Construction of TMEM33 mRNA 3′-untranslated region reporter gene vector and detection of its function
Feng Shengj uan
Lü Xiaowu
Liu Zhen
Li Wenting
Wu Yuj ia
Zhang Yaj ie
Wang Ruichen
Cao ling
Qiu Yabin
Jia Chiyu
Abstract：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.
Keywords：Transmembrane protein 33Luciferase reporter gene vectorMicroRNA-146aInhalation injury
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 204-208 )