Proliferation Of SH-SY5Y Cells Research Articles

Corrigendum to: An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells

Corrigendum to: An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells

Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-β), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Proliferation of SH-SY5Y neuroblastoma cells on confined spaces

Background:Microfluidics offers precise drug delivery and continuous monitoring of cell functions, which is crucial for studying the effects of toxins and drugs. Ensuring proper cell growth in these space-constrained systems is essential for obtaining consistent results comparable to standard Petri dishes. New method:We investigated the proliferation of SH-SY5Y cells on circular polycarbonate chambers with varying surface areas. SH-SY5Y cells were chosen for their relevance in neurodegenerative disease research. Results:Our study demonstrates a correlation between the chamber surface area and SH-SY5Y cell growth rates. Cells cultured in chambers larger than 10 mm in diameter exhibited growth comparable to standard 60-mm dishes. In contrast, smaller chambers significantly impeded growth, even at identical seeding densities. Similar patterns were observed for HeLaGFP cells, while 16HBE14σ cells proliferated efficiently regardless of chamber size. Additionally, SH-SY5Y cells were studied in a 12-mm diameter sealed chamber to assess growth under restricted gas exchange conditions. Comparison with existing methods:Our findings underscore the limitations of small chamber sizes in microfluidic systems for SH-SY5Y cells, an issue not typically addressed by conventional methods. Conclusions:SH-SY5Y cell growth is highly sensitive to spatial constraints, with markedly reduced proliferation in chambers smaller than 10 mm. This highlights the need to carefully consider chamber size in microfluidic experiments to achieve cell growth rates comparable to standard culture dishes. The study also shows that while SH-SY5Y and HeLaGFP cells are affected by chamber size, 16HBE14σ cells are not. These insights are vital for designing effective microfluidic systems for bioengineering research.

Hirschsprung's disease: m6A methylase VIRMA suppresses cell migration and proliferation by regulating GSK3β.

N6-methyladenosine (m6A) is the most abundant mRNA modification in mammals, participating in various biological processes. VIRMA is a key methyltransferase involved in m6A modification. However, the role of VIRMA in Hirschsprung's disease (HSCR) remains unclear. This study aims to investigate the function of VIRMA in HSCR and identify its corresponding regulatory mechanisms. The expression of VIRMA and GSK3β in colon tissues of HSCR was examined using RT-qPCR, Western blot, and Immunohistochemistry. Immunofluorescence detected localization of VIRMA and GSK3β. Cell proliferation was measured by CCK8 and EdU assays, and cell migration was evaluated via cell migration and wound healing assays. The stability of GSK3β mRNA was assessed using the actinomycin D assay and the overall level of m6A in cells was assessed by colorimetric assay. VIRMA was significantly downregulated in narrow-segment colon tissue. Silencing of VIRMA inhibited cell proliferation and migration. VIRMA can inhibit the degradation of GSK3β mRNA and increase the expression of GSK3β. GSK3β was significantly upregulated in narrow-segment colon tissues. Accordingly, our findings showed that GSK3β mediated the VIRMA-driven cell migration and proliferation. VIRMA can inhibit cell migration and proliferation by upregulating the expression of GSK3β, contributing to the onset of HSCR. The expressions of VIRMA were significantly reduced in HSCR, while GSK3β expression was increased in HSCR, and can be used as a molecular marker. VIRMA overexpression promoted the proliferation and migration of SH-SY5Y and HEK-293T cells. VIRMA can inhibit the degradation of GSK3β mRNA and increase the expression of GSK3β.

Circulating miR-30e-3p induces disruption of neurite development in SH-SY5Y cells by targeting ABI1, a novel biomarker for schizophrenia

Schizophrenia (SCZ) represents a set of enduring mental illnesses whose underlying etiology remains elusive, posing a significant challenge to public health. Previous studies have shown that the neurodevelopmental process involving small molecules such as miRNA and mRNA is one of the etiological hypotheses of SCZ. We identified and verified that miR-30e-3p and ABI1 can be used as biomarkers in peripheral blood transcriptome sequencing data of patients with SCZ, and confirmed the regulatory relationship between them. To further explore their involvement, we employed retinoic acid (RA)-treated SH-SY5Y differentiated cells as a model system. Our findings indicate that in RA-induced SH-SY5Y cells, ABI1 expression is up-regulated, while miR-30e-3p expression is down-regulated. Functionally, both miR-30e-3p down-regulation and ABI1 up-regulation promote apoptosis and inhibit the proliferation of SH-SY5Y cells. Subsequently, the immunofluorescence assay detected the expression location and abundance of the neuron-specific protein β-tubulinIII. The expression levels of neuronal marker genes MAPT, TUBB3 and SYP were detected by RT-qPCR. We observed that these changes of miR-30e-3p and ABI1 inhibit the neurite growth of SH-SY5Y cells. Rescue experiments further support that ABI1 silencing can correct miR-30e-3p down-regulation-induced SH-SY5Y neurodevelopmental defects. Collectively, our results establish that miR-30e-3p′s regulation of neurite development in SH-SY5Y cells is mediated through ABI1, highlighting a potential mechanism in SCZ pathogenesis.

Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability

In the quest to unravel the mysteries of neurological diseases, comprehending the underlying mechanisms is supreme. The SH-SY5Y human neuroblastoma cell line serves as a crucial tool in this endeavor; however, the cells are known for its sensitivity and slow proliferation rates. Typically, this cell line is cultured with 10% Fetal Bovine Serum (FBS) supplement. Nu-Serum (NuS), a low-protein alternative to FBS, is promising to advance cell culture practices. Herein, we evaluated the substitution of NuS for FBS to test the hypothesis that an alternative serum supplement can aid and promote SH-SY5Y cell proliferation and differentiation. Our findings revealed that the NuS-supplemented group exhibited a notable increase in adhered cells compared to both the FBS and serum-free (SF) groups. Importantly, cell viability remained high in both sera treated groups, with the NuS-supplemented cells displaying significantly larger cell sizes compared to the SF-treated group. Furthermore, cell proliferation rates were higher in the NuS-treated group, and neuroblast-like morphology was observed earlier than FBS group. Notably, both FBS and NuS supported the differentiation of these cells into mature neurons. Our data supports NuS as an alternative for SH-SY5Y cell culture, with the potential to elevate the quality of research in the neuroscience field.

Magnetoactive Composite Conduits Based on Poly(3-hydroxybutyrate) and Magnetite Nanoparticles for Repair of Peripheral Nerve Injury.

Peripheral nerve injury poses a threat to the mobility and sensitivity of a nerve, thereby leading to permanent function loss due to the low regenerative capacity of mature neurons. To date, the most widely clinically applied approach to bridging nerve injuries is autologous nerve grafting, which faces challenges such as donor site morbidity, donor shortages, and the necessity of a second surgery. An effective therapeutic strategy is urgently needed worldwide to overcome the current limitations. Herein, a magnetic nerve guidance conduit (NGC) based on biocompatible biodegradable poly(3-hydroxybutyrate) (PHB) and 8 wt % of magnetite nanoparticles modified by citric acid (Fe3O4-CA) was fabricated by electrospinning. The crystalline structure of NGCs was studied by X-ray diffraction, which indicated an enlarged β-phase of PHB in the composite conduit compared to a pure PHB conduit. Tensile tests revealed greater ductility of PHB/Fe3O4-CA: the composite conduit has Young's modulus of 221 ± 52 MPa and an elongation at break of 28.6 ± 2.9%, comparable to clinical materials. Saturation magnetization (σs) of Fe3O4-CA and PHB/Fe3O4-CA is 61.88 ± 0.29 and 7.44 ± 0.07 emu/g, respectively. The water contact angle of the PHB/Fe3O4-CA conduit is lower as compared to pure PHB, while surface free energy (σ) is significantly higher, which was attributed to higher surface roughness and an amorphous phase as well as possible PHB/Fe3O4-CA interface interactions. In vitro, the conduits supported the proliferation of rat mesenchymal stem cells (rMSCs) and SH-SY5Y cells in a low-frequency magnetic field (0.67 Hz, 68 mT). In vivo, the conduits were used to bridge damaged sciatic nerves in rats; pure PHB and composite PHB/Fe3O4-CA conduits did not cause acute inflammation and performed a barrier function, which promotes nerve regeneration. Thus, these conduits are promising as implants for the regeneration of peripheral nerves.

An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

MiR-769-3p inhibits cellular proliferation of KSHV-infected SH-SY5Y cells through targeting mTOR.

The infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the most common causes of death in AIDS patients. Our studies have found that KSHV can infect SH-SY5Y cells (named SK-RG) in vivo and mTOR was up-regulated, which results in remarkable enhancement of cell proliferation, migration. But the regulatory role of mTOR in KSHV infected neurons has not yet been fully elucidated. Here, we find that miR-769-3p is decreased in SK-RG cells, which can exert anti-KSHV effect through negatively regulating the expression of mTOR. The knockdown of mTOR or overexpress of miR-769-3p decreased the proliferation, migration ability and cell cycle related protein of SK-RG cells, and the expression of KSHV related genes. In contrast, activating mTOR function by 3BDO treatment weakened the cellular behaviors of miR-769-3p overexpressing cells. Meanwhile, overexpressed miR-769-3p and rapamycin showed a shared inhibition trend in the effects on cell proliferation and motility. Our data indicated that miR-769-3p can inhibit cell proliferation and migration by down regulating mTOR in KSHV infected SH-SY5Y cells, and can be a candidate molecule for anti-KSHV therapy.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy.

JOURNAL/nrgr/04.03/01300535-202409000-00035/figure1/v/2024-01-16T170235Z/r/image-tiff Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy, neurosensory impairments, and cognitive deficits, and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy. The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored. However, the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated. In this study, we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function. Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats. Following transplantation of human placental chorionic plate-derived mesenchymal stem cells, interleukin-3 expression was downregulated. To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy, we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA. We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown. Furthermore, interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy. The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy, and this effect was mediated by interleukin-3-dependent neurological function.

Vinpocetine Exerts Neuroprotective Effects via Downregulating α-Syn in Rotenone-induced Cellular Models of Parkinson’s Disease

Objective: Vinpocetine (Vinp), a derivative of alkaloid vincristine with anti-inflammatory and antioxidant effects, has been shown to have neuroprotective effects in Parkinson's disease (PD). Its role and mechanisms, however, are not fully understood. Therefore, the aim of this study was to investigate the effects and possible mechanisms of Vinp on PD cells. Methods: SH-SY5Y cells were treated with Vinp and then with rotenone to induce a cellular model of PD. The proliferation level and apoptosis rate of SH-SY5Y cells after different treatments were detected by MTT and flow cytometry assays, respectively. Western blot was used to determine the relative protein expression of α-Synuclein (α-Syn) in differently treated cells. Additionally, commercial kits and ELISA were used to determine oxidative stress-related indicators (superoxide dismutase [SOD], malondialdehyde [MDA], and reactive oxygen species [ROS]) and inflammatory factors (tumor necrosis factor α [TNF-α], interleukin-5 [IL-5], and interleukin-1β [IL-1β]) in SH-SY5Y cells after different treatments, respectively. Results: Vinp at different concentrations (5, 10, and 50 µM) had no significant effect on the proliferation and apoptosis of SH-SY5Y cells. For rotenone-induced SH-SY5Y cells, Vinp pretreatment could significantly reduce α-Syn expression, increased cell viability and decreased apoptosis, oxidative stress (downregulation of ROS and MDA levels and upregulation of SOD activity) and inflammation (increased levels of TNF-α, IL-5, and IL-1β). In contrast, overexpression of α-Syn in SHSY5Y cells with Vinp pretreatment and rotenone induction partially reversed the aforementioned protective effects of Vinp, causing a decrease in proliferation, an increase in apoptosis rate, inflammation, and oxidative stress. Conclusion: Vinp exerted neuroprotective effects by downregulating α-Syn to promote proliferation, inhibit apoptosis, and inhibit oxidative stress and inflammation in rotenone-induced SH-SY5Y cells.

Cordycepin, a bioactive compound from Cordyceps spp., moderates Alzheimer’s disease-associated pathology via anti-oxidative stress and autophagy activation

Alzheimer’s causes cognitive dysfunction. This study investigated the neuro-promoting effects of cordycepin on amyloid-beta precursor protein (APP) synthesis in human neuroblastoma SH-SY5Y cells. Cordycepin was found to boost SH-SY5Y cell proliferation and decreased AD pathology. APP, PS1, and PS2 were downregulated whereas ADAM10 and SIRT1 were upregulated by cordycepin. Cordycepin also reduced APP secretion in a dose-dependent manner. Cordycepin alleviated oxidative stress by the upregulation of GPX and SOD, as well as autophagy genes (LC3, ATG5, and ATG12). Cordycepin activity was also found to be SIRT1-dependent. Therefore, cordycepin may relieve the neuronal degeneration caused by APP overproduction, and oxidative stress.

Aumolertinib inhibits proliferation, invasion and migration and promotes apoptosis of neuroblastoma cells by downregulating MMP2 and MMP9 expression

To investigate the effects of aumolertinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), on biological behaviors of neuroblastoma SH-SY5Y cells. CCK-8 assay, colony-forming assay, Transwell assay and flow cytometry were used to assess the effects of 2, 4 and 8 μmol/L aumolertinib on proliferation, survival, migration, invasion and apoptosis of SH-SY5Y cells, and the changes in ultrastructure of the cells were observed using transmission electron microscopy. The protein expressions of Bax, Bcl-2, E-cadherin, vimentin, MMP2, and MMP9 in the treated cells were detected using Western blotting. A nude mouse model bearing subcutaneous SH-SY5Y cell xenograft were treated with aumolertinib (15 mg/kg) or cyclophosphamide (20 mg/kg), and the tumor volume and body mass changes was measured. HE staining was used to observe adverse effects of the treatment on the heart, liver, spleen, lungs and kidneys. Aumolertinib significantly inhibited the proliferation and viability of SH-SY5Y cells (P<0.05) with IC50 of 5.004, 3.728 and 3.228 µmol/L at 24, 48 and 72 h, respectively. Aumolertinib treatment induced obvious apoptosis of the cells, which showed characteristic morphological changes of apoptosis under transmission electron microscope. The treatment also inhibited the invasion and migration abilities of SH-SY5Y cells (P<0.01), up-regulated the expression levels of E-cadherin and Bax and lowered the expression levels of Bcl-2, vimentin, MMP2 and MMP9 (P<0.05). In the nude mouse models, treatment with aumolertinib effectively inhibited the growth of neuroblastoma without causing significant toxicity to the vital organs. Aumolertinib inhibits proliferation, survival, invasion and migration and induces apoptosis in SH-SY5Y cells by downregulating MMP2 and MMP9 expression.

MDMA targets miR-124/MEKK3 via MALAT1 to promote Parkinson’s disease progression

BackgroundParkinson’s disease (PD) is a well-known neurodegenerative disease that is usually caused by the progressive loss of dopamine neurons and the formation of Lewy vesicles. 3,4-Methylenedioxymethamphetamine (MDMA) has been reported to cause damage to human substantia nigra neurons and an increased risk of PD, but the exact molecular mechanisms need further investigation.MethodsMPTP- and MPP+-induced PD cells and animal models were treated with Nissl staining to assess neuronal damage in the substantia nigra (SN) area; immunohistochemistry to detect TH expression in the SN; TUNEL staining to detect apoptosis in the SN area; Western blotting to detect the inflammatory factors NF-κB, TNF-α, IL-6 and mitogen-activated protein kinase kinase kinase 3 (MEKK3); Griess assay for NO; RT‒qPCR for metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and miR-124 expression; Cell proliferation was assessed by CCK-8. Dual luciferase reporter genes were used to verify targeting relationships.ResultsMDMA promoted MALAT1 expression, and knockdown of MALAT1 alleviated the MDMA-induced inhibition of SH-SY5Y cell proliferation, inflammation, NO release, SN neuronal injury, and TH expression inhibition. Both inhibition of miR-124 and overexpression of MEKK3 reversed the neuroprotective effects exhibited by knockdown of MALAT1.ConclusionMDMA promotes MALAT1 expression and inhibits the targeted downregulation of MEKK3 by miR-124, resulting in upregulation of the expression of MEKK3 and finally jointly promoting PD progression.

Effect of Differently Polarized Human Macrophages on the SH-SY5Y Cells Damaged by Ischemia and Hypoxia In Vitro.

Macrophages are the major cells of innate immunity with a wide range of biological effects due to their great plasticity and heterogeneity. Macrophages play a key role in neuroregeneration following nervous tissue injury. However, the neuroregenerative potential of various macrophage phenotypes, including those polarized by efferocytosis, remains unexplored. The aim of this study was to compare the neuroregenerative and neuroprotective activity of soluble factors secreted by variously activated human macrophages on the functions of neural progenitors in an in vitro model of ischemia or ischemia/hypoxia. Macrophages were polarized by interferon-γ (M1), IL-4 (M2a), or interaction with apoptotic cells (M2(LS)). The effect of macrophages conditioned media on the proliferation, differentiation, and survival of SH-SY5Y cells damaged by serum deprivation alone (ischemic conditions) or in combination with CoCl2 (ischemic/hypoxic conditions) was assessed. All studied macrophages stimulated the proliferation and differentiation of SH-SY5Y cells. On day 3, the pro-proliferating effect of M1 and M2 was similar and did not depend on the severity of the damaging effect (ischemia or ischemia/hypoxia), while on day 7 and under ischemic/hypoxic conditions, the effects of M2(LS) exceeded those of M1 and M2a cells. The prodifferentiation effects of macrophages were manifested in both short- and long-term cultures, mainly under ischemic/hypoxic conditions, and were most characteristic of M2(LS) cells. Importantly, the ischemia/hypoxia model was accompanied by the pronounced death of SH-SY5Y cells. Only macrophages with the M2 phenotype demonstrated antiapoptotic activity, and the effect of M2(LS) was higher than that of M2a. The results obtained indicate that human macrophages have neuroprotective and neuroregenerative activity, which is mediated by soluble factors, is most characteristic for macrophages activated by efferocytosis (M2(LS)), and is most prominent under in vitro conditions simulating the combined effect of ischemia/hypoxia.

Silencing of purinergic receptor P2Y2 inhibited enteric neural crest cell proliferation, invasion and migration via suppressing ERK signaling pathway in Hirschsprung disease.

The current study aimed to explore the effect and underlying mechanism of the purinergic receptor P2Y2 in regulating the loss of intestinal neurons and the intestinal neural crest in Hirschsprung's disease (HSCR). Western blotting was used to assess the expression levels of P2Y2 in colon tissues. An in vivo HSCR mouse model was established following treatment with benzalkonium chloride (BAC). We overexpressed or silenced P2Y2 in SH-SY5Y cells, and cell proliferation, migration, and invasion were subsequently investigated by CCK-8, wound healing, and transwell assays, respectively. Additionally, we implemented a xenograft model to assess the impact of P2Y2 on tumor growth as well as the expression of extracellular signal-regulated kinase (ERK). The results showed that the expression of P2Y2 protein in the colon tissues of patients with HSCR was lower than that in the normal colon tissues. P2Y2 expression is downregulated in the colon tissues of mice with HSCR. Additionally, P2Y2 silencing inhibited SH-SY5Y cell proliferation, invasion, and migration. Furthermore, adenosine 5'-triphosphate (ATP, a strong agonist of P2Y2)-induced P2Y2 overexpression enhanced the proliferation, invasion, and migration of SH-SY5Y cells. Immunofluorescence staining and western blot analysis revealed that P2Y2 silencing downregulated phosphorylated (p)-ERK in SH-SY5Y cells. In addition, treatment with PD98059, a p-ERK inhibitor, reversed the effects of ATP on SH-SY5Y cell proliferation, invasion, and migration. Finally, we demonstrated that P2Y2 silencing suppressed tumor growth and decreased p-ERK expression. Overall, the results of the present study suggest that P2Y2 plays an important role in HSCR pathogenesis. P2Y2 silencing inhibited the proliferation, invasion, and migration of nerve cells by suppressing the ERK signaling pathway. P2Y2 silencing could be considered an innovative and possible target for treating HSCR.

Crocin alleviates neurotoxicity induced by bupivacaine in SH-SY5Y cells with inhibition of PI3K/AKT signaling.

Bupivacaine, a common local anesthetic, can cause neurotoxicity and permanent neurological disorders. Crocin has been widely reported as a potential neuroprotective agent in neural injury models. The aim of this study was to investigate the role and regulatory mechanism of crocin underlying bupivacaine-induced neurotoxicity. Human neuroblastoma SH-SY5Y cells were treated with bupivacaine and/or crocin for 24h, followed by detecting cell viability using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The effect of crocin or bupivacaine on SH-SY5Y cell proliferation was measured by Ki67 immunofluorescence assay. The levels of apoptosis-related proteins and the markers in the PI3K/Akt signaling pathway were examined using western blot analysis. The activities of caspase 3, catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were tested using respective commercial assay kits. Flow cytometry analysis was executed for detecting SH-SY5Y cell apoptosis. Crocin attenuated bupivacaine-induced neurotoxicity in SH-SY5Y cells. Meanwhile, crocin inhibited SH-SY5Y cell apoptosis induced by bupivacaine via repressing the activity of caspase-3, reducing Bax expression, and elevating Bcl-2 expression. Moreover, crocin mitigated oxidative stress in SH-SY5Y cells by increasing the content of CAT, SOD, GSH-Px and reducing the content of MDA. Additionally, crocin protected against bupivacaine-induced dephosphorylation of Akt and GSK-3β. The protective effects of crocin against bupivacaine-induced neurotoxicity in SH-SY5Y cells were counteracted by the Akt inhibitor. These results suggested that crocin may exert a neuroprotective function by promoting cell proliferation and suppressing apoptosis and oxidative stress in SH-SY5Y cells. Thus, crocin might become a promising drug for the treatment of bupivacaine-induced neurotoxicity.

Bioinformatics and Experimental Identification of circ_0001535 Associated with Diagnosis and Development of Alzheimer's Disease.

Alzheimer's disease (AD) is a type of disease frequently occurs in the elderly population. Diagnosis and treatment methods for this disease are still lacking, and more research is required. In addition, little is known about the function of the circular RNAs (circRNAs) in AD. In this research, RNA expression data of AD from the Gene Expression Omnibus (GEO) database were downloaded. The expression levels of circRNAs in cerebrospinal fluid samples of healthy participants and AD patients were measured by reverse transcription‑quantitative PCR (RT-qPCR). The diagnosed value of differential expressed circRNAs was analyzed with the Receiver operating characteristic curve (ROC). Pathways related to circ_0001535 were found using gene set enrichment analysis (GSEA) and Metascape. The direct interactions between circ_0001535 and E2F transcription factor 1 (E2F1) or E2F1 and dihydrofolate reductase (DHFR) were verified using Chromatin immunoprecipitation (ChIP) and RNA Binding Protein Immunoprecipitation (RIP) assays. Cell Counting Kit-8 (CCK8) and flow cytometry were used to identify the function of circ_0001535/E2F1/DHFR axis on the proliferation and apoptosis of AD cells. In total, 12 circRNAs have been linked to AD diagnosis. The expression levels of 7 circRNAs differed between AD patients and control groups. Circ_0001535 had the most diagnose value among these circRNAs. Hence, circ_0001535 was regarded as a key circRNA in the present study. E2F1/DHFR axis was predicted to be regulated by circ_0001535. In addition, IP assays experiment results showed that E2F1 could bind to the promoter region of DHFR and be regulated by circ_0001535. In vitro results showed that circ_0001535 overexpression could promote DHFR expression, while E2F1 knock down could inhibit DHFR expression in SH-SY5Y cells. Finally, rescue experiments suggested that circ_0001535 could reduce Aβ25-35-induced SH-SY5Y cell proliferation and facilitate apoptosis through E2F1/DHFR axis. Our research in AD circRNA can offer important information regarding the role of specific circRNAs in the AD environment and point to specific future areas of therapeutic intervention in AD.

Retracted: MicroRNA-29a-3p Regulates SH-SY5Y Cell Proliferation and Neurite Growth through Interaction with PTEN-PI3K/AKT/mTOR Signaling Pathway.

[This retracts the article DOI: 10.1155/2022/8151161.].

TRIM25-mediated ubiquitination of G3BP1 regulates the proliferation and migration of human neuroblastoma cells

Neuroblastoma is one of the most severe malignant tumors and accounts for substantial cancer-related mortality in children. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is highly expressed in various cancers and acts as an important biomarker of poor prognosis. The ablation of G3BP1 inhibited the proliferation and migration of human SHSY5Y cells. Because of its important role in neuroblastoma, the regulation of G3BP1 protein homeostasis was probed. TRIM25, which belongs to the tripartite motif (TRIM) family of proteins, was identified as an interacting partner for G3BP1 using the yeast two-hybrid (Y2H) method. TRIM25 mediates the ubiquitination of G3BP1 at multiple sites and stabilizes its protein level. Then, our study found that TRIM25 knockdown also inhibited the proliferation and migration of neuroblastoma cells. The TRIM25 and G3BP1 double knockdown SHSY5Y cell line was generated, and double knockdown cells exhibited lower proliferation and migration ability than cells with only TRIM25 or G3BP1 knockdown. Further study demonstrated that TRIM25 promotes the proliferation and migration of neuroblastoma cells in a G3BP1-dependent manner. Tumor xenograft assays indicated that the ablation of TRIM25 and G3BP1 synergistically suppressed the tumorigenicity of neuroblastoma cells in nude mice, and TRIM25 promoted the tumorigenicity of G3BP1 intact SHSY5Y cells but not G3BP1 knockout cells. Thus, TRIM25 and G3BP1, two oncogenic genes, are suggested as potential therapeutic targets for neuroblastoma.