| Lung cancer is a malignant tumor with high morbidity and mortality worldwide.Among them,the prevalence of non-small cell lung cancer(NSCLC)accounts for 85 % of the overall prevalence of lung cancer.At present,cisplatin(DDP)is still the first-line chemotherapy drug for most patients with advanced NSCLC and postoperative NSCLC,but it has inherent and acquired chemotherapy resistance,which leads to treatment failure and poor prognosis.Therefore,overcoming cisplatin resistance is the key to cisplatin treatment of NSCLC.Autophagy is a metabolic reaction of normal cells to maintain their homeostasis.Normal cells use autophagy to degrade damaged organelles and redundant proteins,and the nutrients obtained from them are recycled to maintain normal metabolism of cells.However,under certain conditions,tumor cells can use autophagy to ensure that they can survive in adverse environments such as hypoxia,nutritional deficiency,radiotherapy and chemotherapy,and use autophagy to promote tumor cell regeneration,migration and invasion.Therefore,autophagy is also a mechanism leading to drug resistance in tumor cells.Micro RNAs(miRNAs)are a class of small coding RNAs.Many studies have shown that they are not only involved in the regulation of many biological processes such as tumor cell proliferation,apoptosis,epithelial-mesenchymal transition,migration,invasion,autophagy and tumor angiogenesis,but also affect cisplatin chemotherapy resistance.Our previous study found that the expression level of miR-125b-5p(miR-125b)in non-small cell lung cancer A549 / DDP resistant cell line was significantly decreased compared with parental cells.Overexpression of miR-125 b can reverse the chemotherapy resistance of cisplatin by inhibiting autophagy activity,suggesting that the decrease of miR-125 b expression is closely related to the cisplatin chemotherapy resistance of non-small cell lung cancer.Ailanthone(AIL)is a small molecule compound extracted from the bark of Ailanthus altissima.It not only has antibacterial,anti-inflammatory,anti-amoeba,and anti-ascariasis effects,but also has significant anti-tumor effects on prostate cancer,cervical cancer,rectal cancer,melanoma,and leukemia.Studies have also shown that AIL can reduce cisplatin resistance in bladder cancer and gastric cancer through autophagy activity,but the effect of AIL on cisplatin resistance in NSCLC has not been studied.The purpose of this study was to explore whether AIL can reverse cisplatin resistance in NSCLC,and to further explore whether AIL mediates autophagy to reverse cisplatin resistance in NSCLC through miR-125 b expression.Objective :1.To investigate whether ailanthone can affect the resistance of non-small cell lung cancer A549/DDP cells to cisplatin.2.To investigate whether ailanthone reverses the resistance of non-small cell lung cancer A549/DDP cells to cisplatin by affecting the expression of miR-125 b and affecting autophagy.Methods :1.A549/DDP cells were treated with AIL at a gradient concentration of0,0.2,2,20,40,80,160,320 μmol/L for 24 h.The changes of cell viability were detected by MTT assay.A549/DDP cells were treated with AIL at a low gradient concentration of 0,0.2,0.4,0.6,0.8,1,2 μmol/L.MTT assay was used to determine the concentration of AIL in subsequent experiments.A549 and A549/DDP cells were treated with gradient concentrations of 0,0.781,1.563,3.125,6.25,12.5,25 μg/m L DDP and gradient concentrations of 0,25,50,100,200,400,800 μg/m L DDP for 24 h,respectively.MTT assay was used to detect the effect of DDP on the viability of A549 and A549/DDP cells,and the resistance ratio of DDP was calculated.2.The combination index(CI)of AIL combined with DDP was determined by Chou-Talalay method.The concentration of AIL and DDP was determined according to the CI value,and the cultured A549/DDP cells were divided into control group(without drug),DDP(50 μg/m L)group,AIL(0.6μmol/L)group,AIL(0.6 μmol/L)+DDP(50 μg/m L)group.3.Cell cloning and EDU staining were used to detect the cell proliferation of A549/DDP cells after AIL,DDP and AIL+DDP combination.4.The effects of AIL,DDP and AIL+DDP on cell cycle and apoptosis of A549/DDP were detected by flow cytometry.5.The expression of LC3 B was detected by immunofluorescence staining,and the effect of AIL,DDP and AIL+DDP on autophagy of A549/DDP cells was studied.6.Western Blot was used to detect the protein expression levels of apoptosis-related genes: Bcl2,Bax,Caspase3,cycle-related genes: CDK4,Cyclin D1,and drug resistance-related genes: P-gp,MVP.The protein expression levels of AIL reverse cisplatin resistance.The protein expression levels of autophagy-related genes: p62,LC3 B,ATG5 and Beclin1 were detected.To explore the specific mechanism of AIL reversing cisplatin resistance.7.Rapamycin(RAPA),an autophagy activator,was added and divided into control group,AIL group,DDP group,AIL+DDP group and AIL+DDP+RAPA group.MTT assay was used to detect the changes of cell viability after RAPA was added.Western Blot was used to detect the protein expression levels of drug resistance-related genes: P-gp and MVP.Further study of AIL reversing cisplatin resistance through autophagy.8.Lentivirus LV3-NC(A549/DDP-NC)and Hsa-miR-125b-5p mimics(mimics-miR125b)were transfected into A549/DDP cells to establish A549/DDP-NC and mimics-miR125 b stable transfection strains.The transfection efficiency was detected by fluorescence microscopy.Real Time Quantitative PCR(RT-q PCR)was used to detect whether the overexpression of miR-125 b was successfully established.9.The transplanted tumor model of nude mice was constructed with the established A549/DDP-NC and mimics-miR125 b stable transfected strains,and the volume and mass changes of transplanted tumors were monitored.The m RNA expression level of miR-125 b in transplanted tumor tissues was detected by RT-q PCR.To investigate the effect of overexpression of miR125 b on the growth of A549/DDP transplanted tumor.10.Western Blot was used to detect the protein expression levels of autophagy-related genes: p62,LC3 B,ATG5,Beclin1 and BNIP3 L in transplanted tumor tissues,and the protein expression levels of drug resistance-related genes: P-gp and MVP were detected.The protein expression levels of autophagy-related genes: p62,LC3 B,ATG5,Beclin1 and BNIP3 L in transplanted tumor tissues were detected by immunohistochemistry(IHC)DAB staining.To further investigate the effect of miR-125 b on the growth of A549/DDP xenografts through autophagy.11.RT-q PCR was used to detect the m RNA expression level of miR-125 b in A549/DDP cells after AIL,DDP and AIL+DDP combined treatment,and the effect of AIL on miR-125 b in A549/DDP cells was preliminarily discussed.12.A549 / DDP cells were transfected with NC-inhibitor(NC),miR-125b-5p inhibitor(inhibitor-miR125b),and RT-q PCR was used to detect whether the knockdown of miR-125 b was successful.The experimental groups were divided into Control group,AIL+DDP group,AIL+DDP+NC group,AIL+DDP+inhibitor-miR125 b group,and treated with AIL+DDP drugs.MTT assay was used to detect the changes of A549/DDP cell viability in each group.The expression of LC3 B in A549/DDP cells was detected by immunofluorescence staining.To preliminarily study the effect of AIL on autophagy through miR-125 b.13.Western Blot was used to detect the protein expression levels of autophagy-related genes: p62,LC3 B,ATG5,Beclin1,BNIP3 L and drug resistance-related genes: P-gp,MVP in Control group,AIL+DDP group,AIL+DDP+NC group,AIL+DDP+inhibitor-miR125 b group.To further study the effect of AIL on cisplatin resistance by regulating autophagy through miR-125 b.Results:1.The survival rate of A549/DDP cells was significantly decreased when the concentration of AIL was ≥ 2 μmol/L(F=25.37,P < 0.0001).In order to ensure the low toxicity of the drug,A549/DDP was treated with low gradient concentration of AIL,and it was found that 0.6μmol/L was the lowest concentration with statistical significance(F=15.31,P<0.0001),which was used as the concentration of for subsequent experiments.2.After A549 and A549/DDP cells were treated with gradient concentration of DPP,the cell viability of A549 cells was significantly inhibited and the IC50 value was 3.938 μg/ml.The IC50 value of A549/DDP cells was 145.339 μg/ml.Compared with A549 cells,A549/DDP cells were36.95 times more resistant to DDP,indicating that A549/DDP cells were resistant.3.A549/DDP cells were treated with DDP at gradient concentrations of0,25,50,100,200,400,800 μg/ml combined with 0.6 μmol/L AIL,and the IC50 value was 84.268 μg/ml.Compared with the IC50 value of DDP alone,the reversal fold was 1.725,and the reversal fold was >1,indicating that AIL had the effect of reversing DDP resistance.The CI value of AIL combined with DDP was calculated by Chou-Talalay analysis.When the concentration of DDP ≥50 μg/ml,CI<1,indicating that AIL combined with DDP has a synergistic effect.In order to reduce the toxicity of the drug,50 μg/ml DDP with synergistic effect and low concentration was selected for subsequent experiments.4.The results of cell cloning experiments showed that compared with Control group,DDP group and AIL group,the clone formation of AIL+DDP group was the least(F=331.8,P < 0.0001).The results of EDU staining showed that the cell proliferation rate of AIL+DDP group was the lowest compared with Control group,DDP group and AIL group(F=144.7,P <0.0001).5.The effect of AIL on the cell cycle and apoptosis of A549/DDP was detected by flow cytometry.Compared with the Control group,the cells in the AIL group and the AIL+DDP group were significantly blocked in the G1phase(F=410.0,P<0.0001).Compared with the Control group,DDP group and AIL group,the cells in the AIL+DDP group were significantly blocked in the G2 phase(F=209.3,P<0.0001).Compared with Control group,DDP group and AIL group,the total apoptosis rate of AIL+DDP group was significantly increased(F=1683,P<0.0001).6.The fluorescence expression intensity of LC3 B was detected by immunofluorescence.Compared with the Control group and the DDP group,the fluorescence expression intensity of LC3 B in the AIL+DDP group was weakened(F=18.00,P=0.0006).7.Western Blot was used to detect apoptosis and cycle-related genes.The results showed that compared with Control group,DDP group and AIL group,the expression levels of Bcl2(F=386.5,P<0.0001)and Caspase3 in AIL+DDP group were significantly decreased(F=147.2,P<0.0001),and the expression levels of Bax(F=249.3,P < 0.0001)and Cleaved Caspase3(F=645.4,P<0.0001)were significantly increased.Compared with Control group and DDP group,the expression levels of CDK4(F=113.3,P<0.0001)and Cyclin D1(F=149.0,P < 0.0001)in AIL+DDP group increased.Compared with Control group and DDP group,the expression levels of P-gp(F=139.8,P<0.0001)and MVP(F=318.8,P<0.0001)in AIL+DDP group were decreased.Detection of autophagy-related genes Compared with Control group and DDP group,the expression level of P62 in AIL+DDP group increased(F=285.5,P < 0.0001),and the expression levels of LC3B(F=88.42,P<0.0001),ATG(F=15.49,P=0.0011)and Beclin1(F=15.49,P=0.0011)decreased.8.After adding autophagy activator rapamycin(RAPA),the cell viability was detected by MTT.The results showed that compared with AIL +DDP group,the cell viability of AIL+DDP+RAPA group was increased(F=226.5,P < 0.0001).Western Blot was used to detect the protein expression levels of drug resistance-related genes.The results showed that compared with the AIL+DDP group,the expression levels of P-gp(F=98.64,P<0.0001)and MVP(F=54.54,P<0.0001)in the AIL + DDP + RAPA group decreased.This shows that AIL can reverse the resistance of A549/DDP cells through autophagy.9.The transfection efficiency of Hsa-miR-125b-5p mimics lentivirus in A549/DDP cells was 85.252 ± 3.923%.RT-q PCR showed that the m RNA expression level of miR-125 b in mimics-miR-125 b group was significantly up-regulated(F=28.67,P=0.0008),and there was no significant difference between A549/DDP group and A549/DDP-NC group(P=0.9982),indicating successful transfection.10.A nude mouse xenograft model was constructed to monitor the volume and weight of the xenograft.Compared with A549/DDP-NC group,the volume(P=0.0058)and weight(P=0.0011)of transplanted tumor in mimics-miR125 b group were decreased.The m RNA expression of miR-125 b in transplanted tumors was detected by RT-q PCR.Compared with A549/DDP-NC group,the m RNA expression of miR-125 b in transplanted tumors of mimics-miR125 b group was significantly increased(P=0.0001).Western Blot was used to detect autophagy-related genes.Compared with the A549/DDP-NC group,the expression level of p62 in the transplanted tumor of the mimics-miR125 b group increased(P=0.0003),and the expression levels of LC3B(P<0.0001),Beclin1(P=0.0002),ATG5(P=0.0001),BNIP3L(P < 0.0001)decreased;compared with the A549/DDP-NC group,the expression levels of P-gq(P=0.0006)and MVP(P=0.0026)in the transplanted tumor of the mimics-miR125 b group were decreased.11.The autophagy-related genes were detected by DAB coloration in IHC.Compared with the A549 DDP-NC group,the expression level of p62 in the transplanted tumor of the mimics-miR125 b group increased(P=0.0029),and the expression levels of LC3B(P<0.0001),Beclin1(P=0.0234),ATG5(P=0.0011),and BNIP3L(P=0.0076)decreased.12.The results of RT-q PCR showed that the m RNA expression level of miR-125 b in AIL+DDP group was up-regulated compared with Control group,DDP group and AIL group(F=1150,P<0.0001).AIL can up-regulate the expression of miR-125 b in A549/DDP cells.13.A549/DDP cells were transfected with NC-inhibitor(NC),miR-125b-5p inhibitor(inhibitor-miR125b),and RT-q PCR was used to detect successful transfection.Compared with A549/DDP group and NC group,the m RNA expression level of miR-125 b in inhibitor-miR125 b group was decreased(F=129.8,P < 0.0001).Compared with AIL+DDP group and AIL+DDP+NC group,the cell viability of AIL+DDP+inhibitor-miR125 b group was increased(F=3069,P < 0.0001).Immunofluorescence results showed that compared with AIL+DDP group and AIL+DDP+NC group,the fluorescence intensity of LC3 B in AIL+DDP+inhibitor-miR125 b group increased(F=20.07,P=0.0004).Western Blot was used to detect the protein expression of autophagy-related genes and drug resistance-related genes.Compared with AIL+DDP group and AIL+DDP+NC group,the expression level of p62 in AIL + DDP + inhibitor-miR125 b group decreased(F=810.3,P<0.0001).The expression levels of LC3B(F=96.62,P<0.0001),Beclin1(F=35.86,P<0.0001),ATG5(F=80.5,P<0.0001),P-gq(F=318.1,P<0.0001)and MVP(F=19.11,P=0.0005)were increased.The above results indicate that AIL can affect the cisplatin resistance of A549/DDP cells by regulating autophagy through miR-125 b.Conclusion :1.AIL can increase the sensitivity of cisplatin to A549/DDP cells and reverse the resistance of A549/DDP cells to cisplatin.2.AIL reverses cisplatin resistance of A549/DDP cells in non-small cell lung cancer through autophagy;3.miR-125 b inhibits the growth of non-small cell lung cancer A549 /DDP cell xenografts through autophagy.4.AIL mediated autophagy to reverse cisplatin resistance in non-small cell lung cancer A549/DDP cells through regulation of miR-125 b expression;... |
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