SIRT4 Increases The Sensitivity Of Non-small Cell Lung Cancer Cells To Gefitinib By Inhibiting Autophagy

Objective: Lung cancer is one of the malignant tumors with high fatality rate in the world.About 80% of the type of lung cancer is non-small cell lung cancer(Non-small cell lung cancer,NSCLC),and with the development of medicine.The first choice of EGFR tyrosine kinase inhibitors(EGFR-TKIs)for patients with advanced non-small cell lung cancer gene mutations has achieved initial therapeutic results.The main EGFR-TKIs drugs used were gefitinib and erlotinib,in which late-stage cancer patients with 19 thoron deficiencies and no.21 exon L858 R mutations were sensitive to EGFR-TKI and achieved good results.But it also hinders the therapeutic efficacy because of its limited application,primary and secondary drug resistance.Autophagy is a life phenomenon that is widely present in eukaryotic organisms,and cells can remove damaged proteins and organelles by autophagy,and the small molecules produced can be reused to maintain intracellular function and internal environmental stability.In the event of environmental stress such as hunger,lack of growth factors,increased energy demand,or drug stimulation,autophagy mechanisms are quickly created within the cell to resist changes in the external environment or their own needs.In many tumor-related studies,autophagy promotes tumor growth,maintains stability of the environment within tumor cells,provides energy for tumor cell growth,and promotes the survival of tumor cells under chemotherapy drugs.Numerous studies have shown that EGFR-TKIs drugs can induce the occurrence of autophagy.SIRT4 is a member of seven Sirtuins family species with ADP-ribobase transfer assasis and rich in in-line granules.SIRT4 is thought to inhibit tumors by inhibiting the metabolism of mitochondrial glutamine in the DNA damage response.At the same time,SIRT4 inhibits aerobic glycolysis solution sofeticization of tumor cells,SIRT4 transition sensitive cells are more likely to be induced apoptosis by glycolysis edifice inhibitors,while SIRT4 plays a role in anti-cancer in a variety of tumors.The autophagy process also requires the energy metabolism of cells,while SIRT4 plays an important role in the energy metabolism of tumor cells,and EGFR-TKIs induces autophagy in tumor cells,so our research aims to prove that SIRT4 can inhibit e GFR-TKIs-induced lung cancer cells autophagy.In turn,the sensitivity of lung cancer cells to EGFR-TKIs was improved.It provides theoretical basis and feasible method for the clinical treatment of lung cancer.Methods: 1.Lung cancer cell line H1299 and HCC827 in accordance with the company’s scientific research Shanghai cell library to provide the formula and method for culture,to be cell in good condition and in the peerage long-term,with trypsin digestion for resuspension,appropriate density spread in the 6-well plate,overnight,to the cell fully wall,the use of Lipo3000 In accordance with the specification requirements to H1299 cells into THE TRANSFER OF SIRT4 and empty plasmids,to HCC827 into SIRT4 small interference RNA and blank RNA,to regulate SIRT4 overexpression and low expression cells after transfection 48 and 72 hours after detection of SIRT4 expression in a backup.2.Western blot detects the expression of autophagy-related proteins.The well-growing H1299 and HCC827 cells were spread over a 6-well plate at an appropriate density,and after the associated experimental processing,the cells were collected,lysed in the NP-40 mammalian protein extraction reagent containing protease and phosphatase inhibitors,and then centrifuged for 20 minutes at 12000.Cell lyse is collected after centrifugation to determine cell lysate protein concentration.Using SDS-polyacrylamide gel electrophoresis electrophoresis separation protein(20-30 sg),transfer protein to PVDF membrane,place disfigured milk powder in 5% closed 2h,and then imprint with 2% bovine serum albumin / Tris-buffered saline Tween-20 in an overnight incubation at 4 degrees C,with two hours of incubation at room temperature,use ECL to testing protein.3.Immunofluorescent detection detects the formation of autophagy-related protein LC3.The appropriate amount of transfection or interference after the cell spread in the 24-well plate with cell crawl,waiting for the cells to fully paste the wall,add gefitinib treatment 24 h,by pbs clear,4% polyformaldehyde fixed,0.5% Triton X perforation,goatology please close 2h,LC3 B one anti-overnight incubation,fluorescent two anti-light avoidance 2h incubation,DAPI light-avoiding 5min rear seal,observed using a fluorescence microscope.4.CCK-8 detects the survival of cells under the treatment of gefitinib.After the cells after transfection or interference are laid in the 96-well plate in moderation,after the cells are fully walled,use different concentrations of gefitinib to treat the same time or use the same concentration of gefitinib treatment for different times,each hole added 10 sl CCK-8 reagents,after the box to avoid the incubation of 2h,using an enzyme marker to detect 450 nm absorbent value,Draw a survival graph based on the results.5.Cell clonogenic assay detection cell value-added ability under the role of gefitinib.Take the growth state of good cells for transfection and interference exercise,spread in the six-well plate,each hole 1000 cells,waiting for the cells to fully paste the wall after adding the corresponding concentration of gefitinib effect 48 h,replaced with normal culture medium to continue culture for 12 days,ice methanol fixed 15 min,crystal purple staining 15 min,water wash,bacteria drop.6.The flow cytometer detects the apoptosis of cells under the action of gefitinib,transfection or interference of cells,collects cells after 24 hours with the corresponding concentration of gefitinib,and adds the Annexin V10 μl and PI 5 μl reagents produced by Beijing Sizhengbo Company to the cell buffer(500 sl)The room temperature avoids the light incubation for 15 minutes.Streaming cytometer stome detection is used after incubation.7.Radioscope experiment.After collecting processed cells,3% dialdehyde fixed 4h,sent to the university’s cell biology teaching and research room electric mirror room for observation.Results: 1,Western blot results show that compared with the DMSO control group,with the increase of drug concentration,the expression of autophagy-related protein LC3 B increased,the expression of p62 decreased,the drug concentration dependence.The immunofluorescent experiment showed that significant LC3 B speckle formation was observed under the action of gefitinib compared to the DMSO control group.The transmission mirror observed autophagy formation in lung cancer cells under the action of gefitinib compared to the DMSO control group.The results of the CCK-8 experiment showed that the drug toxicity of gefitinib was significantly improved in autophagy inhibitors 3-MA and HCQ.2,Western blot results showed in the role of gefitinib compared with the empty plasmid control group,transfection SIRT4 group autophagy-related protein expression decreased,p62 table greatly increased.Compared with the NC blank control group,the autophagy-related protein LC3 B expression increased and p62 expression decreased in the interfering SIRT4 group.Immunofluorescent experiments showed a decrease in the formation of LC3 B spots in transfection SIRT4 groups compared to the empty plasmid control group.Compared to the NC blank control group,the expression of LC3 B spots in the interferometry SIRT4 group increased.The radioscope observation showed that transfection SIRT4 reduced autophagy formation in cells compared to the empty plasmid control group under the action of gefitinib.3.CCK-8 value-added experiments showed that transfection of SIRT4 increased the toxic effect of gefitinib compared to the empty plasmid control group.Interference SIRT4 reduced the toxic effect of gefitinib compared to the NC blank control group.Apoptosis experiments showed an increase in apoptosis rate of transfection of SIRT4 cells compared to the airborne plasmid control group under the action of gefitinib.Compared with the NC blank control group,the apoptosis rate of interfering SIRT4 cells decreased.Cloning-forming experiments showed that transfection SIRT4 reduced the survival of lung cancer cells compared to the empty plasmid control group under the action of gefitinib.Interference SIRT4 increased the survival of lung cancer cells compared to the NC blank control group.4.Western blot results showed that PI3K-AKT,MEK-ERK,signalpath activity increased,and SIRT4 inhibited the activity of gefitinib-induced MEK-ERK compared to the DMSO control group.5.Western blot results showed an increase in the expression of transfection SIRT4 autophagy-related protein ATG7 compared to the empty plasmid control group,and an increase in the expression of ATG7,a interfering SIRT4 autophagy-related protein,compared to the NC blank control group.Interference with ATG7,p-ERK expression decreased compared to NC blank control group.Conclusion:1.Gefitinib induced lung cancer cells to produce autophagy.2.SIRT4 can inhibit the gefitinib induced autophagy.3.SIRT4 increases the sensitivity of lung cancer cells to gefitinib.4.SIRT4 inhibits the MEK-ERK signal path activated by gefitinib.5.SIRT4 inhibits autophagy by ATG7,p-ERK.