| Objectives:To study the effect of serum selenium level on disease progression of lung cancer patients;to explore the effect of sodium selenite on ferroptosis in lung cancer cells;to clarify the mechanism of sodium selenite-induced ferroptosis in lung cancer cells.Methods:1.Collect 20 serum samples of lung cancer patients in Kunming and 17 serum samples of lung cancer patients in the Xuanwei area from the hospital biological sample bank and use inductively coupled plasma mass spectrometry to detect the serum selenium content of lung cancer patients;Serum selenium levels in patients with lung cancer were stratified into low,medium,and high levels,and the correlation between nutritional status,pathological type,and pathological stage and serum selenium was analyzed;Kaplan-Meier survival analysis was used to evaluate the impact of serum selenium levels on the prognosis of patients.2.After treating lung adenocarcinoma A549 cells and XWLC-05 cells with sodium selenite,the cell proliferation ability was detected by the MTT method and plate cloning assay,and the effect of exogenous sodium selenite on the proliferation ability of lung cancer cells was explored.3.MTT assay was used to detect whether sodium selenite inhibited the proliferation of lung adenocarcinoma A549 cells and XWLC-05 cells in the presence of ferroptosis inhibitor interference and to explore whether sodium selenite inhibited the proliferation of lung cancer cells was related to ferroptosis.4.After treating lung adenocarcinoma A549 cells and XWLC-05 cells with sodium selenite,the DCFH-DA probe detected intracellular reactive oxygen species(ROS)levels and colorimetric method detected cellular malondialdehyde(MDA)content,iron reagent Detecting the level of ferrous iron in cells and observing the ultrastructure of cells by transmission electron microscopy,further clarifying the role of sodium selenite in inducing ferroptosis in lung cancer cells.5.After treatment of lung adenocarcinoma A549 cells and XWLC-05 cells with selenite,RT-qPCR was used to detect the expression of ferroptosis key gene solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)mRNA level expression level,Western Blot method to detect SLC7A11,GPX4 protein expression level,Western Blot method to detect SLC7A11,GPX4 protein expression level after adding ferroptosis inhibitors,to clarify the relationship between sodium selenite-induced ferroptosis in lung cancer cells and SLC7A11-GPX4 axis.6.After treating lung adenocarcinoma A549 cells and XWLC-05 cells with sodium selenite,RT-qPCR was used to detect the key enzyme of lipid metabolism,acyl-Co A synthetase long-chain family member 4(ACSL4),and the key subunit of ferritin,ferritin heavy chain(FTH)and ferritin light chain(FTL)mRNA levels were expressed,and Western Blot detected the protein expression levels of ACSL4,FTH,and FTL.The correlation between sodium selenite-induced ferroptosis,lipid metabolism,and iron metabolism in lung cancer cells was preliminarily explored.Results:1.The average serum selenium level of 37 lung cancer patients was(71.44±3.24)μg/L,which was lower than the normal selenium level(85 μg/L),and the serum selenium level of lung cancer patients in Xuanwei area(59.31±10.59)μg/L was significantly lower than The serum selenium level of patients with lung cancer in Kunming was(81.76±19.93)μg/L(P<0.05);stratified analysis of serum selenium level showed that there was no significant correlation between serum selenium and the nutritional status,tumor pathological type,and pathological stage of the patients(P >0.05);survival analysis showed that the progression-free survival(PFS)of lung cancer patients with low serum selenium levels was shorter than that of patients with high serum selenium levels(P<0.05).2.The results of the MTT experiment showed that compared with the blank control group after 2.0μg/m L sodium selenite treated A549 cells and XWLC-05 cells,the cell proliferation ability decreased(P<0.05);the results of plate cloning experiments showed that 2.0μg/m L sodium selenite could inhibit the colony formation of A549 cells and XWLC-05 cells(P<0.01).3.The results of the MTT experiment showed that compared with the sodium selenite group,the inhibitory effect of sodium selenite on the proliferation of A549 cells and XWLC-05 cells was reversed in the presence of ferroptosis inhibitors(P<0.05).4.The results of the DCFH-DA experiment showed that compared with the blank control group,the ROS levels in A549 cells and XWLC-05 cells had a time-and dose-response relationship with sodium selenite.,the level of ROS gradually increased(P<0.01);the results of the MDA experiment showed that sodium selenite increased the content of MDA in lung cancer cells,and it was positively correlated with the treatment time and concentration of sodium selenite(P<0.01);the iron ion experiment The results showed that 2.0μg/m L sodium selenite increased the level of ferrous iron in cells(P<0.01);transmission electron microscopy showed that 2.0μg/m L sodium selenite made the mitochondria of lung cancer cells smaller and the mitochondrial membrane density increased,Changes characteristic of ferroptosis occurred.5.The results of RT-qPCR showed that compared with the blank control group after2.0μg/m L sodium selenite treated A549 cells,and XWLC-05 cells,the mRNA expression of key ferroptosis genes(SLC7A11,GPX4)was down-regulated(P<0.05);Western Blot results showed that,compared with the blank control group,after 2.0μg/m L sodium selenite treated A549 cells and XWLC-05 cells,the protein expression levels of key genes of ferroptosis(SLC7A11,GPX4)were down-regulated(P< 0.05),when ferroptosis inhibitors were present,the effect of sodium selenite on down-regulating the protein expression of SLC7A11 and GPX4 was reversed(P<0.05).6.The results of RT-qPCR showed that after 2.0μg/m L sodium selenite-treated A549 cells,and XWLC-05 cells,the expression level of the key enzyme ACSL4 mRNA in the cells was up-regulated(P<0.05),and the ferritin subunit FTH mRNA The expression level was down-regulated(P<0.05),while the expression level of FTL mRNA had no significant change(P>0.05);Western Blot results showed that compared with the blank control group,2.0μg/m L sodium selenite treated A549 cells and XWLC-After 05 cells,the protein expression level of ACSL4,a key enzyme of lipid metabolism in the cells,was up-regulated,the protein expression level of FTH was decreased(P<0.05),while the protein expression level of FTL had no significant change(P>0.05).Conclusion(s):Low serum selenium level is one of the factors affecting the poor prognosis of lung cancer patients;a certain amount of sodium selenite can induce ferroptosis in lung cancer cells,and the possible mechanism is through inhibiting the SLC7A11-GPX4 axis and affecting the lipid metabolism pathway and iron metabolism pathway. |
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