MiR-27b-3p靶向ID-3调控骨肉瘤细胞增殖的研究

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摘要目的观察微小RNA-27b-3p(microRNA-27b-3p，miR-27b-3p)与DNA结合/分化抑制蛋白-3(DNA binding/differentiation inhibitory protein-3，ID-3)的靶向关系，分析其对增殖的调控作用。方法应用前瞻性研究方法，选择人骨肉瘤细胞株U2OS，采用双荧光素酶报告基因实验验证miR-27b3p与ID-3的靶向关系。建立空白对照组(NC组)、空载体转染组、miR-27b-3p mimic组、miR-27b-3p mimic和siRNA ID-3共转染组，每组纳入12个培养皿。采用细胞计数试剂盒-8(cell counting kit-8，CCK-8)法检测细胞的活性，采用集落形成实验观察细胞的克隆形成情况。应用Western Blot法检测ID-3和增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)的表达。选择骨肉瘤术后组织共40例，其中男22例，女18例；年龄4~88岁，平均年龄(16.3±4.3)岁。应用实时荧光定量聚合酶链式反应(real time quantitative polymerase chain reaction，RT-qPCR)检测miR-27b-3p的表达，应用免疫组化法检测ID-3和PCNA的表达。结果双荧光素酶报告基因实验发现miR-27b-3p能引起转染pGL-3-ID-3-WT的细胞中荧光素酶活性降低。与空白对照组和空载体转染组比较，miR-27b-3p mimic组细胞活性明显下降，克隆集落数量明显减少，PCNA的表达下降，miR-27b-3p mimic和siRNA ID-3共转染组能逆转上述表达水平。骨肉瘤组织中miR-27b-3p与ID-3、miR-27b-3p与PCNA均具有负相关性，ID-3与PCNA具有正相关性。miR-27b-3p的表达与生存时间相关。结论 miR-27b-3p靶向ID-3负调控骨肉瘤细胞的增殖，MiR-27b-3p低表达可能与不良预后有关。

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关键词 ：骨肉瘤, 微小RNA-27b-3p, DNA结合/分化抑制蛋白-3, 细胞增殖

Abstract：Objective To observe the targeting relationship between microRNA-27b-3p(miR-27b-3p)and DNA binding/differentiation inhibitory protein-3(ID-3)，and analyze its regulatory effect on proliferation on ostersarcoma cells.Methods Prospective research methods was applicated.Osteosarcoma cell line U20S was selected.Targeting relationship between miR-27b-3p and ID-3 was verified by double luciferase reporter gene assay.The blank control group，empty vector transfection group，miR-27b-3p mimic group，miR-27b-3p mimic and siRNA ID-3 co-transfection group were established.Cell activity was detected by CCK-8 method.Clonal formation was detected by cells colony formation experiment.ID-3 and PCNA were detected by Western Blot method.40 cases of osteosarcoma were selected，including 22 males and 18 females.The average age was(16.3±4.3)years old.Expression of miR-27b-3p was detected by real-time fluorescence quantitative PCR.ID-3 and PCNA were detected by immunohistochemistry method.Results MiR-27b-3p could reduce the luciferase activity in pGL-3-ID-3-WT cells.Compared with the blank control group and the empty vector transfection group，the activity of miR-27b-3p mimic group，the number of colonies and the expression of PCNA decreased.Andthey could be reversed in co transfection group.Negative correlation was found between miR-27b-3p and ID-3，miR-27b-3p and PCNA in osteosarcoma.Positive correlation was found between ID-3 and PCNA in osteosarcoma.The expression of miR-27b-3p was correlated with survival time.Conclusion MiR-27b-3p targeting ID-3 negatively regulates the proliferation on osteosarcoma cells.Lower expression of miR-27b-3p may be related to poor prognosis.

Key words： osteosarcoma microRNA-27b-3p-DNA binding/differentiation inhibitory protein-3 cell proliferation

基金资助:河北省重点研发计划指导项目(172302076)

作者简介: 李志强(1981— )，男，副主任医师，河北省沧州市人民医院骨科，061000。

引用本文:

李志强，刘树江，沈朝，郝玉升，吴文杰，苑萌萌. MiR-27b-3p靶向ID-3调控骨肉瘤细胞增殖的研究[J]. 实用骨科杂志, 2021, 27(6): 528-533.
Li Zhiqiang，Liu Shujiang，Shen Chao，et al. Proliferation Regulation of MiR-27b-3p Targeting ID-3 on Osteosarcoma Cells. sygkzz, 2021, 27(6): 528-533.

链接本文:

http://www.sygkzz.com/CN/ 或 http://www.sygkzz.com/CN/Y2021/V27/I6/528

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