The Molecular Mechanism Of Mdig-induced Lung Adenocarcinoma Cell Resistance To Cisplatin

Objective:As one of the most common cancers worldwide,lung cancer is the main cause of cancer deaths due to the high mortality associated with the disease.Among them,13%of lung cancer cases are classified as small cell lung cancer（SCLC）,with a relative5-year survival rate of 6%,and 83%of cases are non-small cell lung cancer（NSCLC）,with a 5-year survival rate of 23%.Chemotherapy is the most common treatment for non-small cell lung cancer.Commonly used drugs include cisplatin（DDP）and taxanes.However,research reports on NSCLC tumor cultures show that the drug resistance rates to DDP and taxanes are approximately 63%and 43%,respectively.Platinum-based chemotherapy has improved the long-term survival rate of patients with non-small cell lung cancer,but it has low efficacy and high toxicity in the short term,and drug resistance is almost inevitable.Therefore,cisplatin（DDP）resistance has become the main cause of death in non-small cell lung cancer（NSCLC）.During chemotherapy,one of the reasons for cisplatin resistance is the emergence of tumor multidrug resistance（MDR）.MDR refers to the acquired resistance of tumor cells to chemotherapeutics with different chemical structures and mechanisms of action.The formation mechanism of MDR is complex,mainly including reduced drug absorption,increased metabolism,target changes,and damage to tumor cell apoptosis pathways.The main mechanism is the high expression of ATP-binding cassette transporters（ABC transporter）on the tumor cell membrane,which leads to the decrease of intracellular drug accumulation.ATP-binding cassette（ABC）transporters are a large number of highly conserved transmembrane proteins.They mediate the active transport of many structurally and functionally diverse substrates through the double-layer structure of the cell membrane.They are divided into 7 subfamilies（ABC-A,ABC-B...,ABC-G）.The ABC transporters associated with MDR,namely ABCB1/P-glycoprotein（P-GP）,ABCG2/breast cancer resistance protein（BCRP）and ABCC1/multidrug resistance-related protein 1（MRP1）,existing in the same kind of cells are distributed asymmetrically.In various studies,ABCB1（MDR1 or Pgp）and ABCG2（BCRP1）are involved in drug resistance.At the same time,studies have shown that activated hedgehog（Hh）signals up-regulate the expression of MDR transporters MDR1（ABCB1 or P-glycoprotein）and BCRP（ABCG2）.Hedgehog（Hh）signaling pathway plays an important role in embryonic development,tissue regeneration and stem cell renewal.The Hh protein family is composed of Sonic（SHH）,Indian（IHH）and Desert（DHH）proteins,which are differentially expressed according to species.In humans,sonic hedgehog（Shh）is the main protein.Combining with 12-transmembrane（TM）receptors-Patched1 and/or Patched2（PTCH1 and PTCH2）leads to the activation of 7-transmembrane smoothing protein（SMO）,thereby activating its downstream signaling pathways.SMO activation activates glioma-related oncogene（GLI）transcription factors（GLI-1,GLI-2 and GLI-3）,thereby activating the expression of downstream target genes,of which GLI-1 is the most important strong transcription activation factor.Studies have confirmed that only SHH in the Hh protein family is related to humans,and the SHH/PTCH/GLI1 pathway has a regulatory effect on the ABC transporters.Mineral dust-inducing gene（MDIG）is a proto-oncogene associated with lung cancer and plays a key role in the biological process of tumorigenesis.The purpose of this study is to determine whether mineral dust-inducing gene（MDIG）is involved in cisplatin（DDP）resistance of lung adenocarcinoma,and to explore its molecular mechanism,and to further explore the role of SHH/PTCH/GLI1 pathway in this process.Methods:In the first part,the drug resistance of lung adenocarcinoma cell line A549 and cisplatin-resistant lung adenocarcinoma cell line A549/DDP was tested by cytotoxicity test（CCK-8 test）to clarify the establishment of drug-resistant cell lines.The expression differences of mdig were detected by reverse transcription quantitative PCR and Western Blotting experiment respectively.Subsequently,the cisplatin resistant adenocarcinoma cell A549/DDP was transfected with lentivirus,and the drug resistance changes were tested again after MDIG overexpression and silenced.Subsequently,the ABC transporters（ABCB1,ABCC1,ABCG2）related to the drug resistance of lung adenocarcinoma were detected by reverse transcription quantitative PCR and Western Blotting experiments.The changes of the above indicators were detected in the cisplatin-resistant adenocarcinoma cell A549/DDP that overexpressed and silenced MDIG.In the second part,the differences in the basic expression of SHH pathway related molecules SHH,PTCH1,and GLI-1 were detected in lung adenocarcinoma cell（A549）and lung adenocarcinoma cell resistant to cisplatin（A549/DDP）through reverse transcription quantitative PCR and Western Blotting experiment.The changes of the above indicators were detected in the adenocarcinoma cisplatin resistant cell A549/DDP that overexpressed and silenced MDIG.In the third part,MDIG-overexpressing A549/DDP cells was selected,and SHH pathway inhibitors were added to detect the changes in drug resistance and the expression of related proteins were detected to further verify the mechanism of MDIG inducing drug resistance in lung adenocarcinoma cells,and to confirm the role of the ABC transporters（ABCB1,ABCC1,ABCG2）and SHH pathway in MDIG-mediated drug resistance of lung adenocarcinoma cells.After the conclusion of the above experiments,the lung adenocarcinoma cell lines A549 and H1299 were further selected for lentivirus transfection,and the stable transfection of A549 and H1299 cell lines with MDIG overexpression and MDIG silenced was established respectively.The above experiments were repeated and the same conclusions were obtained.Finally,the tumor-forming experiment of nude mice was used to verify the promotion effect of MDIG on cisplatin resistance of lung adenocarcinoma cells,and immunohistochemical experiment was used to further detect the tissue expression of key proteins during the regulation process of MDIG on cisplatin resistance of lung adenocarcinoma cells.Results:The first part of the results showed that compared with A549 cells,the IC₅₀value of A549/DDP cells was higher than that of A549 cells,and the MDIG m RNA and the expression of protein in A549/DDP cells were significantly higher than that of A549cells.The inverted fluorescence microscope observed that the A549/DDP cells stably transfected with lentivirus were in good condition,and the transfection efficiency of MDIG overexpression and silenced was verified by RT-q PCR and Western Blotting experiments.The results showed that the LV-MDIG group significantly increased the m RNA and protein expression levels of MDIG in A549/DDP cells;both the LV-mdig-RNAi 1 group and LV-mdig-RNAi 2 group significantly reduced the MDIG m RNA and protein expression level in A549/DDP cells.Compared with the vector group,the IC₅₀value of A549/DDP cells was significantly increased after mdig was overexpressed;after MDIG was silenced,the IC₅₀value was significantly reduced.At the same time,compared with A549 cells,the m RNA and the expression of ABC transporters（ABCB1,ABCC1,ABCG2）in A549/DDP cells were significantly up-regulated.And after MDIG overexpressed,they were significantly up-regulated compared with the control group,and after MDIG silenced,they were significantly lower than the control group.The second part of the results showed that the m RNA of SHH,PTCH1,GLI-1 and expressions of proteins in A549/DDP cells were significantly higher than those in A549cells.The m RNA and protein expression levels of the above-mentioned molecules were significantly up-regulated compared with the control group（vector group）after MDIG was overexpressed,and were significantly lower than the control group after MDIG was silenced.The MDIG-overexpression A549/DDP group（LV-MDIG）was selected subsequently.After the SHH pathway inhibitor cyclopamine was added,the IC₅₀of the inhibitor group（LV-MDIG+cyclopamine group）was significantly lower than that of the control group（LV-MDIG group）.The protein expression levels of related molecules and ABC transporters were significantly lower than those of the control group（LV-MDIG group）.（4）Compared with the control group,the IC₅₀value of A549 and H1299 cells increased significantly after MDIG-overexpression,and decreased significantly after MDIG-silenced in A549 and H1299 cells.The expression of ABC transporters（ABCB1,ABCC1,ABCG2）,SHH,PTCH1 and GLI-1 in A549 and H1299 cells were significantly up-regulated after MDIG-overexpression and down-regulated after MDIG-silenced.After the addition of SHH pathway inhibitor cyclopamine（LV-MDIG+cyclopamine group）,The protein expression levels of GLI1 and ABC transporters（ABCB1,ABCC1and ABCG2）,key molecules of SHH signaling pathway,were down-regulated compared with those of group without pathway inhibitor（LV-MDIG）,and IC50 was also down-regulated.（5）In vivo,the tumor volume of MDIG-silenced A549/DDP stably transfected cell lines in nude mice was significantly smaller than that of the control LV-CON group after 3 weeks of cultivation,and the tumor volume of LV-mdig-RNAi group was reduced after DDP intervention.The results of immunohistochemical experiments showed that ABCB1,ABCG2 and GLI-1 in LV-mdig-RNAi group were significantly lower than those in control LV-CON group.Conclusion:（1）The expression of MDIG was significantly increased in cisplatin resistant cells,and its expression level was related to cisplatin resistance.（2）MDIG can promote the expression of drug-resistant ABC transporters（ABCB1,ABCC1,ABCG2）,thus promoting the extracellular excretion of drugs,and thus inducing cisplatin resistance of lung adenocarcinoma cells.（3）By activating the SHH/PTCH1/GLI-1 signal transduction pathway,MDIG can increase the expression of downstream ABC transporters（ABCB1,ABCC1,ABCG2）,and then induce cisplatin resistance of lung adenocarcinoma cells.