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  • br Journal Pre proof br Figure Overexpression of hsa circ

    2022-04-27


    Journal Pre-proof
    Figure 5 Overexpression of hsa_circ_0007059 impeded lung cancer cell growth
    via suppression of miR-378
    (A) A549 and H1975 Salvinorin A were transfected miR-378 mimic and NC mimic, cell transfection efficiency was evaluated via execution of RT-qPCR. After co-transfection with miR-378 mimic and overexpressed vector of hsa_circ_0007059, (B) cell viability and (C) apoptosis were measured via execution of RT-qPCR, CCK-8 and flow cytometry assays. Protein levels of (D-F) p53, CyclinD1 and (G-I) Bax, Pro-Caspase-3 and Cleaved-Caspase-3 were evaluated via implementation of western blot assay in both A549 and H1975 cells.
    Overexpression of miR-378 reversed the functions of hsa_circ_0007059 in EMT
    process in lung cancer cells
    For further exploration of whether miR-378 joins in modulation of EMT process,
    Journal Pre-proof
    A549 and H1975 cells were transfected with miR-378 mimic and received TGFβ1 exposure. In Figure 6A and 6B, we discovered that the up-regulation of E-cadherin and the down-regulation of Vimentin, Twist1 and Zeb1 (P < 0.001) triggered by overexpressed hsa_circ_0007059 were both eliminated by miR-378 overexpression in A549 cells. Moreover, Figure 6C and 6D disclosed the same results in H1975 cells as that overexpression of miR-378 reversed the functions of hsa_circ_0007059 overexpression in E-cadherin, Vimentin, Twist1 and Zeb1 expression (P < 0.001). These explorations uncovered that miR-378 overexpression abolished the functions of hsa_circ_0007059 in EMT process in lung cancer cells.
    Figure 6 Overexpression of hsa_circ_0007059 repressed EMT process via
    repression of miR-378 in lung cancer cells
    A549 and H1975 cells were co-transfected with miR-378 mimic and overexpressed
    Journal Pre-proof
    vector of hsa_circ_0007059, and subsequently managed with 10 ng/mL TGFβ1 for 48 h. (A and B) Protein levels of E-cadherin, Vimentin, Twist1 and Zeb1 in the disposed A549 cells were evaluated via implementation of western blot assay. (C and D) Protein levels of E-cadherin, Vimentin, Twist1 and Zeb1 in the disposed H1975 cells were detected via utilization of western blot assay. ***P < 0.001.
    Overexpression of hsa_circ_0007059 blocked Wnt/β-catenin and ERK1/2 pathways
    through suppression of miR-378
    Journal Pre-proof
    Figure 7 Overexpression of hsa_circ_0007059 hindered Wnt/β-catenin and
    ERK1/2 pathways via suppression of miR-378
    Discussion
    CircRNAs are a class of RNAs with covalent ring structure, which have been proven to be connected with the stages and metastasis of cancers, and also possess diversified especial biological functions in physiological processes (Ding et al. , 2018, Huang et al. , 2017). In consideration of the high stability and tissue specificity, circRNA is hopeful to become an underlying biomarker for prediction and therapy of cancers (Xia
    Journal Pre-proof
    et al. , 2017). Nonetheless, the influences and modulatory mechanism of circRNA in lung cancer only have a handful of studies reported. In the current research, we probed the functions of hsa_circ_0007059 in lung cancer cell growth and EMT process. Repressed hsa_circ_0007059 was firstly discovered in lung cancer tissues. Function analysis disclosed that overexpression of hsa_circ_0007059 abated cell proliferation, triggered apoptosis and restrained EMT process in A549 and H1975 cells. Additionally, miR-378 was found to ascend in lung cancer tissues but hsa_circ_0007059 overexpression repressed miR-378 expression in A549 and H1975 cells. MiR-378 overexpression evidently reversed the functions of hsa_circ_0007059 in lung cancer cell growth and EMT process. Finally, we discovered that overexpressed hsa_circ_0007059 hindered Wnt/β-catenin and ERK1/2 pathways through repression of miR-378 in A549 and H1975 cells.
    Generally, the existing studies concentrated on circRNAs in lung cancer can be roughly divided into biomarker and functional mechanism studies. In biomarker studies, Yao et al. disclosed that the up-regulation of hsa_circ_100876 was intently linked to lymph node metastasis and tumor stages of lung cancer (Yao et al. , 2017). Luo et al. noticed that the elevation of hsa_circ_0000064 was observed in lung cancer tissues and A549 and H1229 lung cancer cell lines and the exceptional expression was also connected to the clinical characteristics of lymph node metastasis and TNM stage (Luo et al. , 2017). In functional mechanism study, a crucial research corroborated that hsa_circRNA_103809 aggrandized lung cancer cell proliferation and invasion in vitro and accelerated tumor formation in vivo through sponging miR-4302 (Liu et al. ,
    Journal Pre-proof
    2018). In regard to hsa_circ_0007059, it is only reported to restrain cell growth, migration, and invasion in OSCC cell lines of SCC15 and CAL27 (Su et al., 2019). Similar with the aforementioned research, we discovered that hsa_circ_0007059 was declined in lung cancer tissues and hsa_circ_0007059 overexpression refrained cell proliferation, augmented apoptosis and relieved the process of EMT in A549 and H1975 cells. The findings suggested that hsa_circ_0007059 might be a possible biomarker in lung cancer.