重庆医学
重慶醫學
중경의학
CHONGQING MEDICAL JOURNAL
2015年
24期
3316-3318
,共3页
微 RNAs%hsa-miR-335%靶基因%基因调控%CCL11%CCL26%SOX4%双荧光素酶报告基因检测系统
微 RNAs%hsa-miR-335%靶基因%基因調控%CCL11%CCL26%SOX4%雙熒光素酶報告基因檢測繫統
미 RNAs%hsa-miR-335%파기인%기인조공%CCL11%CCL26%SOX4%쌍형광소매보고기인검측계통
MicroRNAs%hsa-miR-335%target gene%gene regulation%CCL1 1%CCL26%SOX4%dual luciferase reporter gene sys-tem
目的：验证人 MicroRNA335(hsa-miR-335)与嗜酸细胞趋化因子1(CCL11)、嗜酸细胞趋化因子3(CCL26)、SOX4的靶向调控关系。方法选择网络在线的生物信息学软件 miRanda 和 TargetScan 共同预测为 hsa-miR-335靶基因的 CCL11、CCL26、SOX4作为研究对象。构建 CCL11、CCL26、SOX4基因3′端非翻译区(3′UTR)双荧光素酶报告基因载体 pMIR-RE-PORT-CCL113′UTR、pMIR-REPORT-CCL263′UTR、pMIR-REPORT-SOX43′UTR；采用 Lipofectamine 2000将真核表达质粒pMIR-REPORT-CCL113′UTR、pMIR-REPORT-CCL263′UTR、pMIR-REPORT-SOX43′UTR、阳性对照质粒分别与 Pre-miRTM miRNA335 Precursor 或阴性对照质粒共转染到293 T7/17细胞；双荧光素酶报告基因检测系统检测 CCL11、CCL26、SOX4基因3′UTR 与 Hsa-mir-335共转染萤火虫(Firefly)和海洋腔肠(Renilla)荧光素酶活性,并与阴性对照对比。结果hsa-miR-335对SOX4的荧光表达有显著的抑制作用(P <0.01),但并不影响 CCL11、CCL26荧光素酶活性(P >0.05)。结论hsa-miR-335可靶向调控 SOX4,而对 CCL11、CCL26的表达无影响。
目的：驗證人 MicroRNA335(hsa-miR-335)與嗜痠細胞趨化因子1(CCL11)、嗜痠細胞趨化因子3(CCL26)、SOX4的靶嚮調控關繫。方法選擇網絡在線的生物信息學軟件 miRanda 和 TargetScan 共同預測為 hsa-miR-335靶基因的 CCL11、CCL26、SOX4作為研究對象。構建 CCL11、CCL26、SOX4基因3′耑非翻譯區(3′UTR)雙熒光素酶報告基因載體 pMIR-RE-PORT-CCL113′UTR、pMIR-REPORT-CCL263′UTR、pMIR-REPORT-SOX43′UTR；採用 Lipofectamine 2000將真覈錶達質粒pMIR-REPORT-CCL113′UTR、pMIR-REPORT-CCL263′UTR、pMIR-REPORT-SOX43′UTR、暘性對照質粒分彆與 Pre-miRTM miRNA335 Precursor 或陰性對照質粒共轉染到293 T7/17細胞；雙熒光素酶報告基因檢測繫統檢測 CCL11、CCL26、SOX4基因3′UTR 與 Hsa-mir-335共轉染螢火蟲(Firefly)和海洋腔腸(Renilla)熒光素酶活性,併與陰性對照對比。結果hsa-miR-335對SOX4的熒光錶達有顯著的抑製作用(P <0.01),但併不影響 CCL11、CCL26熒光素酶活性(P >0.05)。結論hsa-miR-335可靶嚮調控 SOX4,而對 CCL11、CCL26的錶達無影響。
목적：험증인 MicroRNA335(hsa-miR-335)여기산세포추화인자1(CCL11)、기산세포추화인자3(CCL26)、SOX4적파향조공관계。방법선택망락재선적생물신식학연건 miRanda 화 TargetScan 공동예측위 hsa-miR-335파기인적 CCL11、CCL26、SOX4작위연구대상。구건 CCL11、CCL26、SOX4기인3′단비번역구(3′UTR)쌍형광소매보고기인재체 pMIR-RE-PORT-CCL113′UTR、pMIR-REPORT-CCL263′UTR、pMIR-REPORT-SOX43′UTR；채용 Lipofectamine 2000장진핵표체질립pMIR-REPORT-CCL113′UTR、pMIR-REPORT-CCL263′UTR、pMIR-REPORT-SOX43′UTR、양성대조질립분별여 Pre-miRTM miRNA335 Precursor 혹음성대조질립공전염도293 T7/17세포；쌍형광소매보고기인검측계통검측 CCL11、CCL26、SOX4기인3′UTR 여 Hsa-mir-335공전염형화충(Firefly)화해양강장(Renilla)형광소매활성,병여음성대조대비。결과hsa-miR-335대SOX4적형광표체유현저적억제작용(P <0.01),단병불영향 CCL11、CCL26형광소매활성(P >0.05)。결론hsa-miR-335가파향조공 SOX4,이대 CCL11、CCL26적표체무영향。
Objective To identify the targeted-regulating relationship between human MicroRNA335 (hsa-miR-335 )and CCL1 1,CCL26 and SOX4.Methods The potential fragments of hsa-miR-335 target genes CCL1 1,CCL26 and SOX4 were predicted by the bioinformatics analyzing tools online.The 3′untranslated regions(3′UTR)of the CCL1 1,CCL26 and SOX4 were connected to the eukaryotic expression vectors pMIR REPORT.The constructs of pMIR-REPORT-CCL1 13′UTR,pMIR-REPORT-CCL26 3′UTR,pMIR-REPORT-SOX4 3′UTR and positive control were co-transfected with Pre-miRTM miRNA335 Precursor or negative control into 293 T7/1 7 cell line by lipofectamine 2000,respectively.Both Firefly and Renilla luciferase activity were detected by dual luciferase reporter assay system.Results Compared with the negative control group,luciferase assay revealed that has-miR-335 could significantly diminish luciferase activity from SOX4 reporter vector (P <0.01 ),while the suppression of luciferase activity was not found in CCL1 1 or CCL26 reporter vector (P >0.05).Conclusion The results suggested that hsa-miR-335 targeted regu-lated SOX4,but not targeted CCL1 1 and CCL26.
