下调微小RNA-550a-5p靶向调控清道夫受体A类成员3抑制非小细胞肺癌进展

Downregulation of microRNA-550a-5p targetedly regulates scavenger receptor class A member 3 to inhibit progression of non-small cell lung cancer

  • 摘要:
    目的 探讨下调微小RNA(miR)-550a-5p对非小细胞肺癌(NSCLC)细胞增殖、迁移及侵袭的影响及其分子机制。
    方法 将miR-550a-5p抑制物(inhibitor)及其阴性对照(inhibitor NC)片段, 以及清道夫受体A类成员3(SCARA3)小干扰RNA(si-SCARA3)及其阴性对照(si-NC)慢病毒单独或共转染至A549细胞中,并设为inhibitor NC组、miR-550a-5p inhibitor组、miR-550a-5p inhibitor+si-NC(共转染)组和miR-550a-5p inhibitor+si-SCARA3(共转染)组; 采用CCK-8法检测A549细胞增殖活性; 采用平板克隆法检测A549细胞克隆形成能力; 采用划痕法和Transwell小室法检测A549细胞迁移和侵袭能力; 采用酶联免疫吸附测定(ELISA)检测A549细胞上清中基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)表达水平; 采用Annexin V/PI双染法检测A549细胞凋亡水平。
    结果 与癌旁正常组织比较, NSCLC组织中miR-550a-5p表达水平升高, SCARA3 mRNA表达水平降低,差异有统计学意义(P < 0.05)。双荧光素酶报告基因实验证实, miR-550a-5p靶向调控SCARA3表达。与inhibitor NC组比较, miR-550a-5p inhibitor组细胞增殖率、克隆团形成数、迁移及侵袭能力降低,凋亡率升高,差异有统计学意义(P < 0.05)。与inhibitor NC组比较, miR-550a-5p inhibitor组细胞上清中MMP-2和MMP-9表达水平降低, SCARA3蛋白表达水平升高,差异有统计学意义(P < 0.05)。与miR-550a-5p inhibitor组或miR-550a-5p inhibitor+si-NC组比较, miR-550a-5p inhibitor+si-SCARA3组细胞增殖率、克隆团形成数、迁移及侵袭能力升高,凋亡率降低,差异有统计学意义(P < 0.05)。与miR-550a-5p inhibitor组或miR-550a-5p inhibitor+si-NC组比较, miR-550a-5p inhibitor+si-SCARA3组细胞上清中MMP-2和MMP-9表达水平升高, SCARA3蛋白表达水平降低,差异有统计学意义(P < 0.05)。
    结论 下调miR-550a-5p可抑制NSCLC细胞增殖、迁移及侵袭并促进细胞凋亡,其机制可能与靶向上调SCARA3表达有关。

     

    Abstract:
    Objective To investigate the effects of downregulating microRNA (miR)-550a-5p on the proliferation, migration and invasion of non-small cell lung cancer (NSCLC) and to explore its molecular mechanisms.
    Methods The miR-550a-5p inhibitor and its negative control (inhibitor NC), as well as small interfering RNA targeting scavenger receptor class A member 3 (si-SCARA3) and its negative control (si-NC) were individually or co-transfected into A549 cells. These cells were designated as inhibitor NC group, miR-550a-5p inhibitor group, miR-550a-5p inhibitor + si-NC (co-transfection) group and miR-550a-5p inhibitor + si-SCARA3 (co-transfection) group. Cell proliferation was assessed by CCK-8 assay; colony formation ability was evaluated using a plate cloning method; cell migration and invasion were detected by scratch assay and Transwell chamber assay, respectively; levels of matrix metalloproteinase (MMP)-2 and MMP-9 in the supernatant of A549 cells were measured by enzyme-linked immunosorbent assay (ELISA); apoptosis levels were determined by Annexin V/PI double staining.
    Results Compared with adjacent normal tissues, the expression level of miR-550a-5p was increased, while SCARA3 mRNA expression was decreased in NSCLC tissues (P < 0.05). Dual-luciferase reporter assays confirmed that miR-550a-5p directly targeted SCARA3. Compared with the inhibitor NC group, cell proliferation rate, colony formation number, migration and invasion ability of the miR-550a-5p inhibitor group decreased, apoptosis rate increased (P < 0.05). Compared with the inhibitor NC group, the expression levels of MMP-2 and MMP-9 in cell supernatant of the miR-550a-5p inhibitor group were decreased, and the expression levels of SCARA3 protein were increased (P < 0.05). Compared with the miR-550a-5p inhibitor group or the miR-550a-5p inhibitor+si-NC group, cell proliferation rate, clonal colony formation number, migration and invasion ability of the miR-550a-5p inhibitor+si-SCARA3 group were increased, and apoptosis rate was decreased (P < 0.05). Compared with the miR-550a-5p inhibitor group or the miR-550a-5p inhibitor+si-NC group, the miR-550a-5p inhibitor+si-SCARA3 group showed increased expression levels of MMP-2 and MMP-9 in the cell supernatant and decreased SCARA3 protein expression levels (P < 0.05).
    Conclusion Downregulation of miR-550a-5p inhibits the proliferation, migration and invasion of NSCLC cells, and promotes apoptosis, possibly through upregulating SCARA3 expression.

     

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