长链非编码RNA SLCO4A1-AS1靶向微小RNA-615-5p对食管癌细胞增殖、凋亡和炎症因子表达的影响

Effect of long non-coding RNA SLCO4A1-AS1 targeting microRNA-615-5p on the proliferation, apoptosis and inflammatory factors expression in esophageal cancer cells

  • 摘要:
    目的 探讨长链非编码RNA(LncRNA)溶质载体有机阴离子转运蛋白家族成员4A1反义RNA1(SLCO4A1-AS1) 靶向微小RNA-615-5p(miR-615-5p)对食管癌细胞增殖、凋亡和炎症因子表达的影响。
    方法 运用实时荧光定量聚合酶链式反应(RT-qPCR)分析LncRNA SLCO4A1-AS1和miR-615-5p在食管癌组织、细胞系中表达情况。将si-NC、si-LncRNA SLCO4A1-AS1、miR-NC、miR-615-5p模拟物、pcDNA、pcDNA-LncRNA SLCO4A1-AS1、si-LncRNA SLCO4A1-AS1+anti-miR-NC、si-LncRNA SLCO4A1-AS1+anti-miR-615-5p分别转染Eca109细胞。CCK-8和流式细胞术用于检测细胞活力和凋亡率; 平板克隆实验用于检测细胞增殖; 酶联免疫吸附法(ELISA)试剂盒检测培养液中白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平。采用双荧光素酶报告基因法确定LncRNA SLCO4A1-AS1与miR-615-5p的关系。
    结果 食管癌组织、细胞系中LncRNA SLCO4A1-AS1表达上调, miR-615-5p表达下调。抑制LncRNA SLCO4A1-AS1表达后,细胞活力、细胞克隆形成数量以及培养液中IL-6和TNF-α水平降低, miR-615-5p表达量、细胞凋亡率升高,差异有统计学意义(P<0.05)。与miR-NC组比较, miR-615-5p组Eca109细胞中miR-615-5p表达量、Cleaved-caspase-3蛋白水平、凋亡率升高,细胞活力、细胞克隆形成数量、培养液中IL-6和TNF-α水平降低,差异有统计学意义(P<0.05)。与si-LncRNA SLCO4A1-AS1+anti-miR-NC比较, si-LncRNA SLCO4A1-AS1+anti-miR-615-5p组Eca109细胞中miR-615-5p表达量、Cleaved-caspase-3蛋白水平、凋亡率降低,细胞活力、细胞克隆形成数量、培养液中IL-6和TNF-α水平升高,差异有统计学意义(P<0.05)。
    结论 LncRNA SLCO4A1-AS1能够促进食管癌的发生和发展。抑制LncRNA SLCO4A1-AS1能够减少食管癌细胞的增殖,降低炎症因子的表达,诱导癌细胞凋亡。

     

    Abstract:
    Objective To investigate the effects of long non-coding RNA (LncRNA) solute carrier organic anion transporter family member 4A1 (SLCO4A1-AS1) targeting microRNA-615-5p (miR-615-5p) in esophageal cancer cells on cell proliferation, apoptosis, and expression of inflammatory factors.
    Methods Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression of LncRNA SLCO4A1-AS1 and miR-615-5p in esophageal cancer tissues and cell lines. Eca109 cells were transfected with si-NC, si-LncRNA SLCO4A1-AS1, miR-NC, miR-615-5p mimic, pcDNA, pcDNA-LncRNA SLCO4A1-AS1, si-LncRNA SLCO4A1-AS1+anti-miR-NC, and si-LncRNA SLCO4A1-AS1+anti-miR-615-5p. Cell viability and apoptosis rate were measured by CCK-8 and flow cytometry, respectively; cell proliferation was determined by plate cloning assay; and levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the culture medium were measured using an enzyme-linked immunosorbent assay (ELISA) kit. A dual luciferase reporter gene assay was used to determine the relationship between LncRNA SLCO4A1-AS1 and miR-615-5p.
    Results Expression of LncRNA SLCO4A1-AS1 was upregulated, and miR-615-5p expression was downregulated in esophageal cancer tissues and cell lines. After inhibiting LncRNA SLCO4A1-AS1 expression, cell viability, the number of cell clones, and levels of IL-6 and TNF-α in the culture medium decreased, while miR-615-5p expression and apoptosis rate increased (P < 0.05). Compared with the miR-NC group, the miR-615-5p group showed increased miR-615-5p expression, levels of Cleaved-caspase-3 protein, and apoptosis rate, and decreased cell viability, the number of cell clones, and levels of IL-6 and TNF-α in the culture medium (P < 0.05). Compared with the si-LncRNA SLCO4A1-AS1+anti-miR-NC group, the si-LncRNA SLCO4A1-AS1+anti-miR-615-5p group showed decreased miR-615-5p expression, levels of Cleaved-caspase-3 protein, and apoptosis rate, and increased cell viability, the number of cell clones, and levels of IL-6 and TNF-α in the culture medium (P < 0.05).
    Conclusion LncRNA SLCO4A1-AS1 can promote the occurrence and development of esophageal cancer. Inhibiting LncRNA SLCO4A1-AS1 can reduce the proliferation of esophageal cancer cells, decrease the expression of inflammatory factors, and induce apoptosis.

     

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