| ObjectiveLung cancer has the highest incidence and mortality in the world.Due to its complex biological behavior and pathogenesis,the diagnosis and treatment are not timely,and most patients have local progression or metastasis at the initial diagnosis.Therefore,it is important to study the pathogenesis of lung cancer.cancer-associated fibroblasts(CAFs),as the major cell groups in Tumor microenvironment,participate in the regulation of tumor microenvironment by secreting collagen,cytokines,and exosomes.As a kind of small extracellular vesicles,exosomes transport proteins,nucleic acids and other bioactive substances from donor cells to recipient cells to participate in the occurrence and progression of malignant tumors.Elucidate the relationship and molecular mechanism between tumor-associated fibroblast derived exosomes and lung malignancies,which is conducive to further study of the role of tumor microenvironment in the occurrence and progression of tumors,and provide theoretical basis for molecular immune regulation in the treatment of lung cancer.Methods(1)Primary lung cancer-associated fibroblasts(CAFs)and normal fibroblasts(NFs)were isolated from surgically resected lung cancer tissues and adjacent tissues by tissue block adherence method and enzymatic hydrolysis method.Cafs and NFS were routinely cultured in culture medium,and cell morphology was observed under microscope.Western blot and immunofluorescence were used to detect the markers of Fibroblast Activation Protein(FAP)and Smooth muscle actin alpha(SMA alpha).α-SMA),indicating that CAFs and NFs were successfully isolated.(2)The exosomes in the supernatant of lung cancer-associated fibroblasts(CAFs)and normal fibroblasts(NFs)were collected,and the structural characteristics of exosomes were observed by transmission electron microscopy.The exosome marker proteins CD9 and CD63 were detected by Western blot,and then the isolated exosomes were sequenced.The effects of the supernatants on the proliferation of lung cancer cells were detected by CCK-8 assay and plate cloning assay.(3)The expression level of miR-522-3p in four lung cancer cell lines(PC-9,A549,H1975,H1299)and a normal human lung epithelial cell line(BEAS-2B)was detected by real-time PCR according to the sequencing results.(4)The expression level of miR-522-3p in exosomes of fibroblasts transfected with miR-522-3p inhibitor was detected by fluorescence quantitative PCR.The expression levels of miR-522-3p in H1299 cells transfected with CAFs inhibitor-exo exosomes and miR-522-3p inhibitor-exo exosomes were further detected by fluorescence quantitative PCR.(5)The effects of exosome-derived miR-522-3p on the proliferation and cloning ability of lung cancer H1299 cells were detected by CCK-8 method and plate cloning experiment,and then the effects of exosome-derived miR-522-3p on the migration and invasion ability of lung cancer H1299 cells were detected by Transwell.(6)Predicting the binding sites and capabilities between miR-522-3p and RNA-binding domain containing protein 2(ZRANB2)based on online biological information of TargetScanHuman 7.2 software.The binding activity between miR-522-3p and ZRANB2 was then detected using a dual fluorescein reporting assay.(7)The mRNA and protein expressions of ZRANB2 and the vector overexpressing ZRANB2(pcDNA-ZRANB2)were detected by fluorescence quantitative PCR and Western blot,respectively,to verify that miR-522-3p can regulate the expression of ZRANB2 in lung cancer cells.Furthermore,CCK-8 method and Transwell assay were used to detect the regulation of exosome-derived miR-522-3p on the proliferation and migration of lung cancer cells by ZranB2-overexpressed vector(pcDNA-ZRANB2).Results(1)The morphology of cultured cells was long spindle type under phase contrast microscope,which was consistent with the characteristics of fibroblasts.The extracted CAFs expressed α-SMA and FAP by immunofluorescence detection and Western blot,but the expression of α-SMA and FAP in NFs cells was weak,which was consistent with the characteristics of CAFs and NFs,indicating that the extracted CAFs and NFs could be used for subsequent research.(2)The results of exosome morphology using transmission electron microscopy showed that the particle size of the extracted exosomes was between 30 and 100nm,with a double-membrane structure and a "cup" shape,which was consistent with the structural characteristics of exosomes.Western blot detection showed that the extracted exosomes expressed CD9 and CD63,which was consistent with the biological characteristics of exosomes.Compared with NFs,a total of 1723 mirnas were down-regulated and 1485 mirnas were up-regulated in CAFs.These results suggest that these mirnas may be related to the occurrence and development of lung cancer.In addition,CCK-8 assay and plate cloning assay showed that the proliferation ability of lung cancer cells treated with NFs was not significantly changed(P<0.05),while the proliferation ability of CAFs group was significantly increased(P<0.01),indicating that CAFs could significantly promote the proliferation of lung cancer cells(A549 and H1299).(3)miR-522-3p was highly expressed in CAFs exosomes(P<0.01).The expression level of miR-522-3p in four lung cancer cell lines(PC-9 cells,A549 cells,H1975 cells and H1299 cells)was higher than that in normal epithelial cell line(BEAS-2B)(P<0.01).(4)Transfection with the inhibitor-exo of miR-522-3p significantly inhibited the expression of miR-522-3p in tumor fibroblasts(P<0.01).The expression level of miR-522-3p in the cells transfected with CAFs inhibitors-Exo exosomes was significantly increased(P<0.01),and the expression level of miR-522-3p in the cells transfected with miR-522-3p inhibitors-Exo exosomes was significantly decreased(P<0.01).(5)CCK-8 assay(P<0.05)and colony formation assay(P<0.01)showed that transfection of miR-522-3p inhibitors-exo significantly blocked the promoting effect of CAFs inhibitors-exo on lung cancer cell clone formation ability.Transwell assay showed that transfection of miR-522-3p inhibitor-exo significantly blocked the promotion effect of CAFs inhibitor-exo on lung cancer cell migration and invasion(P<0.05).(6)The results of dual-luciferase reporter assay showed that the relative luciferase activity was inhibited after transfection of miR-522-3p mimics in ZRANB2 wild type cells,but there was no significant change in luciferase activity in ZRANB2 mutant cells(P<0.01).(7)Transfection of miR-522-3p inhibitor-exo significantly blocked the inhibitory effect of CAFs inhibitor-exo on ZRANB2 mRNA and protein in lung cancer cells(P<0.01).The expression levels of ZRANB2 mRNA and protein were significantly increased in pcDNA-ZRANB2 transfected lung cancer cells.CCK-8 assay and Transwell assay showed that overexpression of ZRANB2(pcDNA-ZRANB2)significantly blocked the proliferation(P<0.05)and migration(P<0.01)of CAFs inhibitor-exo in lung cancer cells.ConclusionLung cancer-associated fibroblasts(CAFs)can promote the proliferation and migration of lung cancer cells,and the expression level of miR-522-3p in CAFs-derived exosomes is up-regulated,which can act as oncogenes and promote proliferation,invasion and metastasis of lung cancer cells.Further in-depth study of the molecular mechanism shows that miR-522-3p can act as a LncRNA.It can promote the proliferation,invasion and migration of lung cancer cells by inhibiting the expression of RNA-binding domain containing protein 2(ZRANB2).This study is expected to clarify the role of miR-522-3p in the occurrence and development of lung cancer,and to explore its molecular mechanism.The research results provide more basis for further exploring the role and mechanism of CAFs in lung cancer,and lay a good foundation for the diagnosis and treatment of lung cancer. |
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