Regulation Of The Expression Of SLC7A5 Gene In Tumor Cells

Amino acids are the basic elements constituting the protein, while participating in many important cellular metabolic pathways. Abnormal transportion of amino acids can result in serious disease related to absorption and metabolism disorders, thus the amino acid transporters have a pathological significance. In mammals, trans-membrane transport of amino acids is mediated by a variety of amino acid transporters. The reason is that the amino acid is a small molecule polar substance, which is not free to pass the cell membrane, which must be assisted by the corresponding amino acid transport protein on the cell membrane. In the process of tumor cell proliferation, migration and invasion, it also needs a large number of amino acids to provide nutrition. Amino acid transporters in tumor cells can provide the carrier channel, so the amino acid transport protein in tumorigenes and tumor development has a key role and its regulation mechanism has been a hot topic in the field of tumor.L amino acid transporter 1(LAT1), also known as SLC7A5, is an important member of the L amino acid transporter, which is mainly responsible for the transport of large, branched, aromatic neutral amino acids, including some essential amino acids. The literature has indicated that SLC7A5 is highly expressed in some tumor tissues, such as gliomas. In view of SLC7A5's research will be of great significance to the future research of life science, such as medicine, nutrition and so on. In this paper, the expression and regulation of SLC7A5 and its related research are discussed.This project mainly consists of three parts:1. Analysis of tissue expression profile of SLC7A5 gene by using GEO databaseWe analyzed the expression abundance of SLC7A5 gene in different normal tissues which are respectively derived from mouse and human being by using NCBI-GEO database. It was found that SLC7A5 gene was highly expressed in the brain, testis, ovary, placenta and other normal tissues of the mouse and human being, and the expression was less in the lung, heart, liver and other tissues. The results showed that the expression of SLC7A5 gene in these two species was tissue specific, and had the similar expression between species.At the same time, we analyzed the expression of SLC7A5 in human tumor tissues by using this database, and the results showed that the SLC7A5 gene was highly expressed in tumor tissues compared with normal tissues. This suggested that SLC7A5 might be a carcinogenic factor.2. Analysis of biological effects of SLC7A5 gene on tumor cellsTwo different cells, non-small cell lung cancer cell line HI299 and breast cancer cell line MCF-7, were used for RT-PCR experiment, and the expression level of SLC7A5 gene in these two cell lines was initially detected.Then, the recombinant plasmid pcDNA3.1-SLC7A5 which containing SLC7A5 ORF region was successfully constructed.After transfection of the plasmid in these two cell lines, the effects of SLC7A5 gene on cell proliferation and cell cycle were detected by MTT and flow cytometry. The results showed that SLC7A5 could promote the proliferation of H1299 cells and MCF-7 cells, and most of the cells were arrested in S phase, which is the phase of replication. These results suggested that SLC7A5 gene play a role in promoting cell proliferation in tumor cells.Finally, through the over expression of pcDNA3.1-SLC7A5 in H1299 and MCF-7 cells, and in combination with RT-PCR experiments, we investigated the regulation of SLC7A5 gene on cell cycle related genes at the molecular level. The results showed that in H1299 cells, SLC7A5 can promote the expression of cyclin A1, cyclinE1, and upregulate other cell cycle gene expression was not significantly changed; in MCF-7 cells, SLC7A5 can upregulate the expression of cyclinDl, cyclinEl and other gene expression had no significant effects.3. Transcriptional regulation of SLC7A5 gene in tumor cellsUsing bioinformatics software to analyze the SLC7A5 promoter region, it was found that the region has one transcription start site (TSS), and multiple transcription factor binding regions, including the binding sites of smad and p53. So we presume SLC7A5 might be regulated by TGF-?/Smad pathway or p53 signaling.First, the plasmid pGL3-SLC7A5 promoter was constructed and respectively co-transfected with Renilla plasmid into H1299, MCF-7 cells. By dual luciferase reporter gene assay, it was found that the SLC7A5 promoter activity in H1299 cells is higher than that in MCF-7 cells, and may be due to MCF-7 cells with wild type p53 gene and H1299 cell without p53 gene, which suggesting p53 plays a role in regulating SLC7A5 gene.Then, the regulatory role of wild type p53 and mutant p53 on SLC7A5 gene was further studied. pcDNA3.1-mut p53 (H175), pcDNA3.1-mut p53 W248, pcDNA3.1-wt p53 and pcDNA3.1 plasmid were transfected in H1299 and MCF-7 cells, respectively. Through RT-PCR, we confirmed the expression of mutant p53 can promote SLC7A5 and wild type p53 can inhibit expression of SLC7A5 in a certain extent.In addition, after the treatment of exogenous TGF-?1 or TGF-?3 on H1299 and MCF-7 cells respectively, it is comfirmed that SLC7A5 expression level has been increased with enhancement of TGF-?1, TGF-?3 concentration in H1299 cell by RT-PCR. In MCF-7 cell line, SLC7A5 expression level is the highest by treatment of 5ng/ml TGF-?1 or TGF-?3, but SLC7A5 expression declined at the concentration of 10 ng/ml.The results showed that SLC7A5 can respond to the stimulation of TGF-?1/3, while its expression levels vary by different cell types.In summary, we confirm that SLC7A5 gene can promote tumor development; and determine SLC7A5 gene can promote cell proliferation by up regulation of cell cycle related genes expression. TGF-?1/3 and p53 can regulate SLC7A5 gene expression.That is TGF-? in a certain extent promotes SLC7A5 gene expression, and wild type p53 inhibites SLC7A5, mutant p53 can promote the expression of SLC7A5.