The Transcriptional Regulation Mechanism Of Intragenic MiR-135a-1/2

micro RNAs(mi RNAs), a class of short non-coding RNAs that are approximately 22 nucleotides in length, are generally regarded as negative regulators of gene expression and inhibit the translation of m RNA by binding to the 3’ untranslated region(3’UTR) of target m RNAs in animals. Depending on their location in the genome, mi RNAs are classified as either intergenic or intragenic mi RNAs, with the latter including exonic and intronic mi RNAs, which are found in non-coding transcription units(TUs) and protein coding TUs, respectively. Nowadays, the functions of mi RNA have revealed very clearly in both plants and animals, but little information is available on the regulation of mi RNA transcription, and the relation between intragenic mi RNA and the host gene is even known less. On the basis of our previous studies on the roles and functions of mi R-135 a in adipogenesis, this study aimed to explore the transcription mechanism of the mi R-135 a during adipogenesis. In this study, bioinformatics prediction, promoter dual luciferase reporter gene, site-directed mutagenesis, EMSA, Ch IP, Ch IP-q PCR, q-PCR, western blot and other methods were used to study the transcriptional mechanism of mi R-135a-1/2. The main results as follows: 1. Both mi R-135a-1 and mi R-135a-2 are intragenic mi RNAAccording to the NCBI databases, mi R-135a-1 and mi R-135a-2 are located on the Glyctk and Rmst genes, respectively. The antisense strand of mi R-135a-1 is located at the 4th or 5th intron region of Glyctk on Chr9. The sense strand of mmu-mi R-135a-2 is located at the 12 th intron region of Rmst on Chr10. Thus mi R-135a-1 and mi R-135a-2 are intragenic mi RNAs. 2. Intragenic mi R-135a-1 and mi R-135a-2 are independently transcribedSeveral bioinformatics promoter prediction webs were used to predict upstream potential promoter of mi R-135a-1/2, then segmented deletion and promoter dual luciferase reporter gene plasmids were constructed. Furthermore, transiently transfected into 3T3-L1, C3H10T1/2 and BHK cells to measured their double fluorescent activity respective, and to identify the core promoter. Then use bioinformatics to forecast potential transcription factor in this segment, and construct a point mutation plasmid. Then transfected into 3T3-L1 cells to identify whether as the core transcription factor, and then EMSA and Ch IP experiment to verify whether the transcription factor combine with promoters in vitro and in cell culture. The result confirmed that mi R-135a-1/2 is not along with the respective host gene transcription, but have their own unique transcripts. 3. Adipogenic transcription factor STAT5 a upregulates the expression of mi R-135aConstruction of pc DNA3.1-STAT5 a plasmid and transiently transfected into 3T3-L1 cells, then measured the change of mi R-135 a and APC. After overexpression STAT5 a, the expression of mi R-135 a incresed, the expression of APC incresed at the m RNA level but decreased at the protein level. After interference STAT5 a, the expression of mi R-135 a decreased, the expression of APC increased at both the m RNA and protein levels. The results showed that STAT5 a promote the expression of mi R-135 a. 4. A potential negative feedback loop between STAT5 a and APCIn order to explore the relationship between transcription factor STAT5 a and mi R-135 a target APC, 3T3-L1 cells were transfected with the pc DNA3.1-STAT5 a plasmid and adipogenesis was induced for 8 days. The expression time frame of STAT5 a and APC indicated a potential negative feedback between them. These data suggest a negative feedback loop between STAT5 a and APC during 3T3-L1 adipocyte differentiation.In summary, the conclusions of this study are:(1) mi R-135a-1 and mi R-135a-2 are intragenic mi RNA, but does not co-transcribed with host gene, but with an independent transcriptional mechanism.(2) STAT5 a promotes the transcription of mature mmu-mi R-135 a in 3T3-L1 cells by binding to both mi R-135a-1 and mi R-135a-2 promoter elements.(3) STAT5 a and APC have a potential negative feedback loop during 3T3-L1 adipocyte differentiation.