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lncRNA CYTOR/miR-193b-3p/HMGB1轴调控T细胞急性淋巴细胞白血病细胞MOLT-4增殖与凋亡的作用研究

Role of lncRNA CYTOR/miR-193b-3p/HMGB1 axis in regulating the proliferation and apoptosis of T cell acute lymphoblastic leukemia MOLT-4 cell

    摘要
  • 摘要:
    目的 探讨lncRNA CYTOR对T细胞急性淋巴细胞白血病(T-ALL)细胞增殖和凋亡的影响及可能机制。
    方法 体外培养人正常骨髓基质细胞系HS-5和T-ALL细胞系(CCRF-CEM、MOLT-4、CEM-C1), 采用逆转录-实时定量聚合酶链反应(RT-qPCR)法检测细胞中lncRNA CYTOR、miR-193b-3p和HMGB1 mRNA表达; 采用蛋白质印迹(Western blot)法检测细胞中HMGB1蛋白表达。分别转染si-CYTOR、miR-193b-3p mimics、si-HMGB1及共转染si-CYTOR与anti-miR-193b-3p至MOLT-4细胞, 以CCK-8法检测细胞增殖，以流式细胞术检测细胞凋亡, 以Western blot法检测细胞Bax和Bcl-2、cleaved-caspase3蛋白表达。通过双荧光素酶报告基因实验验证lncRNA CYTOR与miR-193b-3p、miR-193b-3p与HMGB1的调控关系。
    结果 与HS-5细胞比较, T-ALL细胞系(CCRF-CEM、MOLT-4、CEM-C1)中lncRNA CYTOR、HMGB1 mRNA及其蛋白表达均升高, miR-193b-3p表达降低，差异有统计学意义(P < 0.05)。下调lncRNA CYTOR、上调miR-193b-3p或下调HMGB1后, MOLT-4细胞增殖受到抑制, Bcl-2蛋白表达降低，凋亡率和细胞中Bax、cleaved-caspase3蛋白表达升高，差异有统计学意义(P < 0.05)。lncRNA CYTOR竞争性吸附miR-193b-3p, HMGB1是miR-193b-3p的靶基因。上调HMGB1表达可逆转lncRNA CYTOR对T-ALL细胞MOLT-4增殖和凋亡的作用。敲低miR-193b-3p可逆转lncRNA CYTOR对MOLT-4细胞增殖和凋亡的作用。
    结论 lncRNA CYTOR在T-ALL细胞系中表达上调，其可能通过竞争性吸附miR-193b-3p进而上调HMGB1的表达来促进T-ALL细胞增殖，并阻碍细胞凋亡，其有可能成为T-ALL治疗的分子靶点。

     

    Abstract:
    Objective To investigate the effect of lncRNA CYTOR on the proliferation and apoptosis of T cell acute lymphoblastic leukemia (T-ALL) cells and its possible mechanism.
    Methods Human normal bone marrow stromal cell lines HS-5 and T-ALL cell lines (CCRF-CEM, MOLT-4 and CEM-C1) were cultured in vitro, and then the expression of lncRNA CYTOR, miR-193b-3p and HMGB1 mRNA in the cells were detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and the expression of HMGB1 protein in the cells were detected by western blot. MOLT-4 cells were transfected with si-CYTOR, miR-193b-3p mimics, si-HMGB1, or co-transfected with si-CYTOR and anti-miR-193b-3p, respectively. And then CCK-8 was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis, and western blot was used to detect the protein expression of Bax, cleaved-caspase3 and Bcl-2 in cells. The dual luciferase reporter gene experiment was used to verify the regulatory relationship between lncRNA CYTOR and miR-193b-3p, miR-193b-3p and HMGB1.
    Results Compared with HS-5 cells, the expressions of lncRNA CYTOR and HMGB1 mRNA and its protein in T-ALL cell lines (CCRF-CEM, MOLT-4 and CEM-C1) were significantly increased, the expression of miR-193b-3p were significantly decreased (P < 0.05). After down-regulating lncRNA CYTOR, up-regulating miR-193b-3p, or down-regulating HMGB1, MOLT-4 cell proliferation was inhibited, the expression of Bcl-2 protein in cells significantly decreased, and the rate of apoptosis and the protein expression of Bax, cleaved-caspase3 in cells were significantly increased (P < 0.05). LncRNA CYTOR competitively adsorbed miR-193b-3p, and HMGB1 was the target gene of miR-193b-3p. Up-regulation of HMGB1 expression reversed the effects of lncRNA CYTOR on the proliferation and apoptosis of MOLT-4 in T-ALL cells. Knockdown of miR-193b-3p reversed the effects of lncRNA CYTOR on the proliferation and apoptosis of MOLT-4 cell.
    Conclusion The expression of lncRNA CYTOR is up-regulated in T-ALL cell lines. It may promote T-ALL cells proliferation and hinder cell apoptosis by competitively adsorbing miR-193b-3p and then up-regulating the expression of HMGB1. It may become a molecular target for T-ALL treatment.

     

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