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Epithelial-mesenchymal transition (EMT) is involved in both physiological and pathological processes. EMT plays an essential role in the invasion, migration and metastasis of tumours. Autophagy has been shown to regulate EMT in a variety of cancers but not in head and neck squamous cell carcinoma (HNSCC). Herein, we investigated whether autophagy also regulates EMT in HNSCC. Analyses of clinical data from three public databases revealed that higher expression of fibronectin-1 (FN1) correlated with poorer prognosis and higher tumour pathological grade in HNSCC. Data from SCC-25 cells demonstrated that rapamycin and Earle's balanced salt solution (EBSS) promoted autophagy, leading to increased FN1 degradation, while 3-methyladenine (3-MA), bafilomycin A1 (Baf A1) and chloroquine (CQ) inhibited autophagy, leading to decreased FN1 degradation. On the other hand, autophagic flux was blocked in BECN1 mutant HNSCC Cal-27 cells, and rapamycin did not promote autophagy in Cal-27 cells; also in addition, FN1 degradation was inhibited. Further, we identified FN1 degradation through the lysosome-dependent degradation pathway using the proteasome inhibitor MG132. Data from immunoprecipitation assays also showed that p62/SQSTM1 participated as an autophagy adapter in the autophagy-lysosome pathway of FN1 degradation. Finally, data from immunoprecipitation assays demonstrated that the interaction between p62 and FN1 was abolished in p62 mutant MCF-7 and A2780 cell lines. These results indicate that autophagy significantly promotes the degradation of FN1. Collectively, our findings clearly suggest that FN1, as a marker of EMT, has adverse effects on HNSCC and elucidate the autophagy-lysosome degradation mechanism of FN1.
Assuntos
Feminino , Humanos , Autofagia , Linhagem Celular Tumoral , Fibronectinas , Lisossomos/metabolismo , Neoplasias Ovarianas , Proteína Sequestossoma-1/metabolismo , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Objective:To investigate the transport pathway and intracellular distribution of the of fluorescent carbon dots(CDs)synthesized by folic acid and polyethyleneimine(PEI)through the membrane of MC3T3-E1 cells and its effect on the cells,and to clarify the mechanism.Methods:The fluorescent CDs with the function of cell imaging were synthesized by hydrothermal method using folic acid and PEI as the raw materials;MTT assay was applied to screen the best concentration of CDs.The MC3T3-E1 cells were divided into blank control group,folic acid group and CDs group.The biocompatibility of CDs was evaluated by the detection of cell cycle,apoptosis and cellular reactive oxygen species(ROS)level.Nystatin as a kind of caveolae inhibitor and nocodazole as a kind of macropinocytosis inhibitor were used to find out the pathway through which the cells took in the CDs.Using the charcteristic of CDs with blue fluorescence stimulated by ultraviolet ray,the organelle probes were used to observe the distribution of CDs.Results:Compared with blank control group,the cells in different concentrations(100-450 mg·L-1)of CDs groups showed no cytotoxicity at 24 h(P>0.05);at 48 h,the cell proliferation rate was reduced to 68.4% of blank control group when the concentration of CDs reached 350 mg · L -1(P<0.05). Compared with blank control group,the percentages of cells in G0phase and G1phase in CDs group were decreased (P<0.05),and the percentage of cells in S phase was increased(P<0.05);the percentages of cells in G2phase and M phase were increased,but there no was significant differences(P>0.05).Compared with blank control group,the apoptotic rates of the cells in folic acid group and CDs group had no significant differences(P>0.05). Compared with blank control group,the intracellular ROS levels in folic acid group and CDs group were significantly decreased(P<0.05).Compared with blank control group,the uptake amount of CDs in the cells was decreased in nystatin group(P<0.05).The blue fluorescence of CDs overlapped with the red fluorescence of mitochondria under an inverted fluorescence microscope,the blue fluorescence of CDs overlapped with the red fluorescence of lysosomes;they didn't overlap completely with the red fluorescence of the endoplasmic reticulum;the blue fluorescence of CDs overlapped poorly with the red fluorescence of Golgi apparatus.Conclusion:CDs perform well in biocompatibility and they can be distributed to different organelles after taken in by the cells.They can be used as a kind of gene carrier in transgenic therapy.
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Objective:To study the lethal effect of vitamin C carbon dots on oral squamous cell carcinoma KB cells, and to clarity its related mechanism.Methods: The KB cells were treated with different concentrations (5, 10, 20, 40 and 80 mg·L-1) of vitamin C carbon dots in vitro as experimental groups, and 0 mg·L-1 vitamin C carbon dots group was used as blank control group.MTT assay was used to detect the proliferation rates of KB cells in various groups;colony formation assay was used to detect the colony formation ability of KB cells;Western blotting was performed to detect the protein expression levels of autophagy related protein LC3 in KB cells in various groups;flow cytometry was used to detect the apoptotic rates of KB cells in various groups.Results: Compared with blank control group, the proliferation rates and colony formation abilities of KB cells in 20, 40 and 80 mg·L-1 carbon dots groups were markedly decreased (P<0.01).Compared with blank control group, the protein expression level of LC3 Ⅱ in 40 mg·L-1 carbon dots group was increased(P<0.05);the apoptotic rate of KB cells was markedly increased(P<0.01).Conclusion: Vitamin C carbon dots can kill the oral squamous cell carcinoma KB cells effectively, suppress the proliferation and impair the colony formation ability of KB cells, which is related to autophagy and apoptosis of KB cells.
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BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are widely used in the field of tissue engineering because of their multi-directional differentiation potential. Micro RNAs play an important role in promoting osteogenic differentiation of BMSCs.OBJECTIVE: To investigate the effect of miR-218 and miR-26a on the osteogenic differentiation of rat BMSCs, and to provide reference for the study on osteogenic differentiation of BMSCs and the clinical application. METHODS: The bone marrow of the femur of Wistar rats was extracted and the BMSCs were isolated and cultured to the 3rd generation. MiR-218, miR-26a and polyethyleneimine (PEI) were mixed in a specific ratio to form a miRNA/PEI complex. Meanwhile, a negative control group was established.RESULTS AND CONCLUSION: (1) Rat BMSCs grew well. (2) The optimal concentration of miRNA mimics was 50 nmol/L by MTT method. (3) The expression of alkaline phosphatase and collagen type I mRNA was significantly increased (P < 0.05). (4) Alkaline phosphatase staining showed that compared with the blank control group and the negative control group, the cytoplasm showed obvious coloring. (5) There were a lot of mineralized nodules shown by alizarin red staining. Therefore, miR-218/PEI complex, miR-26a/PEI complex and miR-218+miR-26a/PEI co-transfection complex could promote the osteogenic differentiation of BMSCs. Under the same conditions, the osteogenesis of miR-26a was slightly better than that of miR-218.
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Objective:To transfect the non-viral vector polyethylenimine (PEI)mediated miR-2861 mimic into the MC3T3-E1 cell line,and to explore the transfection efficiency of PEI/miR-2861 complex and its effects on the proliferation and osteogenesis differentiation in pre-osteoblasts. Methods:The proper amount of PEI was blended with miR-2861 mimic and negative control (NC)separately in a ratio of N∶P=10∶1,and they were divided into experiment group and NC group. The NC/PEI complex acted as the NC group was used to eliminate the interference of osteogenesis from the addition of double-stranded RNA mimic.MTT assay was used to determine the optimal concentration of PEI/miR-2861 mimic complex.The fluorescence imaging technique and bulge-loop RT-PCR were used to detect the transfection efficiency and mRNA expression of miRNA-2861 in the cells with different concentrations (10,30, 50,and 100 nmol · L-1 ), separately.The osteogenesis ability of MC3T3 cells was identified with RT-PCR and Alizarin red staining with the selected concentration of PEI/miR-2861 by transient transfection.Results:Compared with blank control group,the proliferation rates of MC3T3 cells in 100 nmol·L-1 PEI/miR-2861 group was decreased significantly at 72 h (P < 0. 05 ). With the increasing of transfected concentration the transfection efficiency of miRNA/PEI complex was increased gradually.The results of Alizarin red staining and quantitative analysis showed that calcium deposits were more and bigger in experiment group after induced for 21 d,while both in blank control group and NC group they were less.Conclusion:The miRNA-2861 mimic can be effectively transfected into the MC3T3-E1 cell line and expresses with a high level,which is mediated by PEI as the gene vector.miR-2861 mimic has a certain ability of promoting osteogenesis differentiation of MC3T3-E1 cells.
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Pathogen-associated molecular patterns (PAMPs) are conservative molecules associated with groups of pathogens or their products. These molecules are recognized by relevant receptors. PAMPs induce the expression of inflammatory cytokines through the signal cascade. The role of PAMPs in the initiation and development of periodontitis is recently attracting attention. PAMPs induce the expression of inflammatory mediators after they are recognized in the periodontium. This process damages the periodontal soft tissue and osseous tissue, thus resulting in periodontitis. The results of this study will provide an excellent resolution for the treatment of periodontitis by blocking the pathogenic pathway of PAMPs.