MiR-19a-3p regulates the Forkhead box F2-mediated Wnt/β-catenin signaling pathway and affects the biological functions of colorectal cancer cells

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.AIM To explore the expression of microRNA miR-19a-3p and Forkhead box F2(FOXF2)in patients with CRC and the relevant mechanisms.METHODS Sixty-two CRC patients admitted to the hospital were enrolled into the study group,and sixty healthy people from the same period were assigned to the control group.Elbow venous blood was sampled from the patients and healthy individuals,and blood serum was saved for later analysis.MiR-19a-3p mimics,miR-19a-3p inhibitor,miR-negative control,small interfering-FOXF2,and short hairpin-FOXF2 were transfected into HT29 and HCT116 cells.Then quantitative polymerase chain reaction was performed to quantify the expression of miR-19a-3p and FOXF2 in HT29 and HCT116 cells,and western blot(WB)analysis was conducted to evaluate the levels of FOXF2,glycogen synthase kinase 3 beta(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),β-catenin,p-β-catenin,α-catenin,Ncadherin,E-cadherin,and vimentin.The MTT,Transwell,and wound healing assays were applied to analyze cell proliferation,invasion,and migration,respectively,and the dual luciferase reporter assay was used to determine the correlation of miR-19a-3p with FOXF2.RESULTS The patients showed high serum levels of miR-19a-3p and low levels of FOXF2,and the area under the curves of miR-19a-3p and FOXF2 were larger than 0.8.MiR-19a-3p and FOXF2 were related to sex,tumor size,age,tumor-nodemetastasis staging,lymph node metastasis,and differentiation of CRC patients.Silencing of miR-19a-3p and overexpression of FOXF2 suppressed the epithelialmesenchymal transition,invasion,migration,and proliferation of cells.WB analysis revealed that silencing of miR-19a-3p and FOXF2 overexpression significantly suppressed the expression of p-GSK-3β,β-catenin,N-cadherin,and vimentin;and increased the levels of GSK-3β,p-β-catenin,α-catenin,and Ecadherin.The dual luciferase reporter assay confirmed that there was a targeted correlation of miR-19a-3p with FOXF2.In addition,a rescue experiment revealed that there were no differences in cell proliferation,invasion,and migration in HT29 and HCT116 cells co-transfected with miR-19a-3p-mimics+sh-FOXF2 and miR-19a-3p-inhibitor+si-FOXF2 compared to the miR-negative control group.CONCLUSION Inhibiting miR-19a-3p expression can upregulate the FOXF2-mediated Wnt/β-catenin signaling pathway,thereby affecting the epithelial-mesenchymal transition,proliferation,invasion,and migration of cells.Thus,miR-19a-3p is likely to be a therapeutic target in CRC.

Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guid- ance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord iniury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, caspase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental fndings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.

Role of Wnt/β-catenin Signaling Pathway in the Mechanism of Calcification of Aortic Valve

Aortic valve calcification is a common disease in the elderly, but its cellular and molecular mechanisms are not clear. In order to verify the hypothesis that Wnt/β-catenin signaling pathway is involved in the process of calcification of aortic valve, porcine aortic valve interstitial cells（VICs） were isolated, cultured and stimulated with oxidized low density lipoprotein（ox-LDL） for 48 h to induce the differentiation of VICs into osteoblast-like cells. The key proteins and genes of Wnt/β-catenin signaling pathway, such as glycogen synthase kinase 3β（GSK-3β） and β-catenin, were detected by using Western blotting and real-time polymerase chain reaction（PCR）. The results showed that the VICs managed to differentiate into osteoblast-like cells after the stimulation with ox-LDL and the levels of proteins and genes of GSK-3β and β-catenin were increased significantly in VICs after stimulation for 48 h（P0.05）. It is suggested that Wnt/β-catenin signaling pathway may play a key role in the differentiation of VICs into osteoblast-like cells and make great contribution to aortic valve calcification.

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)exhibits high invasiveness and mortality rates,and the molecular mechanisms of HCC have gained increasing research interest.The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development.Recent studies have shown the potential of the protein RING finger and WD repeat domain 3(RFWD3)that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers.AIM To investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways.METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues.Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines.After verifying the silencing efficiency,Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis.Subsequently,cell migration and invasion were assessed by wound healing and transwell assays.In addition,transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis.Next,we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype.Finally,the microarray,ingenuity pathway analysis,and western blot analysis were used to analyze the regulatory network underlying HCC.RESULTS Compared with adjacent tissues,RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage(P<0.05),which indicated a poor prognosis state.RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis,decreased growth,and inhibited the migration in shRNAi cells compared with those in shCtrl cells(P<0.05).Furthermore,the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration.Moreover,the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines.Finally,gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSION We provide evidence for the expression and function of RFWD3 in HCC.RFWD3 affects the prognosis,proliferation,invasion,and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

Involvement of the Wnt signaling pathway and cell apoptosis in the rat hippocampus following cerebral ischemia/reperfusion injury

We investigated the role of the Wnt signaling pathway in cerebral ischemia/reperfusion injury by examining β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region following acute cerebral ischemia/reperfusion. Our results demonstrate that cell apoptosis increases in the CA1 region following ischemia/reperfusion. In addition, β-catenin and glycogen synthase kinase-3β protein expression gradually increases, peaking at 48 hours following reperfusion. Dickkopf-1 administration, after cerebral ischemia/reperfusion injury, results in decreased cell apoptosis, and β-catenin and glycogen synthase kinase-3β expression, in the CA1 region. This suggests that β-catenin and glycogen synthase kinase-3β, both components of the Wnt signaling pathway, participate in cell apoptosis following cerebral ischemia/reperfusion injury.

Functional Insights of Plant GSK3-like Kinases： Multi-Taskers in Diverse Cellular Signal Transduction Pathways

The physiological importance of GSK3-like kinases in plants emerged when the functional role of plant GSK3-like kinases represented by BIN2 was first elucidated in the brassinosteroid （BR）-regulated signal transduction pathway. While early studies focused more on understanding how GSK3-like kinases regulate BR signaling, recent studies have implicated many novel substrates of GSK3-like kinases that are involved in a variety of cellular processes as well as BR signaling. Plant GSK3-like kinases play diverse roles in physiological and developmental processes such as cell growth, root and stomatal cell development, flower development, xylem differentiation, light response, and stress responses. Here, we review the progress made in recent years in understanding the versatile functions of plant GSK3-like kinases. Based on the relationship between GSK3-like kinases and their newly identified substrates, we discuss the physiological and biochemical relevance of various cellular signaling mediated by GSK3-like kinases in plants.

Treated dentin matrix induces odontogenic differentiation of dental pulp stem cells via regulation of Wnt/β-catenin signaling

Treated dentin matrix(TDM)is an ideal scaffold material containing multiple extracellular matrix factors.The canonical Wnt signaling pathway is necessary for tooth regeneration.Thus,this study investigated whether the TDM can promote the odontogenic differentiation of human dental pulp stem cells(hDPSCs)and determined the potential role of Wnt/β-catenin signaling in this process.Different concentrations of TDM promoted the dental differentiation of the hDPSCs and meanwhile,the expression of GSK3βwas decreased.Of note,the expression of the Wnt/β-catenin pathway-related genes changed significantly in the context of TDM induction,as per RNA sequencing(RNA seq)data.In addition,the experiment showed that new dentin was visible in rat mandible cultured with TDM,and the thickness was significantly thicker than that of the control group.In addition,immunohistochemical staining showed lower GSK3βexpression in new dentin.Consistently,the GSK3βknockdown hDPSCs performed enhanced odotogenesis compared with the control groups.However,GSK3βoverexpressing could decrease odotogenesis of TDM-induced hDPSCs.These results were confirmed in immunodeficient mice and Wistar rats.These suggest that TDM promotes odontogenic differentiation of hDPSCs by directly targeting GSK3βand activating the canonical Wnt/β-catenin signaling pathway and provide a theoretical basis for tooth regeneration engineering.

Emerging agents that target signaling pathways to eradicate colorectal cancer stem cells

Colorectal cancer(CRC)represents the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide.The modern concept of cancer biology indicates that cancer is formed of a small population of cells called cancer stem cells(CSCs),which present both pluripotency and self-renewal properties.These cells are considered responsible for the progression of the disease,recurrence and tumor resistance.Interestingly,some cell signaling pathways participate in CRC survival,proliferation,and selfrenewal properties,and most of them are dysregulated in CSCs,including the Wingless(Wnt)/β-catenin,Notch,Hedgehog,nuclear factor kappa B(NF-κB),Janus kinase/signal transducer and activator of transcription(JAK/STAT),peroxisome proliferator-activated receptor(PPAR),phosphatidyl-inositol-3-kinase/Akt/mechanistic target of rapamycin(PI3K/Akt/mTOR),and transforming growth factor-β(TGF-β)/Smad pathways.In this review,we summarize the strategies for eradicating CRC stem cells by modulating these dysregulated pathways,which will contribute to the study of potential therapeutic schemes,combining conventional drugs with CSC-targeting drugs,and allowing better cure rates in anti-CRC therapy.

Expression of hepatic Wnt5a and its clinicopathological features in patients with hepatocellular carcinoma

Backgroud： Wingless-type MMTV integration site family member 5a （Wnt5a） is involved in carcinogenesis.However, little data are available in Wnt5a signaling with hepatocellular carcinoma （HCC）. In thepresent study, we investigated the expression of hepatic Wnt5a in HCC and the role of Wnt5a in HCCprogression and outcome.

Effects of mild intrauterine hypoperfusion in the second trimester on memory and learning function in rat offspring

Mild intrauterine hypoperfusion(MIUH)is a serious pathological event that affects the growth and development of fetuses and offspring.MIUH can lead to growth restriction,low birth weight,neurodevelopmental disorders,and other adverse clinical outcomes.To study the effects of MIUH on learning and memory function in offspring,a model of MIUH was established by placing a coil(length 2.5 mm,diameter 0.24 mm)on the uterine artery and ovarian uterine artery of Sprague-Dawley rats in the second trimester of pregnancy(day 17).Next,120 mg/kg lithium chloride(the MIUH+Li group)or normal saline(the MIUH group)was injected intraperitoneally into these rats.In addition,120 mg/kg lithium chloride(the Li group)or normal saline(the SHAM group)was injected intraperitoneally into pregnant rats without coil placement.The Morris water maze was used to detect changes in learning and memory ability in the offspring at 4 weeks after birth.In the MIUH group,the escape latency and journey length before reaching the platform were both increased,and the number of times that the platform was crossed and the activity time in the target quadrant within 90 seconds were both decreased compared with the SHAM group.Immunofluorescence double staining and western blot assays demonstrated that hippocampal nestin and Ki67(both cell-proliferation-related proteins)expression was significantly downregulated in the MIUH group compared with the SHAM group.Furthermore,western blot assays were conducted to investigate changes in related signaling pathway proteins in the brains of offspring rats,and revealed that glycogen synthase kinase 3β(GSK3β)expression was upregulated andβ-catenin expression was downregulated in the MIUH group compared with the SHAM group.In addition,compared with the MIUH group,the expression levels of p-GSK3βandβ-catenin were upregulated in the MIUH+Li group.These results suggest that MIUH may affect learning and memory function in rat offspring by regulating the GSK3βsignaling pathway.The experimental procedures were approved by Animal Ethics Committee of Shengjing Hospital of China Medical University(approval No.2018 PS07 K)in June 2018.

Regulation of the PI3K/AKT Pathway and Fuel Utilization During Primate Torpor in the Gray Mouse Lemur, Microcebus murinus

Gray mouse lemurs（Microcebus murinus） from Madagascar present an excellent model for studies of torpor regulation in a primate species. In the present study, we analyzed the response of the insulin signaling pathway as well as controls on carbohydrate sparing in six different tissues of torpid versus aroused gray mouse lemurs. We found that the relative level of phospho-insulin receptor substrate（IRS-1） was significantly increased in muscle, whereas the level of phospho-insulin receptor（IR） was decreased in white adipose tissue（WAT） of torpid animals, both suggesting an inhibition of insulin/insulin-like growth factor-1（IGF-1） signaling during torpor in these tissues. By contrast, the level of phospho-IR was increased in the liver. Interestingly, muscle,WAT, and liver occupy central roles in whole body homeostasis and each displays regulatory controls operating at the plasma membrane. Changes in other tissues included an increase in phosphoglycogen synthase kinase 3a（GSK3a） and decrease in phospho-ribosomal protein S6（RPS6） in the heart, and a decrease in phospho-mammalian target of rapamycin（m TOR） in the kidney. Pyruvate dehydrogenase（PDH） that gates carbohydrate entry into mitochondria is inhibited via phosphorylation by pyruvate dehydrogenase kinase（e.g., PDK4）. In the skeletal muscle, the protein expression of PDK4 and phosphorylated PDH at Ser 300 was increased, suggesting inhibition during torpor. In contrast, there were no changes in levels of PDH expression and phosphorylation in other tissues comparing torpid and aroused animals. Information gained from these studies highlight the molecular controls that help to regulate metabolic rate depression and balance energetics during primate torpor.

Effects of scandium chloride on osteogenic and adipogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells(MSCs)are multi-potent cells that are able to differentiate and mature into various types of cells under a certain microenvironment for cell therapy and tissue regeneration.Scandium(Sc),an important rare earth element,recently has been intensively investigated in biomedical fields as well as industrial engineering,and chloride channels have been proven to be able to affect osteogenic differentiation.Thus,it is significant to investigate effects of ScCl_(3)on cell activities of MSCs.In this paper,rat bone MSCs(rBMSCs)were co-cultured with various concentrations of ScCl_(3)(1×10^(-8),1×10^(-6),and 1×10^(-4)mol/L)to evaluate their influence on cell proliferation as well as osteogenic and adipogenic differentiation in vitro.The results indicate that ScCl_(3)promotes the proliferation of rBMSCs initially,which is yet reduced upon ion accumulation.We used immunofluorescence staining,quantitative real time polymerase chain reactions,and assays measuring alkaline phosphatase activity,mineralized deposits,and intracytoplasmic lipids to reveal that rBMSCs treated with ScCl_(3)at concentrations of 1×10^(-8)-1×10^(-6)mol/L can enhance levels of osteogenic differentiation in a dosedependent manner and reduce adipogenic differentiation to a certain degree through Wnt/β-catenin signaling pathway.These results indicate that appropriate concentrations of ScCl_(3)can improve osteogenic differentiation in the lineage commitment of rBMSCs,and thus,promote bone remodeling.This study implies that ScCl_(3) possesses great potentials in the treatment of bone diseases and would provide new strategy of designing composites by SiCl3 doping for biomedical applications in the future.