Preliminary Study On The Role And Mechanism Of Lung Cancer Stem Cells Derived Exosomes Containing MiR-210-3p In The Promotion Of Non Small Cell Lung Cancer Invasion And Metastasis

Objective:Lung cancer is the most common malignant tumor,and its metastasis is one of the primary causes of death of patients with lung cancer.Thus,inhibiting lung cancer metastasis and prolonging the survival of patients with lung cancer are key scientific issues currently addressed by the research scientific community.Lung cancer metastasis is a complex process involving multigene regulation,and studying the cellular and signal transduction molecular networks involved in metastasis is essential.Previously,studies showed that tumor-associated fibroblasts,M2tumor-associated macrophages,granulocyte-like myeloid-derived suppressor cells,mast cells,etc.are closely involved in the regulation of metastatic phenotypes in liver cancer,colon cancer,breast cancer and melanoma.Lung cancer stem cells are a small group of heterogeneous cells found in lung cancer cell lines and lung cancer tissues.They are characterized by their self-renewal ability,high expression of stem markers,enhanced metastatic potential,and resistance to radiotherapy and chemotherapy.Exosomes are extracellular vesicles with a diameter of 30–150 nm wrapped in a lipid bimolecular membrane released by living cells.Exosomes mediate cell–cell communication,including regulation of the phenotype of the transfer recipient cell.Our recent studies found that exosomes derived from glioma stem cells can increase glioma cells invasion potential.Interestingly,regulation of lung cancer metastasis by exosomes released by lung cancer stem cells has not been reported thus far.Microribonucleic acid-210-3p(Micro RNA-210-3p,miR-210-3p)is highly expressed in a variety of malignant tumors and is involved in the regulation of tumor cell invasion and metastasis as well as in other cancer associated processes.Reports have shown that miR-210-3p can promote epithelial-mesenchymal transition(EMT)in human colon and ovarian cancer cells by regulating metastasis-related proteins such as Epithelial cadherin(E-cadherin)and neural cadherin(N-cadherin)to promote tumor cell migration and invasion.Furthermore,recent studies have shown that serum miR-210-3p levels in patients with lung cancer are correlated with the number of lung cancer lymph node metastases,and a previous study from our group discovered that exosomes derived from lung cancer stem cells contain miR-210-3p.Nevertheless,participation of exosomes originating miR-210-3p in the regulation of lung cancer metastasis as well as the possible molecular mechanisms involved need to be further elucidated.In the current study,we further elaborated on the relationship between lung cancer stem cell-derived exosomes and lung cancer metastasis by enriching lung cancer stem cells derived from lung cancer cells,isolating and purifying the exosomes released by lung cancer stem cells,and cocultivating these exosomes with lung cancer cells.We aimed to clarify whether lung cancer stem cell-derived exosomes promote lung cancer metastasis by miR-210-3p mediation and to preliminarily explore the underlying molecular mechanisms of lung cancer metastasis.Materials and Methods:1.Enrichment of lung cancer stem cells and identification of stem characteristics: Serum-free Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12(DMEM/F12)medium supplemented with basic fibroblast growth factor(b FGF)and recombinant human epidermal growth factor(rh EGF)was used to maintain suspension culture method to enrich lung cancer stem cells.We used in vitro tumor ball formation assay and in vivo tumor formation assay to detect lung cancer stem cell self-renewal potential.A transwell assay was used to assess the migration and invasion ability of lung cancer stem cells.Real-time quantitative polymerase chain reaction(RT-q PCR)and Western Blot assays were used to evaluate the expression of lung cancer stem cell stemness genes and metastasis-related proteins.2.The effect of lung cancer stem cell exosomes on the migration and invasion of non-small cell lung cancer: Ultra-high-speed centrifugation was used to separate and purify exosomes derived from lung cancer stem cells.The morphology and structure of exosomes was observed via transmission electron microscopy.We detected the diameter of exosomes using a particle size analyzer.Western blot was used to detect the expression of characteristic exosomes proteins.After coculture of lung cancer stem cell-derived exosomes and lung cancer cells,lung cancer cells were collected for MTT test,cell scratch,and transwell assays to evaluate their proliferation,migration,and invasion,respectively.Western blot was used to detect the expression of metastasis-related proteins on cocultured lung cancer cells.3.The effect of lung cancer stem cells derived exosomes containing miR-210-3p on the migration and invasion of non-small cell lung cancer:RT-q PCR was used to detect the expression level of miR-210-3p in lung cancer stem cells and lung cancer cells.After transfecting miR-210-3p analog(Mi R-210-3p-mimic)and miR-210-3p inhibitor(Mi R-210-3p-inhibitor)in lung cancer stem cells,ultrahigh-speed centrifugation was used to separate and purify the transfected lung cancer stem cell-derived exosomes,and RT-q PCR assay was used to detect the miR-210-3p levels of lung cancer stem cell exosomes.The aforementioned exosomes were cocultured with lung cancer cells,and the lung cancer cells were collected.The cell scratch and transwell invasion assays were then used to evaluate the changes in lung cancer cell migration and invasion ability.The expression of proteins related to cocultured lung cancer cell metastasis was detected via Western blot assay.4.The effect of miR-210-3p downstream target gene BTG2 on the migration and invasion of non-small cell lung cancer: The Wilcox test was used to analyze the expression differences of miR-210-3p in lung cancer and adjacent tissues in the human cancer genome atlas(TCGA)database.Cell FISH experiments were performed to detect the expression and localization of miR-210-3p in lung cancer cells;animal experiments were performed to assess the effects of miR-210-3p on the proliferation of lung cancer-bearing mice and induction of lung metastases in mice.The miR-210-3p downstream target genes were found in the miRTar Base,miRDB,target Scan databases.Transiently transfected miR-210-3p-mimic was used to upregulate the expression of miR-210-3p in lung cancer cells,and RT-q PCR was employed to detect and screen miR-210-3p downstream target genes with significant differential expression.A dual luciferase reporter gene experiment was used to evaluate the regulation of miR-210-3p on downstream target gene B-cell translocation gene 2(BTG2).After upregulating or downregulating the expression of lung cancer cells BGT2,plate clone formation,cell counting kit-8(CCK-8),cell scratch,and transwell assays were used to detect changes in the proliferation,migration,and invasion of lung cancer cells,respectively.A Western blot assay was used to detect the expression levels of metastasis-related molecules in lung cancer cells.Results:1.Lung cancer stem cells have typical Stem cell characteristics:Lung cancer stem cells enriched in serum-free DMEM/F12 medium supplemented with b FGF and rh EGF suspension cultures grow in suspension spheres.After identification of the stem phenotype,lung cancer stem cells have enhanced tumor sphere formation ability and tumorigenicity in nude mice.Lung cancer stem cells highly express stem genes(ALDH1,CD133,Nanog,and Sox2),possess enhanced migration and invasion capabilities while they highly express metastasis-related molecular proteins N-cadherin,vimentin(Vimentin),matrix metalloproteinase 9(Matrix metallopeptidase-9 [MMP-9]),and matrix metallopeptidase-1(Matrix metallopeptidase-1 [MMP-1]).2.Lung cancer stem cells derived exosomes have typical exosomes characteristics : Exosomes derived from lung cancer stem cells are “disk-shaped”vesicles with a diameter of approximately 30–150 nm and express specific markers CD63,CD81,and tumor susceptibility gene 101(tumor TSG101).3.The experimental results of lung cancer stem cell derived exosomes promoting non-small cell lung cancer migration and invasion: After cocultured with different concentrations of lung cancer stem cell-derived exosomes,the proliferation ability of lung cancer cells is enhanced,as well as their migration and invasion capabilities.Moreover,lung cancer cells metastasis-related proteins N-cadherin,Vimentin,MMP-9,and MMP-1 expression levels are upregulated,whereas those of E-cadherin is downregulated.4.The experimental results of lung cancer stem cells derived exosomes containing miR-210-3p promoting non-small cell lung cancer migration and invasion:The expression levels of miR-210-3p in lung cancer stem cells were higher than those of lung cancer cells.After miR-210-3p-mimic was transfected in lung cancer stem cells,their exosomes were isolated,purified,and cocultured with lung cancer cells.Compared with the CON group,lung cancer cell proliferation as well as migration and invasion potentials were enhanced,and lung cancer cell N-cadherin,Vimentin,MMP-9,and MMP-1 expression levels were upregulated,whereas those of E-cadherin expression levels were downregulated.After miR-210-3p-inhibitor transfection of lung cancer stem cells,the isolated and purified lung cancer stem cell exosomes were cocultured with lung cancer cells.Compared with the miR-inhibitor-NC group,lung cancer cell proliferation,migration,and invasion were decreased,and lung cancer cells N-cadherin,Vimentin,MMP-9,and MMP-1expression level were downregulated,whereas those of E-cadherin was upregulated.5.The experimental results of the miR-210-3p downstream target gene BTG2 inhibiting non-small cell lung cancer migration and invasion: Wilcox test analysis of miR-210-3p expression in lung cancer adjacent tissues in the TCGA database showed that the expression levels of miR-210-3p in lung cancer tissues were higher than those of adjacent tissues and that miR-210-3p expression was primarily located in the cytoplasm of lung cancer cells.Furthermore,miR-210-3p can promote xenografts growth and induction of lung metastases in lung cancer-bearing mice.Using database target gene prediction,it was observed that BTG2 is one of the downstream target genes of miR-210-3p.Using dual luciferase gene assay,it was verified that miR-210-3p can directly target and bind to BTG2.After inhibiting lung cancer cell BTG2,lung cancer cell proliferation potential migration and invasion were enhanced,and lung cancer cell E-cadherin expression levels were decreased,whereas those of N-cadherin,Vimentin,MMP-9,and MMP-1 were upregulated.Finally,lung cancer cells overexpressing BTG2 present decreased proliferation,migration,and invasion,and lung cancer cells E-cadherin expression levels were upregulated,whereas those of N-cadherin,Vimentin,MMP-9,and MMP-1 were downregulated.Conclusions:1.Serum-free DMEM/F12 medium supplemented with b FGF and rh EGF suspension culture can enrich lung cancer stem cells derived from lung cancer cells.After the stem phenotype is identified,it has stem cell phenotype characteristics.2.Exosomes derived from lung cancer stem cells have a typical “disk-shaped”vesicle structure with a diameter of approximately 30–150 nm and express exosomal-specific markers CD63,CD81,and TSG101.3.Exosomes derived from lung cancer stem cells can promote proliferation,migration.and invasion of lung cancer cells in vitro and upregulate the expression levels of N-cadherin,Vimentin,MMP-9,and MMP-1 proteins in lung cancer cells;however,they downregulate the expression levels of E-cadherin.4.Exosomes derived from lung cancer stem cells can deliver miR-210-3p to lung cancer cells;promote lung cancer cell migration and invasion;upregulate lung cancer cell N-cadherin,Vimentin,MMP-9,and MMP-1 protein expression levels;and downregulate E-cadherin protein expression levels.5.The miR-210-3p can target BTG2;after downregulating lung cancer cell BTG2,it can promote lung cancer cell migration and invasion;upregulate N-cadherin,Vimentin,MMP-9,and MMP-1 protein expression levels;and downregulate E-cadherin protein expression levels.Lung cancer cells overexpressing BTG2 show decreased migration and invasion;downregulation of the expression levels of N-cadherin,Vimentin,MMP-9,and MMP-1;and upregulation of the expression levels of E-cadherin.