The Study Of Overexpression Of SLC1A5 Regulating Arsenic-induced Cell Proliferation And Self-renewal In Bladder Epithelial Cells

Objective:Arsenic is a metalliod element,which is widely distributed in soil,air,and water.The International Agency for Research on Cancer classifies arsenic as Group 1human carcinogen.Arsenic is related to the occurrence of bladder cancer.Exposure to arsenic through drinking water can increase the incidence and mortality of bladder cancer,but its underlying mechanisms remain elusive.Inorganic arsenic is one of the human carcinogens that does not easily induce tumors in laboratory animals.Therefore,the in vitro cellular malignant transformation model induced by inorganic arsenic is essential for studing the carcinogenic mechanism.Dimethylarsinic acid（DMA）is the predominant arsenic compound detected in human and animal urine,which can induce baldder cancer in rats.Arsenic induce cancer by disrupting the epigenetic control of special sites,resulting in abnormal gene expression.As the most abundant amino acid in plasma,Glutamine（Gln）plays a key role in many processes of cancer cells.Alanine-serine-cysteine transporter 2（SLC1A5）mediates the exchange of amino acids including Gln and is essential for tumor cells to uptake Gln.Enhanced SLC1A5 expression has been observed in various types of cancers,and blocking SLC1A5 to prevent Gln uptake has been shown to successfully inhibit tumor cell proliferation.When transported into the cells through SLC1A5,Gln is metabolized intoα-ketoglutarate,which can activate mTORC1.Once activated,mTORC1regulates translation and cell proliferation by phosphorylation of its substrates p70S6K and4EBP1.Gln uptake is the rate-limiting step in the synthesis of reduced glutathione（GSH）,which is a potent antioxidant that mitigates the oxidative stress generated by the rapid metabolism of cancer cells.Low amounts of reactive oxygen species（ROS）is necessary to maintain high levels ofβ-catenin,plays a crucial role in promoting cell proliferation,drug resistance and maintaining stemness of cancer stem cells（CSCs）.CSCs,a subpopulation of cells retaining embryonic characteristics in tumors,have self-renewal properties and play an important role in promoting tumor growth,metastasis and drug resistance.Therefore,a rat model of sub-chronic sodium arsenite（Na As O₂）and DMA^V exposure was established to study the effects of arsenic exposure on the proliferation of rat bladder epithelial cells and the expression of SLC1A5,mTORC1,andβ-catenin.Establishing a model of long and short-term Na As O₂ treated-normal human bladder epithelial cells（SV-HUC-1）to explore the role of SLC1A5 in the proliferation and self-renewal of Na As O₂treated-SV-HUC-1 cells and related mechanism.This study provide evidence for exploring the mechanism of arsenic-induced bladder cancer.Methods:1.Thirty female F344 rats were randomly divided into 3 groups.Control group:drinking distilled water,Inorganic arsenic exposure group:drinking water containing 87mg/L Na As O₂(50 mg/L As³⁺),Organic arsenic group:drinking water containing 200 mg/L DMA^V.Using HE staining to detect the proliferation of rat bladder epithelial cells.Using immunohistochemical assay to detect the expression level of Ki67 and SLC1A5 in rat bladder epithelial cells.Immunofluorescence assay was used to discover the expression level of SLC1A5,p70S6K,p-p70S6K,4EBP1,p-4EBP1 andβ-catenin in rat bladder epithelial cells.2.SV-HUC-1 cells were treated with 0 or 0.5μM Na As O₂ for 10,20,30,and 40 weeks to establish a long-term inorganic arsenic-treated cell model,and were treated with with 0,0.5,1,2,4,8μM Na As O₂ for 24 hours to establish a short-term inorganic arsenic-treated cell model.Cell viability was determined by CCK-8 assay.Cell clone formation ability was determined by clone formation assay.Cell sphere formation assay was used to detect ability of self-renewal.Western blot was used to detect the protein expression levels of SLC1A5,PCNA,Cyclin D1,CD44,SOX2,OCT4,β-catenin and the phosphorylation levels of p70S6K and 4EBP1 proteins in whole cells,as well asβ-catenin expression levels in the cytoplasm and nucleus.Real time PCR was used to detect the m RNA expression levels of SLC1A5,Cyclin D1,CD44,SOX2 and OCT4.Immunofluorescence assay was used to detect expression level ofβ-catenin.Using GSH assay kit to detect intracellular GSH level.The level of ROS was determined by ROS fluorescent.Cells were cultured with or without Gln,and the above related indicators were detected.Cells were treated with inhibitor or si-RNA of SLC1A5,the above related indicators were determined.Next,we established a stable cell line overexpressing SLC1A5through lentiviral infection,and the above related indicators were detected.The above related indicators were determined after cells were treated with SLC1A5 inhibitors and m TOR agonists.Cells were transfected with si-β-catenin,and the above related indicators were detected.The above related indicators were detected after cells were treated with inhibitor/si-RNA of SLC1A5 andβ-catenin agonist,respectively.Cells overexpressing SLC1A5 were transfected with si-β-catenin,and the above related indicators were detected.The above related indicators were detected after cells were treated with inhibitor/si-RNA of SLC1A5 and ROS scavenger,respectively.Results:1.Compared to the control group,the number of bladder epithelial cells and the rate of Ki67 positive cells were significantly increased in Na As O₂ and DMA^V groups（P<0.01）.The expression levels of SLC1A5,p70S6K,p-p70S6K,4EBP1,p-4EBP1 andβ-catenin were increased significantly in the Na As O₂ and DMA^V groups（P<0.05）.2.After treatment with 0.5μM Na As O₂ for 10 weeks,the uptake of Gln was increased significantly in SV-HUC-1 cells（P<0.01）.As the period of Na As O₂ treatment prolonged,the level of Gln uptake increased.Gln starvation could inhibit cell proliferation and self-renewal of long-term arsenic-treated SV-HUC-1 cells（P<0.05）.3.The m RNA and protein expression levels of SLC1A5 were increased after 4μM Na As O₂ treatment for 24 hours（P<0.05）,and 0.5μM Na As O₂ treatment for 40 weeks significantly increased m RNA and protein expression levels of SLC1A5（P<0.05）.Both inhibitor and si-RNA of SLC1A5 treatment attenuated cell proliferation and self-renewal（P<0.05）,and overexpressing SLC1A5enhanced cell proliferation and self-renewal of long-term arsenic-treated SV-HUC-1 cells（P<0.01）.4.After treatment with 4μM Na As O₂ for 24 h or 0.5μM Na As O₂ for 40 weeks,the phosphorylation levels of p70S6K and 4EBP1 protein were increased（P<0.05）.Both inhibitor and si-RNA of SLC1A5 treatment reduced the phosphorylation levels of p70S6K and 4EBP1（P<0.05）,which were increased induced by overexpression of SLC1A5（P<0.01）.Treatment with dm-αKG increased the phosphorylation level of p70S6K and 4EBP1（P<0.05）,which restored the phosphorylation level of p70S6K and 4EBP1 protein inhibited by inhibitor or si-RNA of SLC1A5（P<0.05）.The m TOR agonist could restore mTORC1 activity inhibited by the SLC1A5 inhibitor（P<0.05）.The cell viability,clone formation,protein expression levels of Cyclin D1 and PCNA were also restored by m TOR agonist treatment（P<0.05）.5.After treatment with 0.5μM Na As O₂ for 40 weeks,the expression level ofβ-catenin in whole cell,cytoplasm and nucleus were significantly increased（P<0.05）.SLC1A5 inhibitor reduced the expression level ofβ-catenin in whole cells,cytoplasm and nucleus（P<0.05）.Overexpression of SLC1A5 further increased the expression levels ofβ-catenin in whole cell,cytoplasm and nucleus of cells treated with long-term inorganic arsenic（P<0.05）.After treatment with si-β-catenin,the cell viability and clone formation were decreased（P<0.01）,and the m RNA and protein expression levels of CD44,OCT4,SOX2,and Cyclin D1 were also decreased（P<0.05）.6.Theβ-catenin agonist could restore the expression ofβ-catenin protein expression inhibited by SLC1A5 inhibitor or si-RNA（P<0.05）.Cell proliferation and self-renewal ability were also improved byβ-catenin agonist（P<0.05）.The si-β-catenin reduced cell proliferation and self-renewal of cells overexpressing SLC1A5（P<0.01）.7.Both the inhibitor and si-RNA of SLC1A5 could reduce the level of GSH and increase the level of ROS in long-term inorganic arsenic-treated SV-HUC-1 cells（P<0.01）.The ROS scavenger NAC effectively reduced intracellular ROS level caused by SLC1A5 inhibitors or si-RNA（P<0.01）,restoring the expression ofβ-catenin protein in whole cells,cytoplasm and nucleus（P<0.05）.Cell proliferation and self-renewal ability were also restored by treatment of NAC（P<0.05）.Conclusion:1.Sub-chronic arsenic exposure induced bladder epithelial hyperplasia,which also induced overexpression of SLC1A5 andβ-catenin,and the activity of mTORC1was elevated.2.Long-term inorganic arsenic treatment increased Gln uptake in SV-HUC-1 cells,and Gln starvation inhibited cell proliferation and self-renewal.Both short-term and long-term inorganic arsenic treatment could induce SLC1A5 overexpression in SV-HUC-1 cells.SLC1A5 mediated cell proliferation by regulating mTORC1 viaα-KG and mediated cell proliferation and self-renewal by regulatingβ-catenin via GSH-ROS.