<rp id="vgn1y"></rp>
<rp id="vgn1y"><object id="vgn1y"></object></rp>

  • <rp id="vgn1y"><ruby id="vgn1y"><input id="vgn1y"></input></ruby></rp>
  • <th id="vgn1y"></th>
      當前位置:首頁 > 智慧健康列表 > 組織工程研究 > 詳細信息
      miR-9 對NRP1 的靶向調控及其在輻射效應中的作用研究
      來源: | 作者: | 發布時間:2013-11-8 18:01:28

      miR-9 對NRP1 的靶向調控及其在輻射效應中的作用研究
      張海芹,董娟聰,金霖霖,高輝,邵立虹,劉麗波,金順子
      基金項目:國家自然科學基金(30870584,81371890);教育部高等學校博士學科點專項科研基金(20120061110063)
      作者簡介:張海芹(1987-),女,碩士研究生,主要從事輻射腫瘤學研究
      通信聯系人:金順子(1965-),女,教授,主要從事輻射免疫學與輻射腫瘤學的研究. 

       (吉林大學公共衛生學院 衛生部放射生物學重點實驗室,長春 130021)
      摘要:目的:構建hsa-miR-9 表達質粒和NRP1-3′UTR 熒光素酶報告質粒,探討miR-9 對NRP1 的靶向調控作用及其在A549 細胞中的輻射效應。方法:利用生物信息學方法預測hsa-miR-9 與 NRP1-3 ′ -UTR 的結合位點; 將PCR 擴增的miR-9 前體序列和
      pcDNA-DEST47 載體連接,構建pcDNA-DEST47-miR-9 表達質粒;將PCR 擴增的 NRP1-310 ′ UTR 序列 和pEZX-MT05 載體經Acc65 I 和Xho I 雙酶切后連接, 構建pEZX-MT05-NRP1-3′UTR 表達質粒;以此質粒為模板,根據突變體引物序列擴增 NRP1-3
      ′UTR 突變序列,構建pEZX-MT05- NRP1-3′UTRMUT 表達質粒。采用雙熒光素酶檢測法驗證miR-9 與NRP1 的靶向關系;采用實時定量 PCR 及 Western blot 分別檢測10 Gy照射后miR-9 的表達水平及對 NRP1 蛋白表達的靶向抑制作用。結果:成功構建 pcDNA-DEST47-miR-9、pEZX-MT05-NRP1- 3′UTR 及pEZX-MT05-NRP1-3′UTR MUT表達質粒,并通過酶切及基因測序得到鑒定;熒光素酶活性實驗結果顯示,miR-9 可以顯著下調野生型NRP1-3′UTR 質粒的熒光素酶活性(t=3.906, p<0.05),而不影響突變型質粒的熒光素酶活性,同時證實miR-9 以外的miRNA(miR-29b)不能抑制野生型NRP1-3′UTR 質粒的熒光素酶活性。實時定量 PCR 結果顯示,miR-9 的表達水平在10 Gy 照射后明顯降低(t=37.319,p<0.01),加入miR-9 mimic 雖然可以抑制輻射引起的miR-9 的表達下調,但還是低于單純mimic 組;Western blot 結果顯示,miR-9 對NRP1 的蛋白表達具有抑制作用,但加入miR-9 mimic 可以阻止輻射誘導的NRP1 表達上調,而加入miR-9 inhibitor 時卻其表達上調。結論:證實miR-9 通過靶向結合 NRP1 基因3'UTR,特異性調控 NRP1 蛋白表達;10 Gy X 射線使A549 細胞中hsa-miR-9 表達下調,從而NRP1 蛋白表達上調,提示在 A549 輻射效應中miR-9 對NRP1 具有靶向調控作用。
      關鍵詞:miR-9;NRP1;A549;靶向調控;電離輻射
      中圖分類號:請查閱《中國圖書館分類法》
      Target regulation of miR-9 on the expression of NRP1 and its role in the study of irradiation effects
      ZHANG Haiqin, DONG Juancong, JIN Linlin, GAO Hui, SHAO Lihong, LIU Libo,
      JIN shunzi
      (College of Public Medicine, Key Laboratory of Radiobiology, Ministry of Health,jilin University,ChangChun 130021)
       Abstract: Objective has-miR-9 recombinant plasmid and NRP1-3'UTR luciferase reporter plasmid was constructed and to explore the effect of miR-9 on the expression of NRP1 and its irradiation effects in A549 cells. Methods Bioinformatics was used to analyze the potential binding sites of sa-miR-9 and NRP1-3′UTR. The precursor of miR-9 was amplified by PCR and the PCR products were cloned into pcDNA-DEST47 vector to construct pcDNA-DEST47-miR-9 expression plasmid. NRP1-3′UTR was amplified by PCR and the PCR products were cloned into Acc65 I and Xho I digestion pEZX-MT05 vector to construct pEZX-MT05-NRP1-3′-UTR expression plasmid ; pEZX-MT05-NRP1-3'UTRMUT expression plasmid was amplifie according to the mutant sequence of pEZX-MT05-NRP1-3'-UTR ; The effect of miR-9 interaction with the 3’-UTR of NRP1 on luciferase activity was detected with a dual luciferase assay system ;the expression level of NRP1 45 protein affected by miR-9 was detected by Western blotting and the level of miR-9 was detected by Real-time PCR after 10 Gy X rays. Results pcDNA-DEST47-miR-9、pEZX-MT05-NRP1-3′UTR and pEZX-MT05-NRP1-3′UTRMUT plasmids were successfully constructed ; and has been identified by restriction analysis and DNA sequencing ; Luciferase reporter experiments suggested that miR-9 could reduce the luciferase activity of wild plasmid (t = 3.906, p<0.05), without affecting the luciferase activity of the mutant plasmid and the luciferase activity was not inhibited in other types of miRNA( miR-29b );The real-time PCR suggested that the level of miR-9 was significantly reduced after 10 Gy X rays (t=37.319,p<0.01); While adding miR-9 mimic can inhibit the expression of radiation-induced down-regulation of miR-9, but still lower than that mimic group ;Western blot suggested that the level of NRP1 was inhibited by miR-9 , while the upregulation of NRP1 induced by irradiation could be suppressed by miR-9 mimic. Conclusion Theses results suggested that miR-9 regulates the expression of NRP1 by targeting the complementary sites and specific regulation of NRP1 protein expression , the level of has-miR-9 was downregulated while the protein level of
      NRP1was upregulate after 10 Gy Xrays , miR-9 plays a role in the process of target regulation of  irradiation on expression of NRP1 protein in A549.I
      Key words: miR-9; NRP1; A549; Targeted regulation; Ionizing radiation

      奇米色8888