Identification Of MiRNAs Regulating Xkrs In Homo Sapiens And Danio Rerio

Programmed cell death(PCD)refers to the autonomic ordered cell death under the certain physiological or pathological conditions.PCD is an active process that involves a series of activation,expression and regulation of genes,including apoptosis and programmed necrosis and autophagy mediated cell death.PCD plays an important role in the process of the body’s normal development and is one of the important pathogenic factors of many common complex diseases such as cancer,autoimmune diseases,inflammation,viral infections and neurodegenerative disorders.C.elegans has special important position in the study of cell apoptosis.Ced(CE11 Death abnormality)gene(ced-1～ced-13)are the core genes to perform apoptosis in C.elegans,with conserved sequences and similar functions in drosophila,humans and other species.In the process of development,the defects of ced-8 gene in C.elegans can lead to cell apoptosis significantly delayed and reduced.According to the results of BLAST,ced-8 gene and CED-8 protein are highly conservative in a variety of far source species(like sponge,nematodes,sea anemones,fruit flies,zebra fish,mice and humans).In human genome,there are multiple ced-8 homologous genes that formed the XK related protein gene family(XK related,xkr),including xkr1-xkr9,xkr-Y and xkr-Y2.microRNA is a kind of endogenous non-coding 18 to 25 nucleotides in eukaryotes.Generally,the microRNAs were single stranded small RNA molecules that can regulate gene expression in the post-transcriptional level.MicroRNAs are widely participated in individual development,organ formation,virus defense,cell differentiation,proliferation and apoptosis.No microRNA that regulates xkr is reported at present.The purpose of our study is to explore expression change of xkr family genes when cell apoptosis occurs,to identify microRNAs regulating xkr family genes in zebrafish and human using computational and experimental approached,and to analyze the conservation of microRNAs-xkr interactions in Homo sapiens and Danio rerio.Human embryonic kidney cells(HEK293T)apoptosis is induced by cisplatin,RT-PCR and Western Blot are used to detect the expression level of xkr family genes when cells undergo apoptosis,respectively.Online computational tools such as ’Target Scan’ are used to predict microRNAs-target gene interactions.MicroRNA overexpression vector,wild type and mutant dual-luciferase reporter gene vector of human and zebrafish xkr 3 ’UTR are constructed.Knockdown effect of target genes is evaluated by the relative lucifersae activity after cotransfecting the vectors into 293T cells.For the positive result of the dual-luciferase reporter gene assay,the expression levels of endogenous xkr genes after transfection of microRNA overexpression vector or microRNAs inhibitors into 293T cells are measured using the qRT-PCR and western blotExpression level of xkrl gene were significantly up-regulated both in mRNA and protein levels when the cell apoptosis occurs.Time effect curve indicates that xkrl reached the highest expression abundance when induced by cisplatin for 18h.Predicted microRNAs-xkr interactions with high score and conservation were involved five human and two zebrafish xkr genes and eight microRNAs,including miR-143/miR-98-xkr1,miR-98/miR-200b/miR-21-xkr8,miR-29b-xkr4,miR-21/miR-200b/miR-29b/miR-129-5p/miR-3 l-xkr5 and dre-miR-29b-xkr6/xkr7.Dual-luciferase reporter gene assay shows that most of the predictions are positive.Negative results include miR-129-5p-xkr6 and miR-98/miR-21-xkr 8.Positive interactions that inhibit the amount of more than 70%by dual-luciferase reporter gene assay are evaluated in cell endogenous transcription level using RT-PCR and qRT-PCR.We successfully identified the following xkr-miRNA interactions:miR-143/miR-98-xkr1 miR-29b-xkr4,miR-21/miR-200/miR-29b-xkr6 and zebrafish xkr6/xkr7-dre-miR-29b,xkr8-miR-200b.Our results show that miR-200b and miR-29b can target multiple xkr genes,and miR-29b can regulate XKR genes in both human and zebrafish.