The Studies On A Human Heart Developmental Candidate Gene KLHL31 On The Left-right Asymmetry

Human KLHL31 is mapped to chromosome 6p12.1,5743bp in cDNA full-length and contains an open reading frame of 1905 nucleotides, which encodes a deduced protein of 634 amino acids. This protein contains a BTB (Broad-Complex, Tramtrack and Bric a brac)domain in N-terminal and a Kelch-repeat domain in C-terminal, and it is specifically expressed in skeletal muscle and cardiac tissue in adult. Previous study used the yeast two-hybrid system to screen a human heart library found that NOMO which related to Nodal signaling pathway is one of the positive clone which can interact with KLHL31. Using co-immunoprecipitation we proved that human KLHL31 interacts with NOMO and the BTB domain of KLHL31 and the C-terminal of NOMO were necessary for mutual binding while the other domains have no interaction.We utilized zebrafish to examine KLHL31’ s roles in the early steps of heart morphogenesis and injected morpholinos to block the translation of the KLHL31 mRNA in zebrafish. These morpholinos caused a severe abnormal development of zebrafish embryos like development, heart rate and the pigment formation slower than wild type; some individuals appeared no heart beat, no eye and mostly organ hypogenesis, finally dead in E4. Morphology observation used the microscope revealed the location of atria and ventricle was abnormal in the slow development zebrafish. We also noticed that these development defects could be rescued by co-injection of mRNA encoding zebrafish KLHL31, suggest KLHL31 plays an important role in the development of zebrafish.Using RT-PCR and Western-blot we detected the expression of nodal signaling pathway marker genes like Lefty2、Pitx2、Nodal in the group of KLHL31 morpholinos injeced, the group of NOMO morpholinos injeced, and the group of KLHL31 and NOMO morpholinos co-injected zebrafish, from the results we deduced that KLHL31 and NOMO play an antagonistic role to the nodal signaling pathway which can influence the development of zebrafish.In muscle differentiation process, the multi-functional mesodermal cells transform into myoblasts, and finally differentiate into contractile muscle cells, the expression of myogenic regulatory factors Myf-5, MyoD, Myogenin and MADS in the differentiation process is highly ordered, and the mechanism of regulation is very complex. In order to find the function of KLHL31 in the complex regulation network, we utilized C2C12 cell line which can differentiate into muscle cells as a model. RT-PCR detection showed in the KLHL31 overexpressed stable cell line the transcriptional activities of myogenesis marker genes MyoD and Myogenin was upregulated at 0 d,2 d,4 d,6 d in the process of differentiation. Luciferase report assays showed that KLHL31 can enhance the activites of muscle-specific gene MCK promoters (MCK4800 and 4RTK), indicate that KLHL31 plays an important role in the myogenesis of C2C12 cells.