Association Analysis Of PIK3CA And AKT3 Genes' Polymorp-Hisms On Growth Speed In Rabbits And The Their Effect On Cell Proliferation Of Rabbit SCs

PI3K-AKT signaling pathway plays an important role in cell growth and survival in a variety of tissues. The members of PI3K (phosphoinositide3-Kinase) family belong to proto-oncogenes, and participate in various signaling pathways. Therefore, it is speculated that PI3K-AKT signaling pathway would play an important role in cell proliferation and survival. In the present study we investigated the polymorphisms of PIK3CA and AKT3 genes and their association with growth speed of rabbits (N=711) in Europe white. Fujian black and Ira rabbits. The rabbit skeletal muscle satellite cells (SCs) were obtained from 1 day old New Zealand rabbit to detect the effect of siRNA-mediated silencing of PIK3CA and AKT3 genes on cell proliferation in rabbit SCs.1. A total of six SNPs of PIK3CA were found on the genomic sequences. They were g.12178A>G, g.12388C>T, g.12605T>C g.12711C>G, c.3068A>G and c.3377G>C. The c.3068A>G was a synonymous mutation and located in the exon20 of PIK3CA. Association analysis indicated that GG genotype individuals had the lowest weight at all growth stages in Fujian and Europe white rabbits. And GG genotype individuals had significant lower ADG of 35-70 days of age than AG individuals.2. Two SNPs of AKT3 were found on the genomic sequences. They were c.1213A>C and g.258389T>C. The c.1213A>C was a synonymous mutation located in the exon10 of AKT3. Association analysis indicated that AC and AA genotype were significantly associated with higher body weight than CC genotype at every growth stage (P<0.05) in Europe white rabbits.3. We designed three siRNA sequences of PIK3CA and AKT3 genes respectively. Transfecting the SCs with selected siRNA in the concentration of 25nM,50nM,75nM and 1 OOnM. and detected the relative transcript level of target genes 48h later. The results indicated that it was in the concentration of 50nM that had the best inhibition efficiency which were 85.16% and 83.93% respectively.4. Transfecting the SCs with either si-PIK3CA-003 or si-AKT3-001, then did the qRT-PCR reaction 48h later to detect the relative expression level of PIK3CA, AKT3, PTEN and FoxO1. The results showed that the expression level of PIK3CA and AKT3 were significantly lower after transfection while the expression level of PTEN and FoxO1 were significantly higher compared with the black group. It indicated that there were negative correlation between the expression level of AKT3 and PTEN/ FoxO1.5. Detect the OD level of SCs in microplate reader with CCK-8 knit after transfection. The OD of the experimental group was significantly lower than the black group (P<0.05). It explained that with the reduction of PIK3CA and AKT3 the proliferation rate of SCs declined as well. So PIK3CA and AKT3 genes could play an key role in the proliferation of SCs.Our results indicated that the polymorphism of both PIK3CA and AKT3 genes are significantly associated with growth speed in rabbits, which would be employed in the breeding program of marker assisted selection. In this study, we found the ability of SCs proliferation were obviously reduced when PIK3CA and AKT3 were down-regulated, it provided molecular breeding markers for improving meat production and quality.