| Objective:1.Detect the expression of CTC-497E21.4 in gastric cancer cells and tissues.To analyze the correlation between the expression of CTC-497E21.4 and the clinicopathological characteristics of gastric cancer patients.The molecular features and subcellular localization of CTC-497E21.4 were retrieved from the database and verified experimentally.2.To investigate whether ferroptosis exists in gastric cancer cells.To analyze the effect of CTC-497E21.4 on ferroptosis in gastric cancer cells.3.To explore the molecular mechanism of CTC-497E21.4 regulating ferroptosis in gastric cancer cells.Methods:1.The relative expression levels of CTC-497E21.4 in gastric cancer cells and tissues were detected by q RT-PCR and the correlation analysis of clinicopathological features was performed.The NCBI website retrieves the chromosomal location of CTC-497E21.4 and predicts the secondary structure.The chromosomal location of CTC-497E21.4 was predicted by lnc ATLAS website,and verified by q RT-PCR and agarose gel electrophoresis.2.The morphological changes of gastric cancer cells treated with erastin were observed by transmission electron microscope.CCK-8 assay was used to detect whether z-vad-fmk,necrostatin-1,chloroquine and ferrostatin-1 could restore erastin-induced ferroptosis in gastric cancer cells.The effects of CTC-497E21.4 on the proliferation of gastric cancer cells were verified by CCK-8 proliferation assay,clone formation assay and Ed U proliferation assay.After interference or overexpression of CTC-497E21.4,the morphological changes of gastric cancer cells were observed by transmission electron microscope.At the same time,gastric cancer cell lines(MKN-45,SGC-7901,AGS)were treated with erastin,the cell viability was detected by CCK-8 assay,the concentration change of malondialdehyde(MDA),the product of membrane lipid peroxidation,was detected by colorimetry.Intracellular lipid reactive oxygen species(ROS)levels were detected by cytometry.3.Molecules on ferroptosis signaling pathway(TFRC,GPX5,SLC1A5,SLC7A11)were screened by q RT-PCR and verified by western blot.The expression of CTC-497E21.4 downstream target gene SLC7A11 in gastric cancer tissues was detected by q RT-PCR.Interference or overexpression of SLC7A11 in gastric cancer cells to detect markers that respond to ferroptosis.The co-transformation of CTC-497E21.4 interference plasmid and SLC7A11 overexpression plasmid was performed to detect the indicators reflecting ferroptosis.The database was screened for binding proteins related to CTC-497E21.4 and SLC7A11 and RIP experiment was carried out to verify.Results:1.The results of qRT-PCR showed that CTC-497E21.4 was highly expressed in gastric cancer cells and tissues(P < 0.01),and was related to the tumor size and differentiation degree of gastric cancer(P < 0.05).CTC-497E21.4 is located at 11p15.3 and has an inner ring structure.CTC-497E21.4 mainly exists in the cytoplasm of gastric cancer cells.2.Mitochondria become smaller and membrane density increased in gastric cancer cells after erastin treatment in transmission electron microscopy.The CCK-8 assay showed that the cell death of gastric cancer cells stimulated by erastin could be recovered by ferroptosis inhibitors.CCK-8,EDU and clone formation experiments showed that the proliferation of gastric cancer cells decreased after interference with CTC-497E21.4(P < 0.05),and the opposite results occurred after overexpression of CTC-497E21.4.At the same time,after interfering with the expression of CTC-497E21.4 in gastric cancer cells,the mitochondria became smaller,the morphological changes of membrane density increased,the survival rate of gastric cancer cells decreased(P <0.001),and MDA and ROS increased(P < 0.05).Overexpression of CTC-497E21.4 had the opposite result.3.qRT-PCR and western blot experiments showed that CTC-497E21.4 is related to SLC7A11 on the ferroptosis pathway.The results of q RT-PCR showed that SLC7A11 was highly expressed in gastric cancer tissues.After interfering with the expression of SLC7A11 in gastric cancer cells,the survival rate of gastric cancer cells decreased(P< 0.01),and the levels of MDA and ROS increased(P < 0.05).Gastric cancer cells co-transfected with CTC-497E21.4 interference plasmid and SLC7A11 overexpression plasmid were constructed,and the results showed that overexpression of SLC7A11 could restore the related ferroptosis indicators caused by interference CTC-497E21.4.Database screening found that IGF2BP1 is an RNA-binding protein between CTC-497E21.4 and SLC7A11,and RIP experiment results also showed that there is a relationship between IGF2BP1 and SLC7A11 interaction.Conclusion:1.CTC-497E21.4 is highly expressed in gastric cancer tissues and cells and is related to tumor size and degree of differentiation.CTC-497E21.4 is located on chromosome 11p15.3,mainly exists in the cytoplasm of gastric cancer cells and has an inner loop structure that can bind to macromolecules.2.Ferroptosis is present in gastric cancer cells.CTC-497E21.4promotes the proliferation of gastric cancer cells and inhibits the occurrence of ferroptosis in gastric cancer cells.3.CTC-497E21.4 may affect SLC7A11 via IGF2BP1 to regulate ferroptosis in gastric cancer cells. |
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