| Colon cancer is the most common malignant tumor of the digestive tract.Because of its lack of highly specific therapeutic targets,its prognosis is often poor and its incidence rate is increasing year by year.Therefore,it is urgent to clarify the molecular mechanism of the occurrence and development of colon cancer cells,establish a highly specific target,and develop a new strategy for the treatment of colon cancer.Studies have shown that glutamine catabolism in most tumor cells is significantly up-regulated.Glutaminase 1（GLS1）is a key enzyme in the catabolism of glutamine.It has abnormally high expression in a variety of tumors and has been shown to be closely related to tumor growth and migration.However,the mechanism by which GLS1 promotes the occurrence and development of colon cancer is still unclear.The purpose of this study is to explore the role of GLS1 in promoting the occurrence and development of colon cancer cells and the specific mechanism of regulating the proliferation and migration of colon cancer cells.This project firstly determined the high expression of GLS1 in colon cancer cells through TCGA database,Western-blot and RT-PCR analysis,and the method of lentiviral infection was used to construct knockdown GLS1 cell lines in colon cancer cells DLD1 and SW480.Subsequently,through MTS,clone formation experiment,wound healing experiment and transwell experiment analysis,it was found that the proliferation and migration ability of colon cancer cells in DLD1 and SW480 cells treated with knockdown of GLS1 and GLS1 inhibitor BPTES was reduced.At the same time,Western-blot analysis found that after inhibiting GLS1,the expression of E-cadherin was up-regulated,and the expression of mesenchymal markers N-cadherin,Vimentin,and Slug were down-regulated.This confirms that GLS1 has a cancer-promoting effect in colon cancer cells.Subsequently,in DLD1 and SW480 cells,the detection of the fluorescence intensity of reactive oxygen species（ROS）found that inhibition of GLS1 increased the level of ROS.Western-blot results showed that inhibition of GLS1 was accompanied by down-regulation of Nrf2 protein expression.Furthermore,cell function experiments and Western-blot detection of epithelial-mesenchymal transition（EMT）marker proteins showed that the use of antioxidant NAC reversed the down-regulation of colon cancer cell proliferation,migration and EMT abilities by GLS1 inhibition.It was suggested that when GLS1 was inhibited,it affects the occurrence and development of colon cancer cells by changing the antioxidant system of colon cancer cells.Subsequently,through the confocal fluorescence detection of GFP-LC3,it was found that the number of autophagosomes was reduced,and it was further discovered by Western-blot that the expression of autophagy marker protein LC3 II was down-regulated and the expression of p62 was increased in DLD1 and SW480 cells treated with sh GLS1 and GLS1 inhibitor BPTES.Treatment of DLD1 and SW480 cells with Nrf2 activator bardoxolone and antioxidant NAC reversed the down-regulation of LC3 II expression and the increase of p62 protein expression caused by inhibiting the expression of GLS1.Finally,cell function experiments and EMT marker protein detection results showed that after using BPTES and GLS1 sh RNAs to inhibit GLS1,the results of SW480 and DLD1 cell proliferation,migration and EMT transformation ability reduction were reversed by the autophagy activator Rapamycin.This indicated that inhibition of GLS1 regulated oxidative stress through the Nrf2 signaling pathway,thereby reducing protective autophagy in colon cancer cells,thereby limiting the growth and migration of colon cancer cells.This study confirmed that GLS1 inhibition decreased autophagy and up-regulated ROS level in colon cancer cells by down-regulating Nrf2 signaling pathway activity,thereby inhibiting the growth and migration of colon cancer cells.This can provide a theoretical basis for precise treatment of colon cancer and open up new research directions. |
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