Clone Of The Promoter Of A Spliced Variant Of Human CD2-associated Protein Genes And The Mechanism Regulated By MicroRNA-548k

Objectives To clone the promoter of a spliced variant of human CD2-associatedprotein genes and clarify whether microRNA-548k is involved in the regulation of the spliced variant at the transcriptional level.Methods The CD2AP002 promoter region was cloned and its activity was identified using a series of 5'deletion promoter plasmids in luciferase reporter assays in HEK293 cells.By scanning gene promoters in silico(RegRNA 2.0)for sequences complementary to known miRNAs,we predicted the potential binding sites of miR-548k in CD2AP002 promoter region.Fluorescence in situ hybridization(FISH)were conducted to profile the distribution of miR-548k in HEK293 cells and we observed the distribution of Argonaute-2(AG02)in HEK293 cells by immunofluorescence staining(IF)assay.Identify whether miR-548k regulate the transcription of CD2AP002 through mutational analysis,transfection of miR-548k mimics/inhibitors and real time quantitative PCR.Chromatin immunoprecipitation assay(ChIP)was used to explore the enrichment of pol II around the promoter region of CD2AP002 gene.Results We has constructed a series of promoter plasmid.Scanning analysis by software revealed two sites highly complementary to miR-548k.The signal of miR-548k and AG02 also dispersed in the nucleus of HEK293 cells via fluorescence in situ hybridization assay and immunofluorescence staining(IF)assay.The induction of CD2AP002 was specific to the sequence of miR-548k and two putative recognition sites were necessary for miR-548k to bind CD2AP002 promoter.Chromatin immunoprecipitation assay(ChIP)affirmed that there were enrichment of pol II around the promoter of CD2AP002.Conclusions The reporter plasmids constructed successfully of CD2AP002 gene promoter showed promoter activity.MiR-548k up-regulates CD2AP002 expression by targeting its promoter,and during the process we find the enrichment of pol II.