构建TRAF6基因3’非编码区荧光素酶报告质粒验证及与潜在靶基因TRAF6的靶向关系

Detection of the targeting relationship between TRAF6 and miR-146-5p by dual luciferase reporter system

背景:前期研究发现miR-146-5p在染氟成骨细胞凋亡时表达上调。目的:构建肿瘤坏死因子受体相关因子6(TNF receptor associated factor 6,TRAF6)基因3’非编码区(3’UTR)荧光素酶报告质粒,利用双荧光素酶报告基因验证miR-146-5p与其潜在靶基因TRAF6的靶向关系。方法:采用生物信息学方法预测mi R-146-5p与TRAF6基因的结合位点。采用PCR技术扩增TRAF6基因3’UTR片段,并克隆至p Yr-MirTarget载体,构建野生型重组双荧光素酶报告质粒。实验分6组:①6a-5p-核苷类似物+TRAF6野生型3’-UTR共转组;②无核苷类似物对照+TRAF6野生型3’UTR共转组(对照组);③miR-146a-5p-抑制剂+TRAF6野生型;④TRAF6野生型3’-UTR转染组;⑤p Yr-MirTarget空质粒转染组;⑥正常细胞组。分别将重组荧光素酶报告质粒与miR-146-5p和核苷类似物共转染Saos-2,同时设置无核苷类似物对照、miR-146-5p抑制剂阴性对照和空载p Yr-MirTarget-W3’UTR、TRAF6-W 3’UTR及正常对照。检测6组细胞中荧光素酶活性。将核苷类似物和miR-146-5pinhibitor以及无核苷类似物对照分别转染人成骨细胞Saos-2,裂解细胞提取蛋白后采用免疫印迹检测TRAF6蛋白表达水平。结果与结论:①共转染miR-146-5p mimics的Saos-2细胞中荧光素酶活性为10.103 0±0.558 5、对照组(无核苷类似物-TRAF63’UTR)荧光素酶活性为13.1400±0.7204、抑制剂(inhibitor)组荧光素酶活性为13.707 1±0.434 8、野生型TRAF6 3’UTR质粒的Saos-2细胞中荧光素酶活性为13.202 1±0.456 5。仅转染空质粒的细胞荧光素梅活性为14.706 2±0.441 6,Saos-2细胞中荧光素酶活性本底值为1.126 4±0.126 2。共转染miR-146-5p mimics组明显降低(F=715.789,P <0.000 1),其他3组间与对照组比较,差异无统计学意义(P> 0.05);②免疫印迹结果显示,与对照组比较,细胞转染miR-146-5p mimics后TRAF6蛋白表达水平明显下调;③结果说明,TRAF6与mi R-146-5p存在靶向关系。

BACKGROUND: Preliminary study has found that miR-146-5 p is up-regulated in osteobIasts induced by sodium fIuoride.OBJECTIVE: To verify the targeting relationship between miR-146-5 p and its potential target gene TNF receptor associated factor 6(TRAF6)using a dual luciferase reporter gene through constructing a luciferase reporter plasmid for the 3’ non-coding region(3’UTR) of TRAF6 gene.METHODS: Bioinformatics methods were used to predict the binding sites of miR-146-5 p and TRAF6 genes. The 3’UTR fragment of TRAF6 gene was amplified by PCR and cloned into pYr-MirTarget vector to construct a wild type recombinant dual-luciferase reporter plasmid. There were six groups:(1) 6a-5p-nucleoside analogs + TRAF6-W 3’UTR co-transfection group;(2) non-nucleoside analogs mimics + TRAF6-W3’UTR co-transfection group(control group);(3) miR-146 a-5p inhibitor + TRAF6-W group;(4) TRAF6-W 3’UTR co-transfection group;(5)empty pYr-MirTarget transfection group;(6) normal cell group. The co-transfection of recombinant luciferase reporter plasmid and miR-146-5 p or nucleoside analogs(mimics) with Saos-2 was made, respectively. At the same time, nucleoside analogs mimics(control group),miR-146-5p inhibitor(negative control group) and empty pYr-MirTarget-W 3’UTR and TRAF6-W 3’UTR were added simultaneously to detect luciferase activity in six groups of cells. Mimics, miR-146-5p inhibitor and nucleoside analogs mimics were transfected to the human osteoblast Saos-2 respectively. The protein expression level was detected by western blot assay after lysing cells to extract protein.RESULTS AND CONCLUSION:(1) Luciferase activity in Saos-2 cells co-transfected with miR-146-5p mimics was 10.103 0±0.558 5;the control group’s(NC mimics-TRAF6 3’UTR) luciferase activity was 13.140 0±0.720 4;the inhibitor group’s luciferase activity was 13.707 1±0.434 8;and luciferase activity in Saos-2 cells of the wild-type TRAF6 3’UTR plasmid was 13.202 1±0.456 5. The luciferase activity of the cells transfected with the empty plasmid alone was 14.706 2±0.441 6. The original luciferase activity in Saos-2 cells was 1.126 4±0.126 2. The number of miR-146-5 p mimics co-transfected significantly decreased(F=715.789, P < 0.000 1). The other three groups were compared with the control group, showing no significant difference(P > 0.05).(2) Western blot assay showed that compared with the control group, the expression level of TRAF6 protein was significantly down-regulated after transfection of miR-146-5 p mimics.(3) To conclude, there is a targeted relationship between TRAF6 and mi R-146-5 p.