CAJ | 학술논문

目的：：根据microRNA-146a(miR-146a)预测靶点构建荧光素酶报告基因重组质粒并进行功能验证,为研究吸入性损伤中 miR-146a通过靶向跨膜蛋白33(TMEM33)对 Toll 样受体/核因子-κB(TLRs/NF-κB)信号通路的调控作用提供前期的工作基础。方法：通过生物学软件预测 TMEM33 mRNA 3′端非翻译区(TMEM33 mRNA 3′-UTR)可能是miR-146a的作用靶点,将设计合成的TMEM33及其突变序列TMEM33-mu序列分别克隆至荧光素酶报告质粒,采用脂质体法将这两种重组荧光素酶报告质粒 pMIR-TMEM33和 pMIR-TMEM33-mu分别与miR146a mimics共转染 HEK-293T细胞,以 pMIR-TMEM33+miR-control 转染 HEK-293细胞作对照,双荧光素酶报告基因系统检测荧光素酶活性。结果：TargetScan 和 miRanda 软件预测显示,miR-146a 与TMEM33 mRNA 3′-UTR存在互补结合位点,构建的荧光素酶报告质粒经酶切及测序鉴定正确。双荧光素酶报告基因检测系统显示,miR-146a对TMEM33 mRNA 3′-UTR 具有靶向作用,pMIR-TMEM33+miR146a mimics组较 pMIR-TMEM33+miR-control 组荧光素酶活性降低62%左右,差异具有统计学意义(P<0.01)；而 miR-146a对TMEM33-mu 3′-UTR无靶向作用,pMIR-TMEM33-mu+miR-146a 组与 pMIR-TMEM33+miR-control组之间差异无统计学意义(P>0.05)。结论：成功构建 TMEM33mRNA 3′-UTR 荧光素酶报告载体,证实 miR-146a对TMEM33 mRNA 3′-UTR具有靶向调控作用,为从基因水平深入揭示吸入性肺损伤发病机制提供实验室数据和方法。
목적：：근거microRNA-146a(miR-146a)예측파점구건형광소매보고기인중조질립병진행공능험증,위연구흡입성손상중 miR-146a통과파향과막단백33(TMEM33)대 Toll 양수체/핵인자-κB(TLRs/NF-κB)신호통로적조공작용제공전기적공작기출。방법：통과생물학연건예측 TMEM33 mRNA 3′단비번역구(TMEM33 mRNA 3′-UTR)가능시miR-146a적작용파점,장설계합성적TMEM33급기돌변서렬TMEM33-mu서렬분별극륭지형광소매보고질립,채용지질체법장저량충중조형광소매보고질립 pMIR-TMEM33화 pMIR-TMEM33-mu분별여miR146a mimics공전염 HEK-293T세포,이 pMIR-TMEM33+miR-control 전염 HEK-293세포작대조,쌍형광소매보고기인계통검측형광소매활성。결과：TargetScan 화 miRanda 연건예측현시,miR-146a 여TMEM33 mRNA 3′-UTR존재호보결합위점,구건적형광소매보고질립경매절급측서감정정학。쌍형광소매보고기인검측계통현시,miR-146a대TMEM33 mRNA 3′-UTR 구유파향작용,pMIR-TMEM33+miR146a mimics조교 pMIR-TMEM33+miR-control 조형광소매활성강저62%좌우,차이구유통계학의의(P<0.01)；이 miR-146a대TMEM33-mu 3′-UTR무파향작용,pMIR-TMEM33-mu+miR-146a 조여 pMIR-TMEM33+miR-control조지간차이무통계학의의(P>0.05)。결론：성공구건 TMEM33mRNA 3′-UTR 형광소매보고재체,증실 miR-146a대TMEM33 mRNA 3′-UTR구유파향조공작용,위종기인수평심입게시흡입성폐손상발병궤제제공실험실수거화방법。
Objective:To construct microRNA-146a(miR-146a)luciferase reporter gene vector according to miR-146a predicted target sequences and to detect its function,in order to provide an initial experimental basis for the study of miR-146a regulating Toll-like receptors/nuclear factor-κB (TLRs/NF-κB)signaling pathway by targeting transmembrane pro-tein 33 (TMEM33)after inhalation injury.Methods:It was predicted by biological software that TMEM33 mRNA 3′-un-translated region (TMEM33 mRNA 3′-UTR)may be a target of miR-146a. TMEM33 and mutant TMEM33 sequence (TMEM3 3-mu)were then designed and synthesized,and they were cloned into luciferase reporter vector. These two lu-ciferase reporter vectors,pMIR-TMEM3 3 and pMIR-TMEM3 3-mu,were respectively transfected into human embryonic kidney-293 cells (HEK 293 cells)together with miR-146a mimic by liposome. pMIR-TMEM33 and miR-control were transfected into HEK293 cells served as control. The luciferase activity was detected by dual luciferase reporter assay sys-tem. Results:TargetScan and miRanda software predicted that miR-146a had the complementary binding sites with 3′-UTR of TMEM3 3 ,and luciferase reporter vector was constructed,therefore the result of sequencing and double digesting of recombined plasmid were completely correct. Dual-luciferase reporter assay showed that miR-146a possesses a target effect on 3′-UTR of TMEM33. Compared to the pMIR-TMEM33+miR-control group,the luciferase activity of the <br> pMIR-TMEM33 + miR146a mimics group was decreased by 62%,with statistically significant difference (P<0.01),while miR-146a had not targeted 3′-UTR of mutant TMEM33. There was no statistically significant difference between pMIR-TMEM33-mu+miR-146a group and pMIR-TMEM33+miR-control group(P>0.05). Conclusions:The luciferase reporter vector containing TMEM33 mRNA 3′-UTR is constructed successfully,and it was found that miR-146a can target TMEM33 mRNA 3′-UTR. The results provide the experiment data for fur-ther disclosing the mechanism of inhalation inj ury on the level of gene expression.