miR-34b-3p targeting FDX1 suppressed the growth and apoptosis of neuroblastoma cells in children
冯祯祯 1赵煜 2黄贵祥 2路翔宇 3王宇 4陈丹1
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作者信息
1. 四川省医学科学院·四川省人民医院，电子科技大学附属医院小儿外科，成都 610072
2. 四川省医学科学院·四川省人民医院，电子科技大学附属医院急诊科，成都 610072
3. 四川省医学科学院·四川省人民医院，电子科技大学附属医院肝胆外科，成都 610072
4. 四川省人民医院灾难医学与急诊医学研究所，成都 610072
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摘要
目的 探究miR-34b-3p在神经母细胞瘤（neuroblastoma，NB）细胞增殖和凋亡过程中的调节作用。 方法 体外培养人正常背根神经节细胞和NB细胞系，搜集NB组织和邻近的正常组织。qRT-PCR检测NB组织和细胞中miR-34b-3p和FDX1的表达；CCK-8法、克隆形成实验和流式细胞术检测NB细胞的增殖和凋亡；双荧光素酶报告基因分析FDX1 mRNA 3'非翻译区（3'-untranslated region，3'-UTR）和miR-34b-3p之间的靶点关系；Western blot检测相关蛋白的表达。采用SPSS 23.0和GraphPad Prism 6.01进行Student'st-test检验。 结果 与对照组比较，miR-34b-3p在NB肿瘤组织和癌细胞系（SK-N-BE、SK-N-SH、SH-SY5Y和LAN-6）中均下调（P<0.05）。与对照组比较，miR-34b-3p过表达抑制了癌细胞SK-N-BE和SH-SY5Y的生长，并诱导凋亡增加（P<0.05）。萤光素酶报告基因证实，miR-34b-3p可以与FDX1 3'-UTR结合，抑制NB细胞中FDX1的表达。此外，FDX1过表达有效逆转了miR-34b-3p对NB细胞生长和凋亡的抑制作用。与对照组比较，miR-34b-3p稳定转染SH-SY5Y细胞抑制了异种移植瘤的生长和肿瘤重量（P<0.05）。 结论 miR-34b-3p可能通过靶向FDX1在NB中发挥肿瘤抑制作用，是一种新颖的NB诊断和治疗的生物标志物。 Objective To explore the regulatory role of miR-34b-3p in the proliferation and apoptosis of neuroblastoma (NB) cells. Methods Human normal dorsal root ganglion cell and NB cell lines were cultured in vitro, and NB tissues and adjacent normal tissues were harvested. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed for detecting the expressions of miR-34b-3p and FDX1 in NB tissues and cells. The proliferation and apoptosis of NB cells were detected by CCK-8 assay, colony formation assay and flow cytometry. Target relationship between ferriredoxin 1 (FDX1) mRNA 3'untranslated region (3'-UTR) and miR-34b-3p was examined by dual-luciferase reporter gene. Western blot was utilized for detecting the expression of related proteins. And SPSS 23.0 and GraphPad Prism 6.01 were utilized for Student's t-test. Results As compared with control group, miR-34b-3p was down-regulated in NB tumor tissues and cell lines (SK-N-BE, SK-N-SH, SH-SY5Y & LAN-6)(P<0.05). An over-expression of miR-34b-3p suppressed the growth of SK-N-BE and SH-SY5Y cells and induced their apoptosis ( P<0.05). Luciferase reporter gene confirmed that miR-34b-3p conjugated with FDX1 3'-UTR and arrested the expression of FDX1 in NB cells. In addition, an over-expression of FDX1 effectively reversed the inhibitory effect of miR-34b-3p on the growth and apoptosis of NB cells. As compared with control group, stable transfection of miR-34b-3p into SH-SY5Y cells suppressed the growth and tumor weight of xenograft tumor (P<0.05). Conclusions miR-34b-3p may play a tumor suppressor role in NB through targeting FDX1. It is a novel biomarker for diagnosing and treating NB.
Abstract
Objective To explore the regulatory role of miR-34b-3p in the proliferation and apoptosis of neuroblastoma (NB) cells. Methods Human normal dorsal root ganglion cell and NB cell lines were cultured in vitro, and NB tissues and adjacent normal tissues were harvested. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed for detecting the expressions of miR-34b-3p and FDX1 in NB tissues and cells. The proliferation and apoptosis of NB cells were detected by CCK-8 assay, colony formation assay and flow cytometry. Target relationship between ferriredoxin 1 (FDX1) mRNA 3'untranslated region (3'-UTR) and miR-34b-3p was examined by dual-luciferase reporter gene. Western blot was utilized for detecting the expression of related proteins. And SPSS 23.0 and GraphPad Prism 6.01 were utilized for Student's t-test. Results As compared with control group, miR-34b-3p was down-regulated in NB tumor tissues and cell lines (SK-N-BE, SK-N-SH, SH-SY5Y & LAN-6)(P<0.05). An over-expression of miR-34b-3p suppressed the growth of SK-N-BE and SH-SY5Y cells and induced their apoptosis ( P<0.05). Luciferase reporter gene confirmed that miR-34b-3p conjugated with FDX1 3'-UTR and arrested the expression of FDX1 in NB cells. In addition, an over-expression of FDX1 effectively reversed the inhibitory effect of miR-34b-3p on the growth and apoptosis of NB cells. As compared with control group, stable transfection of miR-34b-3p into SH-SY5Y cells suppressed the growth and tumor weight of xenograft tumor (P<0.05). Conclusions miR-34b-3p may play a tumor suppressor role in NB through targeting FDX1. It is a novel biomarker for diagnosing and treating NB.
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