The Study Of Autophagy-related MiRNA In Serum Exosomes As A Marker For The Efficacy Of Cisplatin In Non-small Cell Lung Cancer

Lung cancer is the most common malignant tumor with the most cause of human cancer-related deaths for several decades in China.About 80%of them are non small cell lung cancer(NSCLC),and platinum plays a very important role in the treatment.Cisplatin is one of the most widely used of the longest history.However,it is difficult to solve the problem of drug resistance.Although there are a number of studies on the genetic polymorphisms of cisplatin resistant lung cancer tissue,there is not any genetic variation that can be used as an accurate predictor of platinum reactivity in clinical patients.And the tomor tissue is difficult to obtain,the size is limited and there are some heterogeneity,which lead to the limitations and difficulties of clinical application.Therefore,we hope to find a new mechanism of drug resistance and more effective biomarkers to predict and reverse the clinical cisplatin resistance,and provide more ways and means for clinical transformation.It is reported that autophagy may play a role in tumor resistance,but there is still controversy in lung cancer.Here we use the NSCLC cell lines,clinical tumor tissue and blood samples to study the effect of autophagy in response to cisplatin of NSCLC patients and to investigate whether of tumor exosomal miRNA targeting autophagy can be used as a biomarker to predict cisplatin response and provide a theoretical basis for the clinical therapy.Part Ⅰ:The correlation between the level of autophagy and cisplatin response in NSCLCObjective:To detect the level of basal autophagy in NSCLC tissue and cell lines and explore its clinical significance in cisplatin treatment of NSCLC.Method:The levels of autophagy related proteins Atg5 and LC3B-I were detected by immunohistochemistry in 203 cases of NSCLC patients treated with cisplatin based first-line chemotherapy(88 cases of advanced stage and 115 cases after radical resection).The clinical pathological data of 203 patients with NSCLC and the expression level of basal autophagy were analyzed by GraphPad Prism software package.The LC3B protein level of NSCLC cell line was detected by western blot,and IC50 to cisplatin was detected by MTT assay in different NSCLC cell lines.Transfection NSCLC cells of siRNA interference Atg5 gene,lentivirus interference Beclinl gene,and MTT test were performed to check the effect in sensitivity of cells to cisplatin.Results:1.In 88 cases of advanced NSCLC patients,the expression of autophagy related protein Atg5 in cisplatin-resiatant NSCLC(PFS M=6,n=46)was significantly higher than that in cisplatin-sensitivity NSCLC(PFS>6 M.n=42),but autophagy negative associated protein LC3B-Ⅰ was significantly lower in the NSCLC sensitive to cisplatin.2.The expression level of Atg5 was negatively correlated with PFS,and positive correlation was found between LC3B-I and PFS in advanced NSCLC patients.3.In 88 advanced NSCLC patients,the results showed that the high-Atg5 patients had higher progression rates(median PFS:5 months)than the low-Atg5 patients(median PFS:6 months,P=0.0372)while the high-LC3B-I patients had lower progression rates(median PFS:7.5 months than the low-LC3B-I patients(median PFS:4 months,P=0.0045)4.In 115 NSCLC patients after operation,the results showed that the high-Atg5 patients had higher recrudescence rates(median DFS:8 months)than the low-Atg5 patients(median DFS:22 months,P<0.0001),while the high-LC3B-I patients had lower progression rates(median DFS:22 months)than the low-LC3B-I patients(median DFS:6 months,P<0.0001)5.LC3B-II/LC3B-I protein ratio of NSCLC cell lines is different and relative to the response to cisplatin.6.Silencing the Atg5 and Beclinl genes in the NSCLC cells,their response to cisplatin was enhanced.Conclusion:The response of NSCLC to cisplatin is related to the level of basal autophagy,and the higher of the level of autophagy,the lower of the response to cisplatin chemotherapy.Inhibition of autophagy can enhance the sensitivity to cisplatin and improve the therapeutic effect.Basal autophagy level may be used as a biological indicator for predicting the efficacy of cisplatin in NSCLC.Part Ⅱ:The relationship between the exosomal miRNA expression level and cisplatin efficiency of NSCLCObjective:To investigate whether serum exosomal miRNA can predict the reactivity of NSCLC to cisplatin and whether the mechanism is related to autophagy.Method:SRNA sequencing of surum exosomal RNA from cisplatin-sensitive patients(10 cases of mixed serum samples)and cisplatin-resistant patients(10 cases of mixed serum samples)was conduct for analyze miRNA differential expression.Bioinformatic analysis was conducted for the altered miRNA data,including cluster analysis,Gene Ontology(GO)analysis,and pathway analysis dependent on the Kyoto Encyclopedia of Genes and Genomes(KEGG).The qRT-PCR assay was performed to further confirm the changed miRNAs in 170 advanced NSCLC patients.To analyze the relationship between the therapeutic effect of cisplatin and the expression of miRNA,we over expressed or silenced the related miRNA and detected LC3B,p62,Atg5,p-mTOR,p-AKT proteins in NSCLC by Western blot.Meanwhile,MTT assay was conduct to test the reaction to cisplatin.TargetScan software,luciferase reporter gene assay,cell transfection and qRT-PCR were made to find and validate the target gene of the miRNA.Immunohistochemistry test was conduct in 79 cases of advanced NSCLC tissue to verify the effect of target gene in cisplatin sensitivity.Results:1.Advanced NSCLC serum exosomal sRNA sequencing results showed that there were 224 differentially expressed miRNAs in cisplatin-sensitive patients and cisplatin-resisatnt patients,of which 162 cases were higher expressed in sensitive patients(153 cases above 2 times),the remaining 62 cases higher expressed in cisplatin-resisatnt patients(54 cases above 2 times).2.GO analysis and KEGG pathway analysis showed that the expression of miRNA was involved in many biological processes including cell adhesion,tumor cell proliferation and apoptosis,including cisplatin resistance mechanism of autophagy pathway.3.SRNA sequencing results of 6 miRNA with marked difference were further confirmed by qRT-PCR in 170 cases of patients with advanced NSCLC,and 3 of them(hsa-miR-425-3p,hsa-miR-146a-5p,hsa-miR-4755-5p)had significant different expression in 76 cisplatin-resisatnt cases and 94 cisplatin-sensitive cases.4.One hundred and seventy cases of advanced NSCLC patients were divided into high expression group and low expression group according to the median absolute concentration of hsa-miR-425-3p.The results showed that the high-hsa-miR-425-3p patients had higher progression rates(median PFS:4 months)than the low-hsa-miR-425-3p patients(median PFS:8 months,P<0.0001).5.The expression level of hsa-miR-425-3p is lower in the cisplatin-sensitive NSCLC cells than in the cisplatin-resistant NSCLC cells.6.The reaction of the cisplatin-sensitive NSCLC cells to cisplatin decreased after over expression of miR-425-3p.On the contrary,with inhibition of miR-425-3p,the response to cisplatin of resistant NSCLC cells increased and cell killing of cisplatin enhanced.7.After overexpression of miR-425-3p in cisplatin-sensitive NSCLC,the expression of autophagy related protein LC3B-II,Atg5 increased,p62 substrate decreased;negative regulators of p-AKT,p-mTOR protein expression declined,which represented enhancement of autophagic activity.Coincidentally,inhibition of miR-425-3p expression,autophagy related protein reduced,autophagy activity decreased.8.The target gene prediction software TargetScan suggested that miR-425-3p binded to the 3’ UTR region of the AKT1 gene.There were 2 binding sites in the 3’UTR region of 101-124 sequence and 783-802 sequence in AKT1 respectively.9.The results of luciferase reporter assay indicated that miR-425-3p indeed targeted gene AKT1 and binded to its 3 ’UTR region of 783-802 binding site,which played a role in inhibiting the expression of AKT1.Overexpression of miR-425-3p in NSCLC cells,the expression of both AKT1 mRNA and protein decreased,while inhibiting miR-425-3p,the expression of AKT1 increased.10.The results of immunohistochemistry showed that the expression of AKT1 protein in the cisplatin-sensitive group was higher than that in the cisplatin-resistant group in patients with advanced NSCLC.According to the 79 cases,the low-AKT1 patients had higher progression rates(median PFS:3 months)than the high-AKT1 patients(median PFS:6 months,P=0.0042).Conclusion:Has-miR-425-3p targeting AKT1 gene,enhances tumor autophagy.Its level in advanced NSCLC patients’ serum circulating exosomes may serve as a prognosis biomarker of cisplatin,so as to guide the treatment.