The Regulation Of The Upstream Open Reading Frames In The 5' Flanking Sequences Of Human BMPR-â…¡ Gene On Its Gene Expression

Background:The BMPR-Ⅱgene encodes the bone morphogenetic protein receptor typeⅡ(BMPR-Ⅱ) which is a transmembrane serine/threonine kinase receptor.Bone morphogenetic proteins(BMPs) belong to the TGF-βsuper family.BMPs singal play an important role in a number of different cellular processes such as cell proliferation, apoptosis,differentiation,chemotaxis,angiogenesis and matrix production during embryogenic development as well as in adult life.In recent studies,the mutations in BMPR-Ⅱand misexpression of this gene have been found in primary pulmonary hypertension(PPH) and the development and metastasis of several cancers.However, regulation of the expression of BMPRII is poorly understood so far.More and more researchs suggested Up Stream Open Reading Frames(uORF) was essentioal for the regulation of several genes expression.Bioinformatical analysis indicated that BMPR-Ⅱwas a gene rich of uORFs.But till now,there are no reports about whether the uORFs are involved in the regulation of this gene expression.Objective:To study the effect of the uORFs on the expression of BMPR-Ⅱgene and lay the foundation for the mechanism of regulation of BMPR-Ⅱgene expression.Methods:The mutants of five uORFs were amplified from the wild type sequence template(1534bp) which included the promoter and the leader sequences of BMPR-Ⅱgene using overlap extension polymerase chain reaction(PCR).The wild and the mutational fragments were subcloned into the pGL3-Basic vector in order to construct the six recombinant plasmids expressing the luciferase:pGL3-Basic-1534 (wide type),pGL3-Basic-MuORF1,pGL3-Basic-MuORF2,pGL3-Basic-MuORF3, pGL3-Basic-MuORF4 and pGL3-Basic-MuORF5.These six recombinant plasmids was transfected into Hela cells separately and the luciferase activities were analyzed using dual luciferase reporter assay system.Results:Comparing with the pGL3-Basic-1534(wide type),The luciferase activities of the four mutants of uORFs(pGL3-Basic-MuORF1,pGL3-Basic-MuORF2,pGL3-Basic-MuORF4,pGL3-Basic-MuORF5) were increased to some extend. However,the luciferase activity of pGL3-Basic-MuORF3 was decreased. Conclusion:The results suggested that the uORF1,uORF2,uORF4 and uORF5 fragments may regulate the gene expression of the BMPR-Ⅱnegatively and the efficiency of this regulation was correlated with their length and the content of G-C in the uORFs.In addition,the result also implied that the uORF3 may regulate the gene expression of the BMPR-Ⅱpositively.