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Aim To explore the effects of the expression of the transcription faetor Glil of Hedgehog ( Hh ) signaling pathway and the 6-Shogaol mediated Hedge- hog/Glil pathway on the proliferation, invasion and migration in MDA-MB-231 eells of triple negative breast eaneer.Methods MDA-MB-231 eells were transfected by lentiviral vectors to stably overexpressed Glil gene.The overexpression efficiency of Glil was verified by qRT-PCR and Western blot.CCK-8 and EdlJ assays were used to detect the effect of Glil expression and 6-Shogaol on cell viability.Cell scratch assay and Transwell assay were used to detect the ability of migration and invasion.Western blot was used to detect the proteins expression of Hedgehog signaling pathway and other related genes.Results MDA-MB- 231-Glil overexpression cell line was successfully established.When Gli 1 gene was overexpressed, the invasion and migration ability of cells was significantly improved, anrl the expression of Hh signaling pathway gene Glil , EMT marker gene Vimentin, Hippo signaling pathway genes YAP and TEAD4 inereased.When the expression of Glil was inhibited by the Hh/Gli pathway inhibitor Gant61 , the proliferation, invasion and migration abilities were suppressed.When the eells were treated with 6-Shogaol, the abilities of proliferation, invasion and migration were inhibited as well as the proteins expression of Glil , Vimentin, YAP and TEAD4 deereased.Conclusions Glil gene ean promote the invasion and migration of MDA-MB-231 eells.6-Shogaol ean inhibit proliferation, invasion and migration of breast eaneer eells through Hedgehog signaling pathway, suggesting that transcription factor Glil may be one of the targets of 6-Shogaol.
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; Aim To investigate whether the Glil + cells fibrosis is mediated by damaged cardiomyocyte-derived exosomes and explore the possible mechanism. Methods Glil + cells were isolated from mouse heart and identified by flow cytometry. Neonatal rat cardiomyocytes were subjected to normoxic and hypoxic treatment, respectively. Normoxic/hypoxic exosomes were obtained and detected by flow cytometry, nanosight tracficking analysis (NTA), transmission electron microscopy (TEM) and Western blot. Key exosomal miRNA content was determined by RT-qPCR. Then G l i l + cells were co-cultured with normoxic /hypoxic exosomes or transfected with miRNA mimic to confirm the expression level of fibrosis-related proteins. Flow cytometry was used to detect the proportion of G l i l + cells positively expressing both DDR-2 and a-SMA protein. Results G l i l + cells biomarkers, including CD29,CD105 and G l i l, were identified, but CD31, CD34 and CD45 were undetectable. NTA showed that the diameters of exosomes were about 100 nm and exosomal markers CD63, HSP-70, Alix and Flotillin-1 were detectable by Western blot. Further study found that cardiomyocytes produced more exosomal miR-223 (P < 0. 01) under hypoxia treatment. Hypoxic exosomes and miR-223 mimic obviously increased the expression of a-SMA (P < 0. 01, P < 0. 05), DRR-2 (P <0. 01, P < 0. 01) and collagen I (P < 0. 05, P < 0. 01) in G l i l + cells. Conclusions Hypoxic cardiomyocyte-derived exosomes may promote G l i l + cell fibrosis, which may be related to the high expression of miR-223 in exosomes.
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Baihui (DU20)-penetrating-Qubin (GB7) acupuncture can inhibit inflammatory reactions and activate signaling pathways related to proliferation after intracerebral hemorrhage. However, there is no research showing the relationship between this treatment and cell apoptosis. Rat models of intracerebral hemorrhage were established by injecting 60 µL of autologous blood into the right side of the caudate-putamen. Six hours later, the needle traveled subcutaneously from the Baihui acupoint to Qubin acupoint. The needle was alternately rotated (180 ± 10 turns/min) manually along clockwise and counter-clockwise directions. Stimulation lasted for 7 days, and was performed three times each for 6 minutes with 6-minute intervals between stimulations. Rats intraperitoneally receiving Sonic hedgehog pathway activator, purmorphamine (1 mg/kg per day), served as positive controls. Motor and sensory function were assessed using the Ludmila Belayev test. Extent of pathological changes were measured in the perihemorrhagic penumbra using hematoxylin-eosin staining. Apoptosis was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling assay. Expression of smoothened (Smo) and glioma-associated homolog 1 (Gli1) was determined by western blot assay. Our results showed that Baihui-penetrating-Qubin acupuncture promoted recovery of motor and sensory function, reduced the apoptotic cell percentage in the perihemorrhagic penumbra, and up-regulated Smo and Gli1 expression. We conclude that Baihui-penetrating-Qubin acupuncture can mitigate hemorrhage and promote functional recovery of the brain in a rat model of intracerebral hemorrhage, possibly by activating the Sonic hedgehog pathway.
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Objective To up-regulate the expression of Glil gene in periodontal ligament stem cells ( PDLSCs) and to explore the effect of Glil gene on PDLSCs proliferation and osteogenesis differentiation by establishing Glil gene adenovirus vectors. Methods Subcloned Glil to viral backbone vector Adtrack-CMV and transfered the established vector to 293T cells, which was to acquire the virus particles. Trans-fected aim cells,namely PDLSCs,with these virus. Detected its effect on PDLSCs proliferation with CCK-8 assay, and detected the expression of Glil and the bone-related markers ALP and Runx2 through Western blot. Results An adenovirus vector, which were over expressed Glil gene, was successfully constructed and transfected to PDLSCs. Compared with the empty vector group and normal group, the over expressed one had a much slower proliferation rate in CCK-8 assay (P=0. 003). Western blot showed that ALP and Runx2 can be overexpressing os-teogenic differentiated after PDLSCs successfully transfected with the Glil gene. Conclusion Over expressing Glil gene would lead to a much slower proliferation rate in the PDLSCs and an increase of the bone-related markers. It is concluded that Glil can enhance the osteogenic dif-ferentiation capacity in PDLSCs.
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Objective To investigate the expression of Glil and β-catenin,which are central molecules of Hedgehog and Wnt signaling pathway,in colon cancer tissues and cell lines,and evaluate their relationship.Methods RT-PCR and Western blot were used to determine the expression of Glil and β-catenin in colon cancer tissues and cells.Interaction between endogenous Glil and endogenous 3-catenin was examined using immunoprecipitation method.Results Compared to corresponding normal tissues,the expression of Gli1 and β-catenin raised raising with one accord,the average growth rates were 42.69,72.11 respectively (P ＜ 0.05).In colon cancer cell lines,the expression levels of Glil and β-catenin increased,which were 52.54,17.23,5.54 and 5.30,6.34,2.78 (all P ＜ 0.05).Both of them were enhanced in three Wnt aberrant active cells visibly.The immunoprecipitation result indicated that there was an interaction between Glil and β-catenin in colon cancer cells.Conclusion The expression of Glil and β-catenin rise congruously in both colon cancer cells and tissues,and there is an interaction between them.This interaction between two key components of Hedgehog and Wnt signaling may suggest the possible crosstalk manner in these pathways.
