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.

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.

Mutations in components of the Wnt signaling pathway in gastric cancer

AIM: To explore the contribution of AXIN1, AXIN2 and beta-catenin, components of Wnt signaling pathway, to the carcinogenesis of gastric cancer (GC), we examined AXIN1, AXIN2 exon7 and CTNNB1 (encoding beta- catenin) exon3 mutations in 70 GCs. METHODS: The presence of mutations was identified by polymerase chain reaction (PCR)-based denaturing high-performance liquid chromatography and direct DNA sequencing. Beta-catenin expression was detected by immunohistochemical analysis. RESULTS: Among the 70 GCs, 5 (7.1%) had mutations in one or two of these three components. A frameshift mutation (1 bp deletion) in exon7 of AXIN2 was found in one case. Four cases, including the case with a mutation in AXIN2, had frameshift mutations and missense mutations in AXIN1. Five single nucleotide polymorphisms (SNPs), 334 C>T, 874 C>T, 1396 G>A, 1690 C>T and 1942 T>G, were identified in AXIN1. A frameshift mutation (27 bp deletion) spanning exon3 of CTNNB1 was observed in one case. All four cases with mutations in AXIN1 and AXIN2 showed nuclear beta- catenin expression. CONCLUSION: These data indicate that the mutationsin AXIN1 and AXIN2 may contribute to gastric carcino- genesis.

Calycosin-7-O-β-D-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNT signaling pathways

The iso fl avone calycosin-7-O-β-D-glucopyranoside(CG) is a principal constituent of Astragalus membranaceus(AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin(Ocal) m RNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2(BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family(WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.

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.

连翘苷对体内外鼻咽癌血管新生的影响

【目的】观察连翘苷抗鼻咽癌效应及机制。【方法】(1)体外实验:不同浓度(0.625、1.25、2.5、5、10μmol/L)连翘苷作用于体外人脐静脉内皮细胞(HUVECs)、人鼻咽正常上皮细胞系NP69和鼻咽癌细胞系HNE1和SUNE-1后,采用细胞计数试剂盒8(CCK-8)法检测细胞活力,小管形成实验测定HUVECs血管新生活性,Transwell小室实验测定HUVECs侵袭活性,Western Blot法测定HNE1和SUNE-1细胞、HUVECs中血管内皮生长因子A(VEGFA)、血管内皮生长因子受体2(VEGFR2)、Wnt和β-catenin表达水平。(2)体内实验:以连翘苷(10 mg/kg)对鼻咽癌异种移植瘤裸鼠进行干预后,采用血管造影法检测裸鼠鼻咽癌异种移植瘤的血管密度,免疫组织化学法检测鼻咽癌异种移植瘤中CD34、VEGFA和Wnt的表达。【结果】(1)连翘苷(0.625~10μmol/L)对体外HNE1和SUNE-1细胞活力有显著抑制效果,但对体外HUVECs和NP69的细胞活力无显著抑制效果;连翘苷(0.625~10μmol/L)可明显抑制HUVECs血管新生和侵袭活性,VEGFA(20μg/L)可削弱连翘苷对体外HUVECs血管新生和侵袭活性的抑制作用;连翘苷可下调VEGFA、Wnt和β-catenin在HNE1和SUNE-1细胞中的含量;连翘苷可诱导HUVECs中VEGFR2表达下调。(2)连翘苷(10 mg/kg)对裸鼠无明显毒副作用,但可显著抑制鼻咽癌异种移植瘤生长和血管新生,抑制鼻咽癌异种移植瘤组织中的CD34、VEGFA和Wnt的阳性表达。【结论】连翘苷对体内外鼻咽癌血管新生有显著抑制作用,其主要作用机制可能与调控Wnt/β-catenin/VEGFA/VEGFR2信号通路有关。