MiR-27b-3p靶向ID-3调控骨肉瘤细胞增殖的研究

Proliferation Regulation of MiR-27b-3p Targeting ID-3 on Osteosarcoma Cells

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pGL3-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。

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 pGL3-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.