| Research Background and Objectives: Lung cancer is a disease involving multiple genes and complex pathways,and is currently the most common cause of cancer-related death worldwide.Cell morphology and biological characteristics divide lung cancer into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Due to the high incidence and mortality of lung cancer,it is of great significance to find new therapeutic targets and biodiagnostic markers for the clinical diagnosis and treatment of lung cancer.Noncoding RNAs(ncRNAs)have been shown to play an important role in the development of lung cancer.Micro RNAs(miRNAs)are non-coding eukaryotic endogenous small molecule single-stranded RNAs about 20 ～ 24 nt long,which are widely distributed in animals and plants.They are of great significance for biological gene regulatory networks.Mi RNAs are able to pass the 3’ untranslated region(3’UTR)with messenger RNA(m RNA)Binding,which in turn inhibits its translation or directly induces its degradation,thereby regulating gene expression and performing biological functions.A large number of experimental studies have shown that abnormal expression of miRNA is closely related to malignant tumors,and miRNA can also be used as a marker for some tumors to predict the progression and prognosis of tumors including NSCLC.After reviewing the literature,our team found that m-3680-3p has not been studied in non-small cell lung cancer.In this study,we attempted to explore m-3680-3p after overexpression and knockdown of m-3680-3p in non-small cell lung cancer The effects on proliferation,migration,invasion,cell cycle and apoptosis of non-small cell lung cancer,and explore its functional mechanism,so as to provide a basis and target for treatment.Methods: 1.Effect of miR-3680-3p on the proliferation of non-small cell lung cancer cells(1)RT-qPCR detects the expression of miR-3680-3p in the cell line of NSCLC.(2)Mi R-3680-3p was detected in A549,H by CCK-8 experiment and colony formation experiment 1299 Cell proliferation capacity of overexpressed and knocked out cells and negative control(NC)cells,analysis Effect of miR-3680-3p on cell proliferative capacity.(4)Flow cytometry detected the overexpression of miR-3680-3p in A549 and H1299 and reduce apoptosis and cycle changes between knockout cells and NC cells,and analyze the effect of miR-3680-3p on apoptosis ability.2.Effect of miR-3680-3p on migration and invasion of non-small cell lung cancer cells Transwell experiment and cell scratch experiment detected miR-3680-3p overexpression and knockdown in A549 and H1299 The migration and invasion capacity of cells and NC cells were analyzed,and the effect of miR-3680-3p on cell migration ability was analyzed.3.Screening,prediction,and verification of miR-3680-3p downstream target genes(1)miRDB database and Targetscan database predict miR-3680-3 p downstream target gene.(2)RT-PCR detected target genes in A549 and H1299 Overexpression of miR-3680-3p and knockdown of expression changes between miR-3680-3p cells and NC cells were identified to identify downstream target genes.(3)Luciferase experiments verified the binding relationship between miR-3680-3p and downstream target gene CCR6.(4)WB experiments detected the target gene CCR6 in A549 and H1299 Expression changes between medium-overexpression miR-3680-3p and knock-out miR-3680-3p cells and NC cells,Further validation of downstream target genes.4.To explore the mechanism by which miR-3680-3p regulates the proliferation of non-small cell lung cancer cells through CCR6(1)After knocking down miR-3680-3p in A549 and H1299 cells,knockdown CCR6 and pass CCK-8 The experiment and colony formation experiment detected the cell proliferation capacity and the effect of miR-3680-3p on cell proliferation ability by regulating CCR6 was analyzed.(2)After knocking down miR-3680-3p in A549 and H1299 cells,knockdown CCR6 and pass Flow cytometry detected apoptosis and cycle changes of cells,and miR-3680-3p was analyzed for apoptosis by regulating CCR6 Impact.5.To explore the mechanism of miR-3680-3p regulating migration and invasion of non-small cell lung cancer cells through CCR6 After knocking down miR-3680-3p in A549 and H1299 cells,CCR6 was knocked down and passed the transwell experiment Cell scratch assay detected cell migration and invasion ability,and miR-3680-3p was analyzed by regulating CCR6 pairs of cells Impact of migration,invasion capabilities.Experimental results: 1.Effect of miR-3680-3p on the proliferation of non-small cell lung cancer cells(1)Compared with 16 HBE,the expression of miR-3680-3p was reduced in the cell line of NSCLC(P <0.05).(2)CCK-8 experiment and colony formation experiment were found to be overexpressed in A549 and H1299 miR-3680-3p inhibits cell proliferation,while knockdown miR-3680-3p enhances cell proliferation(P<0.05).)。(3)Flow cytometry showed that miR-3680-3p was overexpressed in A549 and H 1299 Promote apoptosis and inhibit the cell cycle;Knockdown miR-3680-3p inhibits apoptosis and accelerates cell cycle(P<0.05).2.Effect of miR-3680-3p on migration and invasion of non-small cell lung cancer cells Transwell experiment and cell scratch assay found overexpression of miR-3680-3p in A549 and H1299 Inhibit cell migration invasion;Knockdown miR-3680-3p promoted cell migration and invasion(P<0.05).3.Screening,prediction,and verification of miR-3680-3p downstream target genes(1)After screening and intersection,the miRDB database and Targetscan database selected the top ten genes as the target genes to be determined,and the RT-PCR results found that PABPC5 was found and CCR6 expression was significantly correlated with miR-3680-3p.Among them,the difference in CCR6 expression was more significant(P<0.05).(2)Luciferase experimental detection found that miR-3680-3p directly bound to CCR6(P<0.05).(3)WB experimental detection found that the expression of CCR6 protein was regulated by miR-3680-3p(P<0.05).4.To explore the mechanism by which miR-3680-3p regulates the proliferation of non-small cell lung cancer cells through CCR6(1)CCK-8 experiment and colony formation experiment found that knocking down CCR6 can partially reverse the knockdown of miR-3680-3p pairs and enhanced cell proliferation capacity(P<0.05).(2)Flow cytometry showed that CCR6 was knocked out in A549 and H1299 It can partially reverse the inhibition of apoptosis and the promotion of the cell cycle by knocking down miR-3680-3p(P<0.05).5.To explore the mechanism of miR-3680-3p regulating migration and invasion of non-small cell lung cancer cells through CCR6 Transwell experiment,cell scratch assay found that CCR6 can be partially knocked out in A549,H1299 Reverse knockdown miR-3680-3p on cell migration and invasion(P<0.05).Conclusion: This study shows for the first time that miR-3680-3p inhibits the proliferation,invasion,migration,and cell cycle of NSCLC cells by downregulating the expression of CCR6,and promoting apoptosis.Notably,miR-3680-3p may also target other genes based on the prediction of the results.Mi R-3680-3p may serve as a biomarker and therapeutic target for NSCLC therapy. |
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