Tissue Distribution Analyses Of FADD And It Regulation Of Cell Migration Through Synergistic Action On HnRNPK And MiR-7a

FADD(Fas-associated death domain), was first recognized as an important adaptin in death receptor-induced apoptosis. Now more attention has focused on its non-apoptotic functions. At present, it has been found FADD plays important functions in cell proliferation, cell cycle, etc. Especially the phosphorylation of Ser191 of FADD, plays important roles in various physiological and pathological conditions.To analyze the evolution of FADD protein in different species and its transcriptional and expressional distribution characteristics in different tissues, protein sequences of FADD in eight main species were compared and analyzed by using the software Mega5.0. The neighbor-joining phylogenetic tree was drawn according to the protein sequence similarity. FADD +/- mice were sacrificed and their brain, liver, kidney, heart, lung, muscle, spleen, stomach and intestine were obtained and then divided into two parts. Half of the tissues were used to extract RNA for RT-PCR, the rest of it were used for Western Blot analyses. The results of FADD expression in FADD +/- mice were compared with that of UniGene EST Profile at both mRNA and protein levels. The protein sequence alignment and evolution law of FADD in different species were analyzed. We found FADD expression was relatively high in lung, heart, stomach, while the expression of FADD in other tissues still remains to be further verified. The present studies revealed that FADD was highly conserved in the process of evolution and it was highly expressed in lung, heart and stomach. These results provided a foundation for further investigation of the biological functions of FADD in different tissues.MicroRNAs are 19-24 nt small non-coding RNA that control expression of target genes through binding to 3’UTR of mRNA, degradating mRNA or preventing protein translation. hnRNPK belongs to the hnRNP family. The hnRNP family is a class of similar structural features of RNA binding protein. hnRNPK also exists in multiple subcellular structures and cellular processes, involved in DNA transcription, RNA processing, transport, cell adhesion, cell cycle and apoptosis. It also plays roles in signal transduction and gene regulation. It has first found that FADD through its phosphorylation, regulated the expression of FAK. It has been found the expression of miR-7a and hnRNPK are upregulated in FADD knockout cell by microRNA microarray and proteomics analyses. The host gene of miR-7a is located in the last intron of hnRNPK, both of them are synchronously transcribed. Analysed by FADD’s overexpression, RNA interference and knockout, we found that FADD, through its phosphorylation, regulated the expression of miR-7a and hnRNPK.In the present study, we studied the role of miR-7a and hnRNPK in cell migration. miR-7a inhibited the expression of FAK when FADD was knocked out. It can inhibit FAK expression by targeting FAK-3’UTR, while FAK is thought through regulating N-WASP promoting cell migration. In addition, hnRNPK, combined with N-WASP, can negatively regulate cell migration. Under different levels of FADD, the changes of cell migration related genes MMP-2 and MMP-9 mRNAs consistent with the change of FAK, while contrary to the change hnRNPK. The synergistic function of miR-7a and hnRNPK may promote FADD’s ability for controlling cell migration.