MTOR Mediated The Mechanism Of EAAT3 Promoting Intestinal Porcine Epithelial Cell Proliferation And Amino Acid Transport

Excitatory amino acid transporter 3(EAAT3)is mainly expressed in the small intestine and is an epithelial type high-affinity anionic amino acid(i.e.,glutamate and aspartate)transporter.The objective of this study was to investigate the effects of EAAT3 on the proliferation of porcine jejunal epithelial cell line(IPEC-J2)and amino acid transport.In this study,Landrace piglets EAAT3 c DNA sequence was cloned by PCR amplification,and then EAAT3-pc DNA3.1+ vector was constructed in order to be transfected in the IPEC-J2 cells and obtained stably transfected cell strains which would be used in the following experiments.Based on the results,designed and synthesized EAAT3 interference fragment in order to gain the IPEC-J2 cells of the effective interference EAAT3 expression.Automatic amino acid analyzer was used to detect the effect of EAAT3 on amino acid transport;the effect of EAAT3 on the cell proliferation of IPEC-J2 was analyzed by MTT assay and cell count;real-time PCR and Western blot were used to analyze the influence of EAAT3 on the key protein of m TOR signaling pathway and glutamate/cystine transporter x CT.Thus,it is expected that this project can laid the foundation for the mechanism of glutamate sensing and transport.The results were as follows:(1)Cloning of pig EAAT3 c DNA and the IPEC-J2 cell lines stably transfected with EAAT3-pc DNA3.1+ were obtainedPig EAAT3 c DNA sequence was cloned by real-time PCR and the cloned c DNA is 1575 bp.EAAT3-pc DNA3.1+ vector was constructed in order to be transfected IPEC-J2 cell lines.To validate whether EAAT3 was overexpressed in the IPEC-J2 cells,immunofluorescence microscopy,a real-time PCR assay and a Western blot analysis were performed.Compared with the control group,the levels of the fluorescence signals,the m RNA and the protein increased(P<0.05)significantly in the overexpression group,which means the cell strains were successfully achieved.(2)The knockdown of EAAT3 expression in the IPEC-J2 cellsThree si RNAs targeting the EAAT3 gene and the unrelated control si RNA were designed and synthesized by High Performance Liquid Chromatography.To validate whether EAAT3 was knocked down in the IPEC-J2 cells,a real-time PCR assay and a Western blot analysis were performed.And,we found that the best interference sequence is si RNA-003 and the best time point for 48 h.(3)Effect of EAAT3 on free amino acid transport and cell proliferation in the IPEC-J2cellsCompared with the control group,the overexpression group increased(P<0.05)markedly the transport of anionic amino acid and cystine.Nonetheless,after the knockdown of EAAT3 expression in the IPEC-J2 cells the transport of aspartate and cystine,as well as glutamate,was significantly reduced(P<0.05).The results of MTT and cell count suggested that the proliferation ability of IPEC-J2 cells were significantly increased(P<0.05)when tranfected with EAAT3-pc DNA3.1+ vector.(4)Effect of EAAT3 on m TOR pathway related protein in the IPEC-J2 cellsCompared with the control group,the overexpression group markedly increased(P<0.05)the protein levels of p-m TOR(Ser2448)and its downstream targets,p-S6K1(Thr389)/p-S6(Ser235)and p-4EBP1(Thr70)/e IF4 E.Interestingly,the phosphorylation of m TOR(Ser2448)and its downstream key proteins were significantly down-regulated(P<0.05)in the knockdown group compared with negative control group.(5)Effect of EAAT3 on ATF4/x CT in the IPEC-J2 cellsCompared with the control group,the overexpression group markedly increased(P<0.05)the expression of x CT both in at the m RNA and the protein level.And,there was a significant difference between the negative control group and the knockdown group.In addition,the protein level of ATF4 is in accordance with the expression of x CT.Conclusion:(1)EAAT3 could promote the intestinal epithelial cell proliferation through transport glutamate activation of m TOR signaling pathway.(2)m TOR signaling pathway could coordinate regulation of glutamate/cystine antiporter x CT to maintain the steady state of metabolic pool glutamate by transporting glutamate and cystine.