| Purpose: At present,the incidence and mortality of lung cancer still ranks first among malignant tumors.In the treatment of lung cancer,platinum-based drug-based combination chemotherapy is the first choice for non-small cell lung cancer,but the increasing resistance of platinum-based drugs has seriously affected the treatment of NSCLC.To this end,this study used human lung adenocarcinoma cisplatin-resistant cell line A549/DDP cells as model cells.First,high-throughput sequencing(RNA-Seq)was used to analyze the differentially expressed genes between the parental A549 cells and GO Terms.Perform functional enrichment analysis of differential genes with KEGG,and screen out the functional partitions of differential genes for cisplatin resistance in NSCLC.Secondly,based on the results ofRNA-Seq screening,explore the molecular mechanism of miRNA-21 targeting tumor cell glycolysis pathway to regulate NSCLC cisplatin resistance.Finally,Shenmai injection,a modern traditional Chinese medicine preparation of shendong drink,was used to intervene A549/DDP cells cultured in vitro,to explore the molecular pharmacological mechanism of shendong drink reversing cisplatin resistance of NSCLC,and to provide laboratory data for the treatment of NSCLC with Yiqi Yangyin method.Materials and methods:Thesis 1(1)Cell culture A549 cells and A549/DDP cells were cultured in DMEM medium containing 10% FBS at 37°C,95% humidity,and 5% CO2.A549/DDP cells were continuously cultured in a low-concentration cisplatin(7 μM)environment to maintain their cisplatin resistance,and were replaced with cisplatin-free medium 2 weeks before the experiment.(2)CCK-8 method to detect the drug resistance index of A549/DDP cells A549 cells and A549/DDP cells in logarithmic growth phase were seeded in 96-well plates and adhered overnight.Remove the culture medium and add DMEM culture medium containing different concentrations of cisplatin to interfere with the cells for 24 h.Add 10 μL of CCK-8solution to each well.After incubating for 4 h at 37 ℃,detect the absorbance at OD460 nm.The calculation formula calculates the IC value.(3)Determination of cell glucose and lactate content of A549 and A549/DDP cells in logarithmic growth phase,collect the cells after trypsinization,add 0.1 m L of lysis buffer for every 1×106 cells to lyse the cells,and dilute the working solution with the sample The solution was mixed at 37 oC,reacted for 20 min,and measured OD530 nm.(4)RNA-Seq sequencing analysis A549 and A549/DDP cells were extracted by trizol to obtain totalRNA,a sequencing library was constructed,and the library was quantified by the Real Time PCR method,RNA-Seq expression analysis,PCA and correlation of gene expression levels Analysis,gene/transcript differential expression analysis,functional gene and related signal pathway enrichment analysis,analysis of the expression profile of differential genes in A549/DDP cells.(5)Real Time PCR verifies the accuracy ofRNA-Seq results Collect A549 cells and A549/DDP cells,extract totalRNA,reverse transcription reaction and Real Time PCR reaction to verify the expression of differential genes.Thesis 2(1)miRNA-21 sponge transfection cell experiment A549/DDP cells were seeded into a24-well plate with a cell density of 60-80%.Mix the Golden Tran DR reagent and miRNA-21NC/miRNA-21 sponge immediately Add to the cell surface,culture for 56 h,then perform mRNA or protein detection.(2)Determination of glycolysis level of A549/DDP cells by miRNA-21 sponge combined with cisplatin.Collect and lyse A549/DDP cells,mix the working solution of glucose and lactic acid detection reagents with the sample diluent,and place it in a 37 oC incubator.React for 20 min,and measure the OD530 nm value.(3)Determination of protein expression of A549/DDP cell-related signal pathways by miRNA-21 sponge combined with cisplatin.Collect A549/DDP cells intervened by cisplatin and miRNA-21 sponge alone or in combination,and lyse the cells to extract the total protein of each group of cells.The BCA method was used to determine the total protein content of each group of cells,and the differences in protein expression levels were analyzed by Western blotting and Image J software.(4)Detection of LC3 B fluorescent spots: Fix A549/DDP cells with 4% paraformaldehyde,add 1% Tritonx-100 solution to infiltrate the cells,add LC3 B antibody and incubate overnight at 4 ℃,and finally add FITC-labeled rabbit Ig G secondary antibody to incubate 1 h,observe and take pictures under a fluorescence microscope.(5)Observe the cell death by transmission electron microscope.After trypsinizing A549/DDP cells,the cells were collected by centrifugation,and 2.5%glutaraldehyde was added overnight.After washing with phosphate buffer,adding acidic fixative for fixing for 2 h,and washing with phosphate buffer.Carry out gradient dehydration,embed and infiltrate with acetone and embedding agent,carry out sectioning,and dye the sections with uranyl acetate and lead citrate,and finally observe and photograph by transmission electron microscope.Thesis 3(1)The determination of A549/DDP cell viability by Shendong drink A549/DDP cells were seeded in 96-well plates,the culture medium was removed after adherence overnight,and DMEM culture medium containing different concentrations of Shendong drink was added to interfere with the cells for 24 h.Add 10 μL of CCK-8 solution to each well,incubate at 37 ℃ for 4 hours,detect the absorbance at OD460 nm,and calculate the IC value according to the modified Kou formula.(2)Determination of glycolysis level of A549/DDP cells by Shendong drink combined with cisplatin.Collect and lyse A549/DDP cells,mix the working solution of glucose and lactic acid detection reagent with sample diluent,and react in 37 oC incubator 20 min,measure the OD530 nm value.(3)Determination of the protein expression of A549/DDP cell related signal pathways by Shendong drink combined with cisplatin.Collect A549/DDP cells intervened by cisplatin and Shendong drink alone or in combination,and lyse the cells to extract the total protein of each group of cells.BCA method The total protein content of each group of cells was determined,and the difference in protein expression level was analyzed by immunoblotting method and Image J software.(4)Cell Ed U proliferation detection 4% paraformaldehyde fixes A549/DDP cells,adds glycine solution to neutralize the formaldehyde,adds 0.5% Triton X-100 permeation solution,then adds Ed U staining,and finally adds Hoechst 33342 counterstaining,and places Observe and get pictures under DAPI and CY3 fluorescence modules.Results:Thesis 11.There are differences between A549 cells and A549/DDP cells in morphology,cisplatin sensitivity and glycolysis level.Observed under high magnification,there is a significant difference in morphology between cisplatin-resistant A549/DDP cells and their parent A549 cells.Compared with A549 cells,the volume and intercellular space of A549/DDP cells are significantly increased.Different concentrations of cisplatin were used to interfere with A549 cells and A549/DDP cells.The IC50 values of the two cell lines obtained by the CCK-8 method were 37.8 μM and63.3 μM,respectively.The resistance index(RI)of A549/DDP cells is 1.67,which means that compared with A549 cells,A549/DDP cells have higher cisplatin resistance.In addition,the IC5,IC10 and IC20 values of A549/DDP cells were 6.6,12.3 and 23.3 μM,respectively.By measuring glucose consumption and lactic acid production,the glycolysis level of A549/DDP cells was significantly higher than that of A549 cells.2.RNA-Seq data analysis and functional enrichment analysis of differentially expressed genes.High-throughput sequencing(RNA-Seq)was used to compare the differential gene expression of A549/DDP cells and their parent A549 cells.Compared with A549 cells,there are about 2,200 differentially expressed genes up-regulated in A549/DDP cells and about4,300 differentially expressed genes down-regulated.Through the method of Real Time PCR,the first 8 up-regulated genes and the first 8 down-regulated genes of the differentially expressed genes in A549/DDP cells were further verified.The results showed that they were basically consistent with theRNA-Seq sequencing results.In addition,to study the regulatory pathways involved in these differential genes,we conducted enrichment analysis through GO Terms and KEGG and showed that A549/DDP cells have significant changes: cell cycle,DNA replication,metabolic pathways,and enzyme activities.3.The carbohydrate metabolism pathway regulates the construction of ceRNA network and miRNA.Use Target Scan and miRNA software to predict microRNA targets with significantly different sugar metabolism genes(PKM2,LDHA,ALDOA,ENO1,ENO2,HK1,PGAM1),and construct a ceRNA network diagram.In addition,we also used Pubmed database,Embase database and CBM database to mine miRNA-21 may be related to NSCLC cisplatin resistance.Thesis 21.miRNA-21 is highly expressed in A549/DDP cells.Real Time PCR results showed that miRNA-21 was highly expressed in cisplatin-resistant A549/DDP cells.Compared with A549 cells,the expression of miRNA-21 in A549/DDP cells was up-regulated by 5 times.2.miRNA-21 regulates the glycolysis level of A549/DDP cells through PI3K/Akt/m TOR/HIF-1α signaling pathway.The previous results have proved that miRNA-21 is highly expressed in A549/DDP cells.In order to further study the function of miRNA-21 in A549/DDP cells,we constructed miRNA-21 sponge(plasmid microRNA suppressor vector)to inhibit The expression of miRNA-21.As measured by the glucose and lactate detection kit,compared with miRNA-21 NC and cisplatin alone intervening in A549/DDP cells,miRNA-21 sponge combined with cisplatin to intervene in A549/DDP cells can reduce glucose consumption,pyruvate production and lactate production.In addition,the down-regulation of miRNA-21 combined with cisplatin by Western blot method can significantly reduce the glycolysis rate-limiting enzyme PKM2 and LDHA protein expression in A549/DDP cells.miRNA-21 sponge combined with cisplatin can significantly down-regulate the expression of p-PI3 K,p-Akt(Thr308),p-Akt(Ser473),m-TOR and HIF-1α protein.3.miRNA-21 sponge promotes A549/DDP cell death.Firstly,by fluorescence staining observation of LC3 B,it was found that cisplatin,miRNA-21 sponge and miRNA-21 sponge combined with cisplatin interfered with A549/DDP cells,and the cells had different degrees of autophagy.When miRNA-21 sponge combined with cisplatin interfered with A549/DDP The largest number of autophagosomes are produced in cells.Secondly,the nucleus was stained by DAPI and found that cisplatin,miRNA-21 sponge and miRNA-21 sponge combined with cisplatin interfered with cisplatin-resistant A549/DDP cell nuclei and nuclear debris appeared around the nucleus,and miRNA-21 sponge combined with cisplatin interfered with A549/DDP The largest number of nuclear fragments are produced.Finally,through transmission electron microscopy,when cisplatin and miRNA-21 sponge interfere with A549/DDP cells alone,the increase of autophagosomes and autophagolysosomes can be clearly observed.When miRNA-21 sponge combined with cisplatin interfered with A549/DDP cells,A549/DDP cells appeared necroptotic.In addition,by Western blot detection,miRNA-21 sponge combined with cisplatin interfered with A549/DDP cell apoptosis-related proteins cleave caspase-3 and Bax expression significantly increased,while the expression of Bcl-2 decreased;autophagy-related proteins LC3 B and ATG7 Expression has also increased significantly.Thesis 31.The expression of key enzymes of sugar metabolism in A549/DDP cells increased.Based on the results of high-throughput sequencing,Real Time PCR and Western blot were used to verify the key enzymes in the glycolysis pathway of tumor cells.The results showed that HK2,PKM1/2,PKM2,GLUT1 and LDHA in A549/DDP cells The expression of mRNA and protein increased significantly.2.Shendong drink inhibits the growth of A549/DDP cells.Shendong drink has a toxic effect on A549/DDP cells,and the concentration is positively correlated.The CCK-8 method showed that the IC50 of Shendong drink on A549/DDP cells was 40.0 mg/m L,IC5,IC10 and IC20 were 10.0,15.0 and 20.0 mg/m L,respectively.3.Shendong drink enhances the cytotoxicity of cisplatin on A549/DDP.Shendong drink has enhanced toxicity to cisplatin.When the concentration of cisplatin combined with Shendongyin was 10.0,15.0 and 20.0 mg/m L,the IC50 value of cisplatin on A549/DDP cells decreased from 63.6 μM to 21.0 μM,20.3 μM and 13.7 μM,the reversal index rose from 3.1 to 3.5 and 4.6,respectively.When the concentration of cisplatin combined with Shendong drink was 20.0 mg/m L,compared with cisplatin alone,it could significantly reduce the glucose consumption and lactic acid production of A549/DDP cells.In addition,it was found by Western blot that Shendong drink combined with cisplatin can inhibit the protein expression of HK2,PKM1/2,GLUT1 and PDH in A549/DDP cells.4.Shendong drink enhances the toxicity of cisplatin through the PI3K/Akt/m TOR/c-Myc pathway.Shendong drink combined with cisplatin can significantly reduce the protein expression of p-Akt(Ser473),p-Akt(Thr308),p-m TOR(Ser2448)and c-Myc in A549/DDP cells compared with cisplatin alone..In order to confirm that Shendong drink enhances the toxicity of cisplatin through the PI3K/Akt/m TOR/c-Myc pathway,inhibitors at different sites of the PI3K/Akt/m TOR/c-Myc pathway were selected for intervention.The results showed that after LY294002,rapamycin and 10058-F4 were used to intervene in A549/DDP cells respectively,the results proved that Shendong drink enhanced the toxicity of cisplatin through PI3K/Akt/m TOR/c-Myc pathway.5.Cell proliferation and apoptosis detection.The combined intervention of Shendong drink and cisplatin reduces the proliferation rate of A549/DDP cells by about 60% compared with cisplatin alone.In addition,the expression levels of apoptosis-related proteins cleave caspase-3 and Bax were significantly reduced,and the expression level of Bcl2 was significantly increased by Western blot.Conclusion:1.Transcriptomics data showed that the level of glucose metabolism in cisplatin-resistant A549/DDP cells increased.2.miRNA-21 inhibits glycolysis of A549/DDP cells through PI3K/Akt/m TOR signaling pathway and increases the sensitivity of NSCLC to cisplatin.3.Shenmai injection regulates PI3K/Akt/m TOR signaling pathway,inhibits glycolysis of A549/DDP cells and increases the sensitivity of NSCLC to cisplatin. |
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