| Objective: Colorectal cancer is one of the most common malignant tumors in the digestive system.The treatment effect of advanced colorectal cancer is not good,so it is necessary to find a new treatment method to improve the treatment effect.Cell ferroptosis is a new type of cell death found in recent years.This discovery provides a new way for the treatment of advanced cancer patients,that is,to induce cell ferroptosis to remove tumor cells.Erastin is a classic drug to induce ferroptosis of tumor cells,but the induction effect on different colon cancer cell lines is not clear;in addition,many molecular mechanisms in the process of cell ferroptosis are not clear,which needs further study.In the previous study,we found that the expression level of SPINK4 in colon cancer was significantly higher than that in normal colon tissue,indicating that SPINK4 was involved in the pathogenesis of colon cancer.Therefore,this paper will explore the following contents: 1.Study the induction effect of Erastin on different colon cancer cell lines,select the appropriate cell lines and the optimal induction time and concentration;2.Study the expression level of SPINK4,VIL1,NF-κB,MYD88 in the process of ferroptosis of colon cancer cells induced by Erastin,and preliminarily clarify the role of SPINK4/VIL1 and NF-κB/MYD88 pathway in cell ferroptosis.Methods: 1.SW620,Lo Vo and SW480 colon cancer cell lines were cultured to the logarithmic growth stage,then Erastin was used to induce ferroptosis in each cell line.The morphological changes and ferroptosis indexes(iron content,GSH,MDA and GPx4)of each cell line were detected,and the most suitable ferroptosis cell line was clearly.(2)the most suitable ferroptosis cell was treated by using different concentrations(10 μ m,20 μ m,30 μ m)of Erastin and different intervention time(12 h,24 h,48 h)to induce ferroptosis.The optimal concentration and time of ferroptosis of colon cancer cells induced by Erastin were found out by the change of indexes.(3)the cell line was treated with or without Erastin,then the proliferation rate was detected by CCK8 method,The effect of Erastin on the migration and apoptosis of colon cancer cells was tested by wound healing test and flow cytometry,respectively.2.(1)Detection of SPINK4 expression in different colon cancer cell lines.The expression of SPINK4 m RNA and protein was detected by RT-PCR and Western-blot respectively,and the changes of SPINK4 expression after ferroptosis in colon cancer cells were studied;(2)the binding relationship between SPINK4 and VIL1 was studied by bioinformatics methods using public database prediction,and the binding relationship between VIL1 and NF-κB,NF-κB was studied.The protein expression of VIL1,NF-κB,NF-κB1 and MYD88 were detected using Western-blot after cell ferroptosis.(4)The expression of VIL1,NF-κB,NF-κB 1and MYD88 were detected by Western-blot after overexpression of SPINK4 in colon cancer cells induced by Erastin,and the change of ferroptosis was also tested.Results: 1.After the treatment of SW620,Lo Vo and SW480 cells by Erastin,the ferroptosis of each cell line occurred,and the changes of iron content,GSH,MDA and GPx4 in Lo Vo cells were consistent with the characteristics of ferroptosis,which showed that Lo Vo cells were the most stable and the most suitable cell line for Erastin induced ferroptosis,so Lo Vo cells were selected for follow-up experiments;2.Using 20 μ M Erastin to induce Lo Vo for 24 hours,the changes of iron content,GSH,MDA and GPx4 in the cells reached the most significant values,suggesting that the optimal concentration and time of Erastin and intervention were used to induce Lo Vo cells;(3)the optimal concentration of Erastin was used to induce Lo Vo cells ferroptosis,and compared with the uninduced cells,CCK8 method showed that the proliferation rate of Lo Vo cells after ferroptosis decreased significantly(P < 0.05)The results of wound healing test and flow cytometry showed that there was no significant difference in migration ability and apoptosis rate between the cells with and without ferroptosis(P > 0.05).2.RT-PCR and Western-blot results showed that SPINK4 is the highest expressed in Lo Vo cells and the expression level of SPINK4 m RNA and protein decreased significantly after the ferroptosis of Lo Vo cells induced by Erastin,which indicated that SPINK4 was involved in the process of ferroptosis.Bioinformatics method predicted that there was a binding relationship between SPINK4 and VIL1,and there also was a correlation between SPINK4 and VIL1 in colon cancer(P < 0.01);bioinformatics method also predicted that there was a sequence binding site between VIL1 promoter and NF-κB subunit NF-κB1,and there was also a significant correlation between VIL1 and NF-κB1 in colon cancer(P < 0.01);(3)Western-blot method showed that The expression levels of VIL1,NF-κB,NF-κB 1 and MYD88 in Lo Vo cells induced by Erastin were lower than those in without treatment of Erastin(P < 0.05).The overexpression of SPINK4 was significantly increased after lentivirus transfection,which indicated that the overexpression plasmid of SPINK4 was successfully transfected,and then the Lo Vo cells were induced by Erastin.Western-blot showed that after overexpression of SPINK4,the expression levels of VIL1,NF-κB,NF-κB1 and MYD88 protein levels in the cells were significantly higher than those in the cells without overexpression of SPINK4(P < 0.05).Conclusion: 1.Erastin can induce ferroptosis in many colon cancer cells,and the effect is best on Lo Vo cells;2.The expression of SPINK4 was decreased in the process of ferroptosis of Lo Vo cells induced by Erastin,and the protein levels of VIL1,NF-κB,NF-κB1 and MYD88 were the similar as that of SPINK4;3.There may be protein binding relationship between SPINK4 and VIL1,and between VIL1 and NF-κB1.In the process of ferroptosis of Lo Vo cells,the changes of SPINK4 expression can affect the expression of VIL1,NF-κB,NF-κB 1 and MYD88,suggesting that SPINK4/VIL1 may participate in the ferroptosis induced by Erastin through the regulation of NF-κB/MYD88 signaling pathway. |
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