title: [Effects of Lin28a and Lin28b on let-7 family activity].
abstract: In this report, we study the effects of over-expression of Lin28a and Lin28b on let-7 family activity in HeLaS3. Firstly, we constructed pAAV2neo-Lin28a and pAAV2neo-Lin28b to express Lin28a and Lin28b, respectively. Then, pAAV2neo-Lin28a and pAAV2neo-Lin28b were transfected into HeLaS3, selected with G418 and obtained cell lines, HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b, to express Lin28a and Lin28b stably. Thereafter, we constructed eight plasmid vectors for detection of let-7 family activity based on pAAV2neo-Gluc-(Fluc). These vectors were further packaged into recombinant adeno-associated viral vectors (rAAV) which were used as sensors, nominated as Asensors, to detect inhibition activity of miRNA at post-transcriptional level. Subsequently, with HeLaS3 as a control, we assayed expression levels of Lin28a and Lin28b by Western blot, detected expression levels of let-7 family by QRT-PCR, and tested let-7 family activity by Asensors in HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b. Results demonstrated that both HeLaS3/pAAV2neo-Lin28a and HeLaS3/pAAV2neo-Lin28b could express Lin28a and Lin28b effectively. Compared with HeLaS3, the expression level of let-7 family except let-7e declined in HeLaS3/pAAV2neo-Lin28a. But declining extent among members of let-7 family was different. The let-7 family activity also decreased while the decreasing extent varied among members. Furthermore, the activity level was not consistent with its expression level for the same member in let-7 family. Compared with HeLaS3, both expression level and activity level of let-7 family in HeLaS3/ pAAV2neo-Lin28b were decreased. However, the decreasing extent of let-7 family expression changes was larger than that of HeLaS3/pAAV2neo-Lin28a while the decreasing extent of activity changes was similar. In this study, we established a method to detect and compare post-transcriptional inhibition level mediated by miRNA complementary targets. We firstly clarified the effect of Lin28a and Lin28b on let-7 family activity profile and found that this effect was not the same as that at expression level of let-7 family, suggesting that it was more comprehensive to understand miRNA regulation roles to detect both miRNA expression and activity. This paves a way for further research on mechanism of regulation of let-7 family.
authors.affiliation: Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College, Wenzhou 325035, China.
authors.forename: Xue-Rong
authors.initials: XR
authors.lastname: Liu
authors.forename: Wen-Hong
authors.initials: WH
authors.lastname: Tian
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Dong
authors.forename: Xiao-Zhe
authors.initials: XZ
authors.lastname: Wu
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Lv
authors.forename: Xiao-Bing
authors.initials: XB
authors.lastname: Wu
mesh_terms: D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D016601:RNA-Binding Proteins
chemical_list: D004268:DNA-Binding Proteins; C516326:LIN28B protein, human; D035683:MicroRNAs; D016601:RNA-Binding Proteins; C487931:mirnlet7 microRNA, human