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.

Neuroprotection mediated by the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage

The Wnt/Frizzled signaling pathway participates in many inflammation-linked diseases. However, the inflammatory response mediated by the Wnt/Frizzled signaling pathway in experimental subarachnoid hemorrhage has not been thoroughly investigated. Consequently, in this study, we examined the potential role of the Wnt/Frizzled signaling pathway in early brain injury in rat models of subarachnoid hemorrhage.Simultaneously, possible neuroprotective mechanisms were also investigated. Experimental subarachnoid hemorrhage rat models were induced by injecting autologous blood into the prechiasmatic cistern. Experiment 1 was designed to examine expression of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. In total, 42 adult rats were divided into sham(injection of equivalent volume of saline), 6-, 12-, 24-, 48-, 72-hour, and 1-week subarachnoid hemorrhage groups. Experiment 2 was designed to examine neuroprotective mechanisms of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. Rats were treated with recombinant human Wnt1(rhwnt1), small interfering Wnt1(siwnt1) RNA, and monoclonal antibody of Frizzled1(anti-Frizzled1) at 48 hours after subarachnoid hemorrhage. Expression levels of Wnt1, Frizzled1, β-catenin, peroxisome proliferator-activated receptor-γ, CD36, and active nuclear factor-κB were examined by western blot assay and immunofluorescence staining. Microglia type conversion and inflammatory cytokine levels in brain tissue were examined by immunofluorescence staining and enzyme-linked immunosorbent assay. Our results show that compared with the sham group, expression levels of Wnt1, Frizzled1, and β-catenin were low and reduced to a minimum at 48 hours, gradually returning to baseline at 1 week after subarachnoid hemorrhage. rhwnt1 treatment markedly increased Wnt1 expression and alleviated subarachnoid hemorrhage-induced early brain injury(within 72 hours), including cortical cell apoptosis, brain edema, and neurobehavioral deficits, accompanied by increasing protein levels of β-catenin, CD36, and peroxisome proliferator-activated receptor-γ and decreasing protein levels of nuclear factor-κB. Of note, rhwnt1 promoted M2-type microglia conversion and inhibited release of inflammatory cytokines(interleukin-1β, interleukin-6, and tumor necrosis factor-α). In contrast, siwnt1 RNA and anti-Frizzled1 treatment both resulted in an opposite effect. In conclusion, the Wnt/Frizzled1 signaling pathway may participate in subarachnoid hemorrhage-induced early brain injury via inhibiting the inflammatory response, including regulating microglia type conversion and decreasing inflammatory cytokine release. The study was approved by the Animal Ethics Committee of Anhui Medical University and First Affiliated Hospital of USTC,Division of Life Sciences and Medicine, University of Science and Technology of China(approval No. LLSC-20180202) in May 2017.

Ubiquitin-like modifier activating enzyme 2 promotes cell migration and invasion through Wnt/β-catenin signaling in gastric cancer

AIM To investigate the function and mechanism of ubiquitinlike modifier activating enzyme 2(Uba2) in progression of gastric cancer(GC) cells.METHODS Uba2 level in patients with GC was analyzed by Western blotting and immunohistochemistry. MTT and colony formation assays were performed to examine cell proliferation.Flow cytometry was used for cell cycle analysis.Wound healing and Transwell assays were conducted to examine the effects of Uba2 on migration and invasion.Expression levels of cell cycle-related proteins, epithelial-mesenchymal transition(EMT) biomarkers, and involvement of the Wnt/β-catenin pathway was assessed by Western blotting. Activation of the Wnt/β-catenin pathway was confirmed by luciferase assay.RESULTS Uba2 expression was higher in GC than in normal tissues.Increased Uba2 expression was correlated with tissue differentiation, Lauren's classification, vascular invasion,and TNM stage, as determined by the analysis of 100 GC cases(P < 0.05). Knock-down of Uba2 inhibited GC cell proliferation, induced cell cycle arrest, and altered expression of cyclin D1, P21, P27, and Bcl-2, while upregulation of Uba2 showed the opposite effects. The wound healing and Transwell assays showed that Uba2 promoted GC cell migration and invasion. Western blotting revealed alterations in EMT biomarkers, suggesting the role of Uba2 in EMT. Furthermore, the luciferase reporter assay indicated the involvement of the Wnt/β-catenin signaling pathway as a possible modulator of Uba2 oncogenic functions.CONCLUSION Uba2 plays a vital role in GC cell migration and invasion,possibly by regulating the Wnt/β-catenin signaling pathway and EMT.

NETO2 promotes melanoma progression via activation of the Ca^(2+)/CaMKⅡ signaling pathway

Melanoma is the most aggressive cutaneous tumor.Neuropilin and tolloid-like 2(NETO2)is closely related to tumorigenesis.However,the functional significance of NETO2 in melanoma progression remains unclear.Herein,we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients.Disrupting NETO2 expression markedly inhibited melanoma proliferation,malignant growth,migration,and invasion by downregulating the levels of calcium ions(Ca^(2+))and the expression of key genes involved in the calcium signaling pathway.By contrast,NETO2 overexpression had the opposite effects.Importantly,pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis.Overall,this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression,indicating that targeting NETO2 may effectively improve melanoma treatment.

Exogenous FGF－2 improves biological activity of endothelial progenitor cells exposed to high glucose conditions

Purpose: To investigate the effects of exogenous basic fibroblast growth factor-2(FGF-2) on the biological activity of endothelial progenitor cells(EPCs) exposed to high glucose conditions. Materials and Methods: 1) Bone marrow EPCs from C57BL/6 mice were isolated and cultured in vitro. EPC purity was identified by flow cytometry and immunofluorescence staining. 2) Apoptosis was detected by TUNEL assay. Migration and tube formation ability was detected by Transwell chamber and Matrigel assays, respectively. The expression and activation of β-catenin was detected by Western blot. 3) Doppler flowmetry was used to detect the effect of FGF2 on blood flow recovery in ischemic hind limbs of mice. Results: 1) FGF-2 treatment reversed high glucose induced growth inhibition of EPCs. FGF-2 treatment also increased migration and tube formation ability of EPCs even in high glucose conditions. 2) Western blot analysis demonstrated that the percentage of activated β-catenin/total β-catenin in the high glucose group were significantly lower than that in the control group, while FGF-2 treatment reversed high glucose induced β-catenin inhibition. 3) In vivo experiments demonstrated that the blood flow recovery in ischemic hind limbs of mice was significantly improved after FGF-2 treatment. Conclusion: Exogenous FGF-2 could play a role in the functional repair of damaged EPC exposed to high glucose conditions, via the activation of the Wnt/β-catenin signaling pathway.

Yiguanjian decoction enhances fetal liver stem/progenitor cell-mediated repair of liver cirrhosis through regulation of macrophage activation state

AIM To investigate whether Yiguanjian decoction(YGJ) has an anti-liver cirrhotic effect and whether it regulates hepatic stem cell differentiation.METHODS A rat model of liver cirrhosis was established via subcutaneous injection of carbon tetrachloride(CCl4) for8 wk. From the beginning of the ninth week, the rats received 2-acetylaminofluorene(2-AAF) by oral gavage and a DLK-1+ fetal liver stem/progenitor cell(FLSPC)transplant or an FLSPC transplant in combination with YGJ treatment for 4 wk. In vitro, lipopolysaccharide(LPS)-activated macrophages were co-cultured with WB-F344 cells, and the differentiation of WB-F344 cells was observed in the presence and absence of YGJ treatment.RESULTS FLSPC transplantation improved liver function and histopathology, and inhibited the activation of the noncanonical Wnt signaling pathway, while activating the canonical Wnt signaling pathway. YGJ enhanced the therapeutic effects of FLSPCs and also promoted the liver regeneration differentiation of FLSPCs into hepatocytes.In vitro, LPS-activated macrophages promoted the differentiation of WB-F344 cells into myofibroblasts, and the canonical Wnt signaling was inhibited while the noncanonical Wnt signaling was activated in WB-F344 cells.YGJ suppressed the activation of macrophages and then inhibited non-canonical Wnt signaling and promoted canonical Wnt signaling.CONCLUSION YGJ enhances FLSPC-mediated repair of liver cirrhosis through regulation of macrophage activation state, and YGJ in combination with stem cell transplantation may be a suitable treatment for end-stage liver cirrhosis.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

The ornithine-urea cycle involves fumaric acid biosynthesis in Aureobasidium pullulans var.aubasidani,a green and eco-friendly process for fumaric acid production

The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming.In this study,the engineered strains of A.pullulans var.aubasidani were found to be suitable for green fumaric acid producer.Removal and complementation of the relevant genes showed only the ornithine-urea cycle(OUC)was involved in high level fumarate biosynthesis which was controlled by the Ca^(2+)signaling pathway.Removal of both the GOX gene encoding glucose oxidase and the PKS1 gene encoding the polyketide synthase for 3,5-dihydroxydecanoic acid biosynthesis and overexpression of the PYC gene encoding pyruvate carboxylase made the strain e-PYC produce 88.1±4.3 g/L of fumarate at flask level and 93.9±0.8 g/L of fumarate during the fed-batch fermentation.As a yeast-like fungal strain,it was very easy to cultivate A.pullulans var.aubasidani DH177 and their mutants in the bioreactor and to edit its genomic DNAs to enhance fumarate production.It was found that 2 mol of CO_(2) could be fixed during a maximal theoretical yield of 2 mol of fumarate per mole of glucose consumed in the OUC.Therefore,the OUC-mediated fumarate biosynthesis pathway in A.pullulans var.aubasidani was a green and eco-friendly process for the global sustainable development and carbon neutrality.