigenomic regulatory network enhances NOTCH signaling and thereby contributes to the oncogenic properties of T2E+ PCa.

Cochlea cell-specific marker expression upon in vitro Hes1 knockdown.

NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

HES1 promotes breast cancer stem cells by elevating Slug in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. TNBC is enriched with breast cancer stem cells (BCSCs), which are responsible for cancer initiation, cancer progression and worse prognosis. Our previous study found that HES1 was overexpressed and promoted invasion in TNBC. However, the role of HES1 in modulating BCSC stemness of TNBC remains unclear. Here, we found that HES1 upregulates Slug both in transcriptional level and in protein level. HES1 also has a positive correlation with Slug expression in 150 TNBC patient samples. TNBC patients with high HES1 and Slug levels show worse prognosis in both progression-free survival and overall survival analyses. Survival analyses indicate that the effects of HES1 on survival prognosis may depend on Slug. Furthermore, we reveal that HES1 is a novel transcriptional activator for Slug through acting directly on its promoter. Meanwhile, HES1 knockdown reduces BCSC self-renewal, BCSC population, and cancer cell proliferation in TNBC, whereas overexpression of Slug restores the oncogenic function of HES1, both in vitro and in vivo, suggesting that HES1 performs its oncogenic role through upregulating Slug. Taken together, HES1 promotes BCSC stemness properties via targeting Slug, highlighting that HES1 might be a novel candidate for BCSC stemness regulation in TNBC and providing new clues for identifying promising prognostic biomarkers and therapeutic targets of TNBC.

MYEOV increases HES1 expression and promotes pancreatic cancer progression by enhancing SOX9 transactivity.

Emerging evidence indicates that myeloma overexpressed (MYEOV) is an oncogene and plays crucial roles in multiple human cancers. However, its roles in the development of pancreatic ductal adenocarcinoma (PDAC) remain elusive. Here, we provide evidence of essential roles of MYEOV in the development and progression of PDAC. In tumor specimens derived from pancreatic cancer patients, MYEOV was overexpressed and associated with poor prognosis. In addition, MYEOV expression in PDAC was upregulated through promoter hypomethylation. MYEOV depletion impaired metastatic ability and proliferation of PDAC cells both in vitro and in vivo, whereas its overexpression had the opposite effect. Mechanistic investigations revealed that MYEOV interacted with SRY-Box Transcription Factor 9 (SOX9), a well-known oncogenic transcription factor in PDAC. This interaction occurred mainly in the nuclei of PDAC cells and increased transcriptional activity of SOX9. Furthermore, MYEOV promoted the expression of Hairy and enhancer of split homolog-1 (HES1), a SOX9 target gene, by enhancing SOX9 DNA-binding ability to the HES1 enhancer without affecting the protein level and subcellular localization of SOX9. HES1 knockdown partly abrogated the oncogenic effect of MYEOV. Our findings suggest that MYEOV could be a potential prognostic biomarker and therapeutic target for PDAC.

HES1 and HES4 have non-redundant roles downstream of Notch during early human T-cell development.

In both mouse and human, Notch1 activation is the main initial driver to induce T-cell development in hematopoietic progenitor cells. The initiation of this developmental process coincides with Notch1-dependent repression of differentiation towards other hematopoietic lineages. Although well described in mice, the role of the individual Notch1 target genes during these hematopoietic developmental choices is still unclear in human, particularly for HES4 since no orthologous gene is present in the mouse. Here, we investigated the functional capacity of the Notch1 target genes HES1 and HES4 to modulate human Notch1-dependent hematopoietic lineage decisions and their requirement during early T-cell development. We show that both genes are upregulated in a Notch-dependent manner during early T-cell development and that HES1 acts as a repressor of differentiation by maintaining a quiescent stem cell signature in CD34+ hematopoietic progenitor cells. While HES4 can also inhibit natural killer and myeloid cell development like HES1, it acts differently on the T- versus B-cell lineage choice. Surprisingly, HES4 is incapable of repressing B-cell development, the most sensitive hematopoietic lineage with respect to Notch-mediated repression. In contrast to HES1, HES4 promotes initiation of early T-cell development, but ectopic expression of HES4, or HES1 and HES4 combined, is insufficient to induce T-lineage differentiation. Importantly, knockdown of HES1 or HES4 significantly reduces human T-cell development. Overall, we show that the Notch1 target genes HES1 and HES4 have non-redundant roles during early human T-cell development which may relate to differences in mediating Notch-dependent human hematopoietic lineage decisions.

PDK1 Regulates Transition Period of Apical Progenitors to Basal Progenitors by Controlling Asymmetric Cell Division.

The six-layered neocortex consists of diverse neuron subtypes. Deeper-layer neurons originate from apical progenitors (APs), while upper-layer neurons are mainly produced by basal progenitors (BPs), which are derivatives of APs. As development proceeds, an AP generates two daughter cells that comprise an AP and a deeper-layer neuron or a BP. How the transition of APs to BPs is spatiotemporally regulated is a fundamental question. Here, we report that conditional deletion of phoshpoinositide-dependent protein kinase 1 (PDK1) in mouse developing cortex achieved by crossing Emx1Cre line with Pdk1fl/fl leads to a delayed transition of APs to BPs and subsequently causes an increased output of deeper-layer neurons. We demonstrate that PDK1 is involved in the modulation of the aPKC-Par3 complex and further regulates the asymmetric cell division (ACD). We also find Hes1, a downstream effecter of Notch signal pathway is obviously upregulated. Knockdown of Hes1 or treatment with Notch signal inhibitor DAPT recovers the ACD defect in the Pdk1 cKO. Thus, we have identified a novel function of PDK1 in controlling the transition of APs to BPs.

Hes1 Knockdown Exacerbates Ischemic Stroke Following tMCAO by Increasing ER Stress-Dependent Apoptosis via the PERK/eIF2α/ATF4/CHOP Signaling Pathway.

Apoptosis induced by endoplasmic reticulum (ER) stress plays a crucial role in mediating brain damage after ischemic stroke. Recently, Hes1 (hairy and enhancer of split 1) has been implicated in the regulation of ER stress, but whether it plays a functional role after ischemic stroke and the underlying mechanism remain unclear. In this study, using a mouse model of ischemic stroke via transient middle cerebral artery occlusion (tMCAO), we found that Hes1 was induced following brain injury, and that siRNA-mediated knockdown of Hes1 increased the cerebral infarction and worsened the neurological outcome, suggesting that Hes1 knockdown exacerbates ischemic stroke. In addition, mechanistically, Hes1 knockdown promoted apoptosis and activated the PERK/eIF2α/ATF4/CHOP signaling pathway after tMCAO. These results suggest that Hes1 knockdown promotes ER stress-induced apoptosis. Furthermore, inhibition of PERK with the specific inhibitor GSK2606414 markedly attenuated the Hes1 knockdown-induced apoptosis and the increased cerebral infarction as well as the worsened neurological outcome following tMCAO, implying that the protection of Hes1 against ischemic stroke is associated with the amelioration of ER stress via modulating the PERK/eIF2α/ATF4/CHOP signaling pathway. Taken together, these results unveil the detrimental role of Hes1 knockdown after ischemic stroke and further relate it to the regulation of ER stress-induced apoptosis, thus highlighting the importance of targeting ER stress in the treatment of ischemic stroke.

Overexpression of Notch3 is associated with metastasis and poor prognosis in osteosarcoma patients

BackgroundNotch signaling abnormalities are associated with the development of various tumors, including hematopoietic and epithelium-derived tumors. However, the role of Notch signaling in tumors originating from mesenchymal cells is unclear. The effect of Notch3 expression on the prognosis of osteosarcoma and its role and mechanism in osteosarcoma cells have never been reported.Materials and methodsIn this study, we performed a clinicopathological analysis of 70 cases of osteosarcoma, with primary focus on survival. Osteosarcoma cell lines MTH and U2OS were used. After knockdown of Notch3 by lentiviral transfection and siRNA, the cell cycle, cell viability, and wound healing capacity were assessed. Subsequently, the Transwell assay was performed, and the expression levels of hairy and enhancer of split-1 (Hes1) and matrix metalloproteinase 7 (MMP7) were detected by RT-PCR and Western blot assay. The expression of MMP7 was also detected after knockdown of Hes1. Animal experiments were performed by injecting the cell lines MTH of Notch3 knockdown into mice tail veins and comparing the development of lung metastasis with the control group.ResultsComparison of survival curves showed that Notch3 expression significantly impacts patient survival. Additionally, multivariate analysis revealed that Notch3 is an independent prognostic factor for osteosarcoma. In in vivo experiments, osteosarcoma-associated pulmonary metastasis in nude mice was reduced after Notch3 silencing. The expression of downstream effector molecule, Hes1, and that of the invasion and metastasis-associated proteolytic enzyme, MMP7, were reduced, and MMP7 was further decreased by Hes1 knockdown in in vitro experiments.ConclusionNotch3 is a prognostic factor for osteosarcoma and might regulate its invasion and metastasis through the downstream target gene Hes1 and effector MMP7.

MicroRNA-374b promotes the proliferation and differentiation of neural stem cells through targeting Hes1

Accumulating evidence has documented that microRNAs (miRNAs) are critical regulators of neural stem cell (NSC) proliferation and differentiation. MiRNA-374b (miR-374b) has been reported to play an important role in regulating various cellular processes, such as proliferation and differentiation. However, whether miR-374b is involved in NSC proliferation and differentiation remains unclear. In this study, we investigated the potential role of miR-374b in regulating NSC proliferation and differentiation to elucidate the underlying molecular mechanism. Our results showed that miR-374b expression was significantly upregulated during NSC differentiation. Functional experiments showed that overexpression of miR-374b promoted NSC proliferation and differentiation to neurons. By contrast, miR-374b inhibition showed the opposite effect. Hairy and enhancer of split 1 (Hes1), a master regulator of neurogenesis, was predicted as a potential target gene of miR-374b by bioinformatics analysis. Dual-luciferase reporter assays showed that miR-374b could directly target the 3′-untranslated region of Hes1. Further experiments showed that miR-374b negatively regulated the mRNA and protein expression of Hes1 in NSCs. Moreover, overexpression of Hes1 significantly reversed the miR-374b overexpression-mediated effect on NSC proliferation and differentiation. In addition, knockdown of Hes1 abrogated the miR-374b inhibition-mediated effect on NSC proliferation and differentiation. Taken together, these results demonstrate that miR-374b regulates the proliferation and differentiation of NSCs through targeting Hes1 and suggest that miR-374b is a potential target for modulating NSC-mediated neurogenesis.

A personalized, multiomics approach identifies genes involved in cardiac hypertrophy and heart failure

A traditional approach to investigate the genetic basis of complex diseases is to identify genes with a global change in expression between diseased and healthy individuals. However, population heterogeneity may undermine the effort to uncover genes with significant but individual contribution to the spectrum of disease phenotypes within a population. Here we investigate individual changes of gene expression when inducing hypertrophy and heart failure in 100 + strains of genetically distinct mice from the Hybrid Mouse Diversity Panel (HMDP). We find that genes whose expression fold-change correlates in a statistically significant way with the severity of the disease are either up or down-regulated across strains, and therefore missed by a traditional population-wide analysis of differential gene expression. Furthermore, those “fold-change” genes are enriched in human cardiac disease genes and form a dense co-regulated module strongly interacting with the cardiac hypertrophic signaling network in the human interactome. We validate our approach by showing that the knockdown of Hes1, predicted as a strong candidate, induces a dramatic reduction of hypertrophy by 80–90% in neonatal rat ventricular myocytes. Our results demonstrate that individualized approaches are crucial to identify genes underlying complex diseases as well as to develop personalized therapies.

Inhibition of Hes1 enhances lapatinib sensitivity in gastric cancer sphere-forming cells.

It has been considered that the neurogenic locus notch homolog protein (Notch) signaling pathway serves an essential role in cellular differentiation, proliferation and apoptosis. However, the function of the Notch signaling pathway in gastric cancer stem cells (GCSCs) and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) sensitivity remains unclear. The present study aimed to delineate the role of the Notch1 signaling pathway in GCSCs and lapatinib sensitivity. Sphere-forming cells were separated from human gastric cancer MKN45 parental cells. The sphere-forming cells exhibited characteristics of CSCs and higher Notch1 expression compared with that of parental cells. To investigate the role of the Notch1 signaling pathway in GCSCs, the expression of transcription factor Hes1 (Hes1) was knocked down using small interfering RNA against Hes1. It was observed that Hes1 expression was significantly downregulated in knocked down cells. The inhibition of Hes1 suppressed the properties of CSCs, as indicated by significant decreases in the expression of the transcription factor sex determining region Y-box 2, epithelial cell adhesion molecule and the homeobox protein Nanog and reduced spheroid colony formation. In addition, epithelial-mesenchymal transition was significantly impaired in sphere-forming cells following Hes1 knockdown. Furthermore, the inhibition of Hes1 effectively enhanced lapatinib sensitivity in sphere-forming cells. These results suggest that sphere-forming gastric cancer cells possess the characteristics of CSCs, and that the Notch1 signaling pathway serves an essential role in the maintenance of CSCs and lapatinib sensitivity.

Activation of STAT3/HIF-1α/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN

BackgroundResistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events. Material and methodsWe have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance. ResultsWe found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells. ConclusionThe impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein. General significanceThese findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.