中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (27): 4397-4401.doi: 10.3969/j.issn.2095-4344.1392

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

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

张亚楼1，邓  强2，周杨俊杰2，赵  阳1，郭  琼1，蒋稥菊3，岳明明1，陈  龙1，马文静4   

  1.  (1新疆医科大学基础医学院组织胚胎学教研室，新疆维吾尔自治区乌鲁木齐市  830011；2新疆医科大学第一附属医院骨科中心，新疆维吾尔自治区乌鲁木齐市  830011；3新疆医科大学克拉玛依学院，新疆维吾尔自治区克拉玛依市  834000)；4新疆医科大学中心实验室，新疆维吾尔自治区乌鲁木齐  830011)
  • 收稿日期:2019-03-27 出版日期:2019-09-28 发布日期:2019-09-28
  • 通讯作者: 邓强，博士，主任医师，新疆医科大学第一附属医院骨科中心，新疆维吾尔自治区乌鲁木齐市 830011
  • 作者简介:张亚楼，男，1976年生，重庆市人，汉族，2010年新疆医科大学毕业，博士，副教授，主要从事骨细胞凋亡研究。
  • 基金资助:

    国家自然科学基金项目(81460481)，项目负责人：张亚楼；新疆自然科学基金(2015211c040)，项目负责人：邓强

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

Zhang Yalou1, Deng Qiang2, Zhou Yangjunjie2, Zhao Yang1, Guo Qiong1, Jiang Xiangju3, Yue Mingming1, Chen Long1, Ma Wenjing4   

  1.  (1Department of Histology and Embryology, Basic Medical College of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China; 2Orthopedic Center, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China; 3Karamay College of Xinjiang Medical University, Karamay 834000, Xinjiang Uygur Autonomous Region, China; 4Central Laboratory of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China)
  • Received:2019-03-27 Online:2019-09-28 Published:2019-09-28
  • Contact: Deng Qiang, MD, Chief physician, Orthopedic Center, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • About author:Zhang Yalou, PhD, Associate professor, Department of Histology and Embryology, Basic Medical College of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • Supported by:

     the National Natural Science Foundation of China, No. 81460481 (to ZYL); the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2015211c040 (to DQ)

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摘要：

文章快速阅读：

文题释义：
双报告基因检测系统：荧火虫和海肾荧光素酶检测的组合，匹配相应载体，提供了双遗传报告基因应用的理想检测系统。一个报告基因活性的改变，与基因表达的特定实验条件相关，而第二个报告基因的组成性活性则提供内对照，使实验值可以正态化。
肿瘤坏死因子受体相关因子：是肿瘤坏死因子超家族和Toll样/白细胞介素1受体超家族重要的接头分子，在天然免疫和获得性免疫中发挥重要作用。TRAFs包括7 个密切相关的蛋白，其中，TRAF6是脂多糖、TNF等刺激信号的下游分子，可以和多种激酶结合。
摘要
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miR-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pYr-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；⑤pYr-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pYr-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13.140 0±0.720 4、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miR-146-5p存在靶向关系。

关键词: 微小RNA, 肿瘤坏死因子家族, 双荧光素酶, TRAF6基因, 荧光素酶

Abstract:

BACKGROUND: Preliminary study has found that miR-146-5p is up-regulated in osteobIasts induced by sodium fIuoride.
OBJECTIVE: To verify the targeting relationship between miR-146-5p 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-146-5p 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-W 3'UTR co-transfection group (control group); (3) miR-146a-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-146-5p 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-146-5p 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-146-5p mimics. (3) To conclude, there is a targeted relationship between TRAF6 and miR-146-5p.

Key words: microRNA, tumor necrosis family, dual luciferase, TRAF6 gene, luciferase

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