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circVRK1靶向miR-4428调控急性淋巴细胞白血病KOCL44细胞增殖及凋亡的分子机制研究

Molecular Mechanism of circVRK1 Regulating the Proliferation and Apoptosis of Acute Lymphoblastic Leukemia KOCL44 Cells by Targeting miR-4428

  • 摘要:
    目的 分析circVRK1和miR-4428在急性淋巴细胞白血病(acute lymphoblastic leukemia, ALL)细胞增殖和凋亡中的关系。
    方法 体外培养ALL细胞KOCL44,实验分组为:pcDNA、pcDNA-circVRK1、anti-miR-NC、anti-miR-4428、si-NC、si-circVRK1、pcDNA-circVRK1+miR-NC和pcDNA-circVRK1+miR-4428组。qRT-PCR检测细胞circVRK1和miR-4428的表达水平;CCK-8法、流式细胞术分别检测细胞增殖及凋亡;双荧光素酶报告实验检测circVRK1与miR-4428的靶向关系〔实验分为circVRK1野生型报告质粒(WT-circVRK1)+miR-NC、WT-circVRK1+miR-4428、circVRK1突变报告质粒(MUT-circVRK1)+miR-NC和MUT-circVRK1+ miR-4428组〕;Western blot检测Ki-67、cleaved caspase-3、cleaved caspase-9蛋白表达量。
    结果 相较于pcDNA组,pcDNA-circVRK1组的circVRK1表达上调(P<0.05);与转染pcDNA或者转染anti-miR-NC相比,转染pcDNA-circVRK1或anti-miR-4428后,KOCL44细胞活力和Ki-67蛋白表达降低(P<0.05),凋亡率和cleaved caspase-3、cleaved caspase-9蛋白水平增加(P<0.05);circVRK1可负向调控miR-4428的表达,但该作用仅在转染WT-circVRK1的组别中体现;与pcDNA组相比,pcDNA-circVRK1组miR-4428表达降低(P<0.05);与si-NC组相比,si-circVRK1组miR-4428表达增加(P<0.05);与共转染pcDNA-circVRK1+miR-NC相比,共转染pcDNA-circVRK1+miR-4428后细胞活力升高(P<0.05),Ki-67蛋白表达增加(P<0.05),凋亡率和cleaved caspase-3、cleaved caspase-9蛋白水平降低(P<0.05)。
    结论 circVRK1过表达可通过下调miR-4428表达而减弱ALL细胞增殖能力及诱导细胞凋亡。

     

    Abstract:
    Objective To elucidate the role of circVRK1 and its interaction with miR-4428 in regulating proliferation and apoptosis in acute lymphoblastic leukemia (ALL) cells.
    Methods KOCL44 ALL cells were cultured in vitro, and experimental groups included pcDNA, pcDNA-circVRK1, anti-miR-NC, anti-miR-4428, si-NC, si-circVRK1, pcDNA-circVRK1+miR-NC, and pcDNA-circVRK1+miR-4428. The expression levels of circVRK1 and miR-4428 were detected using qRT-PCR. CCK-8 assays and flow cytometry were used to assess cell proliferation and apoptosis, respectively. The dual luciferase reporter assays were employed to investigate the interaction between circVRK1 and miR-4428, with groups categorized as WT-circVRK1+miR-NC, WT-circVRK1+miR-4428, MUT-circVRK1+miR-NC, and MUT-circVRK1+ miR-4428. Western blotting was utilized to detect the expression levels of Ki-67, cleaved caspase-3, and cleaved caspase-9 proteins.
    Results Compared to the pcDNA group, circVRK1 expression was up-regulated in the pcDNA-circVRK1 group (P<0.05). Compared to transfection with pcDNA or anti-miR-NC, transfection with pcDNA-circVRK1 or anti-miR-4428 led to decreased cell viability and Ki-67 protein levels in KOCL44 cells (P<0.05), and increased apoptosis rates and levels of cleaved caspase-3 and cleaved caspase-9 (P<0.05). circVRK1 was found to negatively regulate miR-4428 expression, with this effect observed only in the WT-circVRK1 group. miR-4428 levels were lower in the pcDNA-circVRK1 group compared to the pcDNA group (P<0.05) and higher in the si-circVRK1 group compared to the si-NC group (P<0.05). Co-transfection with pcDNA-circVRK1+miR-4428 resulted in increased cell viability (P<0.05) and Ki-67 expression (P<0.05), and decreased apoptosis rates and levels of cleaved caspase-3 and cleaved caspase-9 (P<0.05) compared to co-transfection with pcDNA-circVRK1+miR-NC.
    Conclusion Overexpression of circVRK1 reduces the proliferation ability of acute ALL cells and induces cell apoptosis by downregulating miR-4428 expression.

     

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