| Objectives: Lung cancer is one of the most common malignant tumors in the world,its high mortality rate brings a burden to public health services that cannot be ignored.Therefore,the research on lung cancer is still a hot spot in the field of cancer.miRNAs(micro RNAs)are small non-coding RNAs that can regulate gene expression at the posttranscriptional level by inhibiting messenger RNA translation or promoting m RNA degradation.In recent years,the role of exosomes in the occurrence and development as well as the screening,diagnosis,treatment,and prognosis of a variety of cancers has gradually become a research hotspot.Exosomes are vesicles with a diameter of about 30-120 nm secreted by living cells.Exosomes are extracellular vesicles secreted by living cells with diameters ranging from 30 to 120 nm.Because exosomes can not only directly participate in the transmission of information between cells,but also participate in the transport of substances between cells,exosomes are considered as important carriers of intercellular communication,immune regulation,and circulating biomarkers for disease diagnosis and prognosis.Exosomes contain nucleic acid components such as active proteins and RNA,and exosomes have a variety of physiological functions.In addition,it has a certain impact on tumor biology,immune nervous system,tissue repair,etc.Therefore,exosomes have potential clinical application value in the field of cancer.Our previous study found that miR-140-3p was differentially expressed in human blood exosomes.We planned to explore the effect of intracellular miR-140-3p on the biological function of non-small cell lung cancer cells by regulating EGLN3 gene through functional experiments.To further explore the role of exosomal miR-140-3p in the progression of NSCLC(non-small cell lung cancer)by regulating EGLN3/ MAPK(Mitogen-Activated Protein Kinase)signal axis.Methods: Part Ⅰ: The miRNA expression matrix of NSCLC tissues from TCGA(The Cancer Genome Atlas)database was analyzed to obtain the differentially expressed miRNAs.A total of 14 serum exosomal samples from 7 healthy subjects and 7 patients with non-small cell lung cancer were sequenced and analyzed to obtain differentially expressed exosomal miRNAs.The down-regulated miRNAs in both lung cancer tissue and serum exosomes were obtained by intersection,and the most down-regulated miR-140-3p was selected for subsequent analysis.In order to explore the role of exosomal miR-140-3p in the progression of NSCLC,we first investigated the regulatory effect of miR-140-3p in NSCLC.The relationship between miR-140-3p expression level and clinical characteristics(age,gender,pathological classification,survival status,clinicopathological stage,T stage,N stage,M stage)of non-small cell lung cancer patients in TCGA database was explored.The expression level of serum exosomal miR-140-3p in 66 healthy subjects and 66 patients with non-small cell lung cancer,and the relationship between the expression level of exosomal miR-140-3p and the clinical information of patients(age,gender,pathological classification,clinicopathological stage,T stage,M stage,lymph node metastasis)was explored.Firstly,the expression level of miR-140-3p in NSCLC cells and normal cells was analyzed by q RT-PCR(quantitative real-time PCR).NSCLC cells A549 and SK-MES-1 were transfected with miR-140-3p mimics and miR-140-3p mimics-NC(Negative Control).MTS proliferation assay was utilized to detect the proliferation ability of cells in different treatment groups.Different groups of cells were treated with Annexin V-APC/7-AAD reagent according to the instructions of the kit,and the percentage of cell apoptosis in different treatment groups was detected by flow cytometry,to explore the effect of miR-140-3p on the proliferation and apoptosis of NSCLC cells.The Starbase v2.0bioinformatics website was used to predict the biological pathways that miR-140-3p might be involved in.Part Ⅱ: Investigating the molecular mechanism of miR-140-3p regulating the proliferation and apoptosis of NSCLC cells.The potential target genes of miR-140-3p were firstly predicted by five bioinformatics websites: Target Scan,Tarbase,miRDB,miRWalk and Starbase v2.0.The intersection of the results predicted by five websites was obtained by Venn diagram.The ENCORI website was used to explore the relationship between the intersection genes and the expression level of miR-140-3p,and the genes negatively correlated with the expression level of miR-140-3p were selected for subsequent research.The expression levels of candidate genes in NSCLC tissues in TCGA database were analyzed,and the expression levels of these genes in NSCLC cells were analyzed by q RTPCR.miR-140-3p mimics and miR-140-3p mimics-NC were transfected into A549 cells and SK-MES-1 cells,and the expression levels of the candidate genes in different treatment groups were analyzed by q RT-PCR.Genes whose expression levels were inhibited by miR-140-3p overexpression were screened for subsequent studies.Western Blot and dual luciferase reporter assay were utilized in the study to verify the targeted binding of miR-140-3p to EGLN3 gene and inhibition of its expression.The relationship between EGLN3 gene expression level and clinical information(age,gender,survival status,clinicopathological stage,TNM stage)of NSCLC patients in TCGA database,and the relationship between EGLN3 gene expression level and prognosis of patients were explored.OE-EGLN3(over expression)and OE-NC plasmids were transfected into NSCLC cells A549 and SK-MES-1.MTS assay was used to detect the proliferation of cells in different treatment groups.Different groups of cells were treated with Annexin VAPC/7-AAD reagent according to the instructions of the kit,and the percentage of cell apoptosis in different treatment groups was detected by flow cytometry,to explore the effect of EGLN3,the target gene of miR-140-3p,on the proliferation and apoptosis of NSCLC cells.Western Blot was used to explore the expression level of EGLN3 gene in different treatment groups of recovery experiment,MTS proliferation assay was used to detect the proliferation of cells in different treatment groups.Different groups of cells were treated with Annexin V-APC/7-AAD reagent according to the instructions of the kit,and the percentage of cell apoptosis in different treatment groups was detected by flow cytometry,to explore the regulation of miR-140-3p on the expression of target gene EGLN3 and its effect on the function of NSCLC cells.GSEA(Gene Set Enrichment Analysis)was used to predict the possible biological pathways involved in EGLN3.Western Blot was used to explore the expression levels of downstream protein molecules of MAPK signaling pathway in cells with different EGLN3 gene expression levels.Part Ⅲ: Investigating the molecular mechanism of BEAS-2B cells transferring miR-140-3p to NSCLC cells through exosomes and causing changes in cell function.Transmission electron microscopy,NTA(Nanoparticle Tracking Analysis)and Western Blot were used to verify the exosomes extracted from the serum of the study subjects.The expression level of miR-140-3p in exosomes derived from human normal lung epithelial cells and NSCLC cells were detected by q RT-PCR.The ability of non-small cell lung cancer cells to take up exosomes containing miR-140-3p secreted by BEAS-2B cells was verified by cell co-culture,q RT-PCR and GW4869 treatment.Exosomes derived from BEAS-2B cells were extracted and co-cultured with non-small cell lung cancer cells.MTS proliferation assay was used to explore the proliferation ability of cells in the control group and the co-culture group.The cells in control group and co-culture group were treated according to the instructions of Annexin V-APC/7-AAD apoptosis detection kit,the percentage of apoptotic cells in different treatment groups was detected by flow cytometry,to explore whether BEAS-2B cells can transfer exosomes to NSCLC cells and cause functional changes in NSCLC cells.After transfection of miR-140-3p mimics and miR-140-3p mimics-NC into BEAS-2B cells,exosomes were extracted,and the expression levels of miR-140-3p in BEAS-2B cells and exosomes after transfection were detected by q RT-PCR.The transfected exosomes were co-cultured with A549 cells and SK-MES-1cells,and the expression level of EGLN3 gene was verified by q RT-PCR and Western Blot.MTS assay was used in the study to explore the proliferation of A549 cells co-cultured with different exosome miR-140-3p expression levels.The cells co-cultured with different exosomal miR-140-3p expression levels were treated according to the instructions of Annexin V-APC/7-AAD apoptosis detection kit,and the percentage of cell apoptosis in different treatment groups was detected by flow cytometry,to explore the effect of exosomal miR-140-3p on EGLN3 gene expression and proliferation and apoptosis of NSCLC cells.In addition,Western Blot was utilized to verify the expression levels of downstream protein molecules of MAPK signaling pathway in cells co-cultured with different exosomal miR-140-3p expression levels.Results: Part Ⅰ: Serum exosomes of 14 subjects were sequenced and analyzed.A total of182 differentially expressed exosomal miRNAs were obtained,including 71 up-regulated exosomal miRNAs and 111 down-regulated exosomal miRNAs.The differentially expressed miRNAs in NSCLC tissues from TCGA database were intersected with sequencing results.Three down-regulated exosomal miRNAs were obtained from the intersection: let-7a-5p,miR-140-3p and miR-27a-5p.Among them,miR-140-3p expression was down-regulated by the largest fold,and subsequent studies were conducted on it.The results showed that in TCGA database,the expression level of miR-140-3p was statistically different between patients with different T stages and different genders.q RTPCR results showed that the expression of miR-140-3p was down-regulated in the serum derived exosomes of NSCLC patients compared with healthy controls.And q RT-PCR results showed that the expression of miR-140-3p was down-regulated in non-small cell lung cancer cells(A549 cells and SK-MES-1 cells)compared with BEAS-2B cells.After transfection of miR-140-3p mimics and miR-140-3p mimics-NC into A549 cells and SKMES-1 cells,the expression of miR-140-3p was up-regulated in miR-140-3p mimics group.MTS assay showed that the proliferation ability of NSCLC cells was inhibited after the upregulation of miR-140-3p expression.Apoptosis assay showed that the apoptosis ability of NSCLC cells was promoted after the up-regulation of miR-140-3p expression.Therefore,the expression of miR-140-3p is down-regulated in NSCLC cells,and overexpression of miR-140-3p can cause changes in cell proliferation and apoptosis.Starbase v2.0bioinformatics website predicted that miR-140-3p might be involved in the MAPK signaling pathway.Part Ⅱ: The intersection of the results predicted by Target Scan,Tarbase,miRDB,miRWalk and Starbase v2.0 obtained 26 possible target genes of miR-140-3p,of which SMIM13,HBP1,EGLN3 and HMGXB4 were negatively correlated with the expression level of miR-140-3p.q RT-PCR results showed that the expression of HBP1 and EGLN3 genes was up-regulated in NSCLC cells compared with BEAS-2B cells.After transfection of miR-140-3p mimics and miR-140-3p mimics-NC into A549 cells and SK-MES-1 cells,the expression of EGLN3 gene in cells was inhibited,so EGLN3 was a possible target gene of miR-140-3p.Dual luciferase reporter assay verified that miR-140-3p targeted the 3 ’-untranslated region of EGLN3 gene.Western Blot results demonstrated that miR-140-3p could inhibit the expression of EGLN3 gene at protein level,indicating that EGLN3 gene was the target gene of miR-140-3p.Among NSCLC patients from TCGA database,the expression level of EGLN3 gene in Stage III+IV group was higher than that in Stage I+II group,and the expression level of EGLN3 gene in T2 and T3 stage was higher than that in T1 stage,and the prognosis of patients with high EGLN3 expression was poor.The results of q RT-PCR and Western Blot demonstrated that the expression of EGLN3 gene in SKMES-1 cells and A549 cells was up-regulated in OE-EGLN3 group after transfected with OE-EGLN3 and OE-NC plasmids.MTS assay showed that cell proliferation ability of nonsmall cell lung cancer cells was promoted after EGLN3 overexpression,and apoptosis assay showed that the apoptosis ability of NSCLC cells was inhibited after EGLN3 overexpression.EGLN3 as a target gene of miR-140-3p is up-regulated in NSCLC cells,and overexpression of EGLN3 can cause changes in proliferation and apoptosis of NSCLC cells.Cell proliferation and apoptosis recovery experiments showed that EGLN3 gene could partially reverse the effect of miR-140-3p on the proliferation and apoptosis of nonsmall cell lung cancer cells.GSEA prediction results showed that EGLN3 gene was enriched in MAPK signaling pathway.Western Blot results showed that the activity of MAPK signaling pathway was inhibited after EGLN3 gene overexpression,the protein expressions of MAPK7,JNK,MAP2K4 and p38 were down-regulated.Therefore,EGLN3 gene expression is inhibited by miR-140-3p and affects the proliferation and apoptosis of NSCLC cells by regulating the MAPK signaling pathway.Part Ⅲ: q RT-PCR results showed that the expression of miR-140-3p in exosomes derived from NSCLC cells was down-regulated compared with BEAS-2B cells.After BEAS-2B cells and cell culture medium were co-cultured with NSCLC cells,the expression level of miR-140-3p in NSCLC cells was up-regulated.Western Blot results showed that the ability of BEAS-2B cells to secrete exosomes was inhibited by GW4869.q RT-PCR results showed that the expression of miR-140-3p in BEAS-2B cells was upregulated,the expression of miR-140-3p in cell-derived exosomes was down-regulated.These results indicated that miR-140-3p could be delivered by exosomes secreted by BEAS-2B cells.The exosomes extracted from BEAS-2B cells were co-cultured with nonsmall cell lung cancer cells.The q RT-PCR results demonstrated that the expression level of miR-140-3p was up-regulated in the co-culture group.The MTS proliferation test results showed that the cell proliferation ability of the co-culture group was inhibited,and the apoptosis test results showed that the apoptosis ability of the co-culture group was promoted.These results suggest that BEAS-2B cell-derived exosomes can induce functional changes in NSCLC cells.After transfection of miR-140-3p mimics and miR-140-3p mimics-NC into BEAS-2B cells,the exosomes were extracted,and the q RT-PCR results showed that the expression of miR-140-3p in BEAS-2B cells and exosomes was up-regulated.The transfected exosomes were then co-cultured with A549 cells and SKMES-1 cells,and the expression of EGLN3 gene was down-regulated according to the results of q RT-PCR and Western Blot.MTS proliferation assay showed that the cell proliferation ability was inhibited in the exosome miR-140-3p mimics co-culture group.The results of apoptosis assay showed that the apoptosis ability of the cells in the exosomal miR-140-3p mimics co-culture group was promoted.These results indicated that BEAS-2B cell-derived exosomal miR-140-3p could be absorbed into NSCLC cells and cause changes in cell proliferation and apoptosis.Western Blot results demonstrated that the activity of MAPK signaling pathway was activated after co-culture with exosome miR-140-3p mimics.Conclusions: Part I: miR-140-3p expression was down-regulated in serum exosomes of NSCLC patients,tissues of NSCLC patients and NSCLC cells in TCGA database.Cell proliferation of NSCLC cells was inhibited after up-regulation of miR-140-3p expression,and cell apoptosis was promoted.Part II: miR-140-3p inhibited the expression of EGLN3,and the up-regulation of EGLN3 gene expression promoted cell proliferation and inhibited cell apoptosis of NSCLC cells.Overexpression of EGLN3 partially reversed the effects of miR-140-3p overexpression on the proliferation and apoptosis of NSCLC cells.Part III:The up-regulation of exosomal miR-140-3p inhibits the proliferation of non-small cell lung cancer cells and promotes cell apoptosis.Exosomal miR-140-3p/EGLN3/MAPK signaling axis regulates NSCLC progression. |
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