The Study Of TRIP13 Promotes Cisplatin Resistance In Lung Cancer Via Autophagy Activation

Lung cancer still dominates the morbidity and mortality among malignant tumors.Cisplatin-based chemotherapy,as the first-line clinical drug regimen for lung cancer,is particularly important for prolonging the survival of lung cancer patients.However,acquired resistance to cisplatin remains a major cause of poor treatment outcomes in lung cancer.Therefore,elucidating the mechanisms behind cisplatin resistance is crucial to provide new effective therapeutic strategies for lung cancer.Cell autophagy is also known as type II programmed cell death.During autophagy,cell protection mechanisms are activated to provide cancer cells with the nutrients needed to increase energy metabolism and remove toxic reactive oxygen species and misfolded proteins.Tumor cells survive and eventually develop drug resistance.High levels of autophagic activity are prevalent in cisplatin-resistant tumor cells and are thought to promote tumor cell survival.More and more studies have shown that blocking autophagy can effectively increase the sensitivity of lung cancer cells to cisplatin.TRIP13(Thyroid hormone receptor interactor 13)is a member of the AAA(+)ATPase superfamily,which is involved in a series of cellular processes,including spindle assembly checkpoint signaling,DNA break repair and recombination.Studies have found that overexpression of TRIP13 in bladder cancer reduces sensitivity to anticancer drugs such as cisplatin and doxorubicin.The role and mechanism of TRIP13 in mediating drug resistance in lung cancer are still unclear.This study intends to study the regulation of TRIP13 on cisplatin resistance in lung cancer,and explore the molecular mechanism of TRIP13 in regulating cisplatin sensitivity in lung cancer.Objective: 1.To explore the regulatory role of TRIP13 on cisplatin sensitivity in non-small cell lung cancer;2.To explore whether TRIP13 can regulate cisplatin resistance through autophagy and the possible molecular mechanism.Methods: TCGA database was used to analyze the expression of TRIP13 in lung cancer;the relationship between TRIP13 and the prognosis of lung cancer platinum drug therapy was analyzed by survival.Transfect TRIP13 and empty plasmid into A549 cells,transfer TRIP13 small interfering RNA and blank control RNA into A549/DDP cells,up-regulate or down-regulate the expression of TRIP13,and detect the expression of TRIP13 after 72 hours of transfection for future use;Western Blot was used to detect the expression of autophagy and apoptosis-related proteins;MTS method was used to detect the IC50 of cells treated with cisplatin;colony formation assay was used to detect the proliferation ability of cells treated with cisplatin.Results:1.TRIP13 is upregulated in lung cancer and is associated with poor prognosis of platinum-based drugs.2.TRIP13 can regulate the drug sensitivity of lung cancer cells to cisplatin: to verify the drug resistance of A549/DDP cell line,the drug resistance index(RI)was determined to be 5.4,and the drug resistance index is greater than 5,and followup experiments can be carried out.A549 cells were transfected with TRIP13 plasmid,and the expression of TRIP13 protein in the cells was significantly increased;at the same time,the drug sensitivity of A549 cells to cisplatin was significantly lower than that of the control group.After TRIP13 interference was performed on A549/DDP cells,the expression of TRIP13 protein in the cells was significantly reduced;at the same time,the drug sensitivity of the cells to cisplatin was significantly higher than that of the control group.In the presence or absence of cisplatin,the levels of apoptosis-related proteins were detected by WB after overexpression and knockdown of TRIP13 in A549 and A549/DDP cells,respectively.The results showed that TRIP13 could effectively inhibit cisplatininduced cell drug resistance.3.TRIP13 promotes the level of autophagy in lung cancer cells.WB results showed that after transfection of TRIP13 in A549 cells,the level of LC3Ⅱ/Ⅰ was up-regulated and P62 was down-regulated.After the interference of TRIP13 in A549/DDP cells,the level of LC3Ⅱ/Ⅰ was down-regulated and P62 was up-regulated.After overexpression of TRIP13,combined with late autophagy inhibitor CQ,the level of LC3Ⅱ/LC3Ⅰ was further up-regulated,indicating smooth flow of autophagy.4.TRIP13 mediates the resistance of lung cancer cells to cisplatin through autophagy.MTS experiments showed that compared with the empty plasmid control group,transfection of TRIP13 could reduce the drug toxicity of cisplatin,and this effect could be reversed by the autophagy inhibitor CQ.Compared with the blank control group,interfering with TRIP13 can increase the drug toxicity of cisplatin,and this effect can be reversed by the autophagy activator rapamycin;colony formation assays also showed similar results,and interfering with TRIP13 reduced the cytotoxic effect of cisplatin.Clonogenicity in response to cisplatin treatment,which was reversed by the autophagy activator rapamycin.Using cisplatin alone or in combination with chloroquine to treat A549 cells in the control group or the overexpression group,Western Blot detected the changes of apoptosis-related proteins,and the reduction of apoptosis induced by TRIP13 was reversed by CQ.A549/DDP cells transfected with sh NC or sh TRIP13 were treated with cisplatin alone or in combination with rapamycin,and the results showed that rapamycin effectively antagonized the increased apoptosis induced by knockdown of TRIP13.Conclusion:1.TRIP13 inhibits cisplatin-induced apoptosis in lung cancer cells and promotes the resistance of lung cancer cells to cisplatin.2.TRIP13 activates autophagy in lung cancer cells.3.TRIP13 induces the resistance of lung cancer cells to cisplatin via the activation of autophagy pathway.