环状RNA_0005273对食管癌细胞生物学行为的影响及可能机制

Influence of circular RNA_0005273 on biological behavior of esophageal cancer cells and its possible mechanism

  • 摘要:
    目的 探讨环状RNA_0005273(circ_0005273)对食管癌细胞生物学行为的影响及其可能作用机制。
    方法 分别采用实时荧光定量聚合酶链反应(qRT-PCR)与蛋白质印迹法(Western blot)检测食管癌组织、癌旁组织、人食管癌细胞EC109中circ_0005273、微小RNA-1200(miR-1200)基因表达量与信号转导与转录活化因子3(STAT3)蛋白表达量。正常培养EC109细胞并设为对照组,另将si-NC、si-circ_0005273、miR-NC、miR-1200、si-circ_0005273+anti-miR-1200分别转染至EC109细胞并设为si-参照组、si-circ组、miR-参照组、miR-1200组、si-circ+anti组。分别采用噻唑蓝(MTT)法、平板克隆形成实验、流式细胞术和Transwell实验检测各组细胞增殖抑制率、集落形成数、细胞凋亡率和迁移细胞数; 采用双荧光素酶报告基因实验分析miR-1200与circ_0005273、STAT3间的靶向调控关系。
    结果 食管癌组织中circ_0005273、STAT3蛋白表达量高于癌旁组织, miR-1200表达量低于癌旁组织,差异有统计学意义(P < 0.05); si-circ组miR-1200表达量、细胞增殖抑制率、凋亡率均高于对照组、si-参照组, circ_0005273表达量、STAT3蛋白表达量、集落形成数和迁移细胞数均低于或少于对照组、si-参照组,差异有统计学意义(P < 0.05); miR-1200组STAT3蛋白表达量、集落形成数、迁移细胞数均低于对照组、miR-参照组, miR-1200表达量、细胞增殖抑制率、凋亡率均高于对照组、miR-参照组,差异有统计学意义(P < 0.05)。circ_0005273可靶向调控miR-1200, 且miR-1200可靶向调控STAT3。si-circ+anti组STAT3蛋白表达、集落形成数、迁移细胞数高于si-circ组,细胞增殖抑制率、凋亡率低于si-circ组,差异有统计学意义(P < 0.05)。
    结论 circ_0005273在食管癌组织中呈高表达,干扰circ_0005273表达可通过上调miR-1200表达和下调STAT3表达而发挥抑制食管癌细胞增殖、迁移的作用,并诱导细胞凋亡。

     

    Abstract:
    Objective To explore the influence of circular RNA_0005273 (circ_0005273) on biological behavior of esophageal cancer cells and its possible mechanism.
    Methods The genetic expression levels of circ_0005273, microRNA-1200 (miR-1200) and protein expression level of signal transducer and activator of transcription 3 (STAT3) in esophageal cancer tissues, paracancerous tissues and human esophageal cancer cells EC109 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot respectively. EC109 cells were normally cultured and named as control group, and the si-NC, si-circ_0005273, miR-NC, miR-1200 and si-circ_0005273+anti-miR-1200 were respectively transfected into EC109 cells and named as si-reference group, si-circ group, miR-reference group, miR-1200 group and si-circ+anti group. Methyl Thiazolyl Tetrazolium (MTT) assay, colony formation assay, flow cytometry and Transwell assay were used to detect inhibition rate of cell proliferation, the number of colony formation, cell apoptosis rate and the number of cell migration; the targeted regulatory relationships of miR-1200 with circ_0005273 and STAT3 were analyzed by dual-luciferase reporter assay.
    Results The protein expression levels of circ_0005273 and STAT3 in the esophageal cancer tissues were significantly higher than those in the paracancerous tissues, while the expression level of miR-1200 was significantly lower than that in the paracancerous tissues (P < 0.05); the expression level of miR-1200, inhibition rate of cell proliferation and cell apoptosis rate in the si-circ group were significantly higher than those in the control group and si-reference group, while the expression level of circ_0005273, protein expression level of STAT3, the number of colony formation and the number of migrating cells were significantly lower than those in the control group and si-reference group (P < 0.05); the protein expression level of STAT3, the number of colony formation and the number of migrating cells in the miR-1200 group were significantly lower than those in the control group and miR-reference group, while the expression level of miR-1200, inhibition rate of cell proliferation and apoptosis rate were significantly higher than those in the control group and miR-reference group (P < 0.05). The circ_0005273 was able to regulate miR-1200 and miR-1200 was able to regulate STAT3 by targeted therapy. Protein expression level of STAT3, the number of colony formation and the number of migrating cells in the si-circ+anti group were significantly higher than those in the si-circ group, while inhibition rate of cell proliferation and apoptosis rate were significantly lower than those in the si-circ group (P < 0.05).
    Conclusion The circ_0005273 is highly expressed in esophageal cancer tissues, and interference of circ_0005273 can inhibit the proliferation and migration of esophageal cancer cells as well as induce cell apoptosis by up-regulating expression of miR-1200 and down-regulating expression of STAT3.

     

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