| Objective: to improve inhibitory efficiency by tandem expressing Tu D and to optimize the plasmid construction by introducing multiple-PCR, golden-gate conling,etc., which improve the construction efficiency.Methods and Results: in terms of the construction, a part of Tu D fragment is first cloned to the generic template plasmid and receptor plasmid. the other part of Tu D fragment containing MBS is obtained through two steps of PCR; then it is inserted into the receptor plasm Id by golden-gate cloning to create a dTuD plasm Id. Luciferase report assays prove that the dTuD plasmid can inhibit the miRNA-223 activity and its inhibition efficiency is higher than the Tu D-miR-223. In addition, the expression level of free target miRNAs is evaluated by q RT- PCR in 293 A cells transfected with dTuD-miR-20 a, dTuD-miR-92 a, dTuD-miR-195 and dTuD-miR-497, respectively.The results show that dTuD efficiently, dose-dependently and specifically suppresses target miRNA without disrupting other family members. A small inhibitor library consisting of 88 dTuDs was construed by our novel method and performed to find out the miRNAs involved in AP-1 pathway. Finally, we proved that p LVX-dTuD can reduce the expression level of miR-1 and its m RNA target Myogeninenic regulatory factor Myogenin in mice.Conclusions: in this study, the dTuD plasmid construction efficiency is improved by optimizing the plasmid construction method, and we prove that the inhibitory efficiency of dTuD on miRNA is higher than that of Tu D; dTuD can effectively and specifically inhibit miRNA; dTuD can suppress the activity of miRNA in vitro and in vivo. Finally,we identifed 3 miRNA related to AP-1 pathway way through a dTuD libraryBackground: the development of modern society makes the increasing requirements for pork quantity and higher quality and traditional industry faces with the development challenges. Myogeninenic differentiation is an important process of muscle growth and development, including the cell proliferation, fusing, APoptosis,etc. micro RNA(mi RNA), a kind of important regulatory factor, participate in the whole process of Myogeninenic differentiation. Thus, research on the role of mi RNA in Myogeninenic differentiation has vital significance on revealing the molecular mechanism of muscle development.Objective: mi RNA expression profiling was accomplished in order to Identify the mi RNA involved in porcine development, among which mi R-195/497 was selected as potential target and We tried to clarify the molecular mechanism of mi R-195/497 influencing Myogeninenic differentiation.Methods and Results: the LDM from “Duroc× Landrace× Yorkshire” pigs with different growth days are isolated to detect 273 porcine mi RNAs expression. Among them, 24 mi RNAs were not detected. In the other 225 mi RNAs, 42 mi RNAs are increased or reduced over 2.5 times. Over expression mi R-195/497 significantly inhibited the proliferation of C2C12 cells and promoted Myogeninenic differentiation,contrary to inhibition of mi R-195/497. mi R-195/497 directly targeted to HMGA13’UTR and down-regulated its expression, which reduced the downstream Id protein family, and ultimately influences Myogeninenic differentiation. At last, we proved that the expression of mi R-195/497 is regulated by methylation in C2C12.Conclusions: the study performed a systematic analysis on mi RNAs in Myogeninenic differentiation and clarified the molecular mechanism of mi R-195/497 involved in Myogeninenic differentiation through mi R-195/497-HMGA1-Id regulation pathway,which is also regulated by methylation. The study prov Ides a first systemically analysis of novel mi RNAs detected related to Myogeninenic differentiation. |
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