The Regulatory Effects And The Mechanisms Of MiR-885-3p/Aurora-A On Chemosensitivity Of Human Lung Adenocarcinoma Cells

Background and ObjectiveLung cancer is the most malignant tumor with morbidity and mortality in China in recent years,with an increase rate of 3-5%per year,which accounts for 22.7%of malignant tumor classification.Lung cancer is divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),which covers squamous cell carcinoma,adenocarcinoma,large cell carcinoma,etc,accounting for 70-80%of lung cancer.Since it is difficult to diagnose the disease at the onset,more than 2/3 of the patients have local or distant metastasis,surgical opportunities are limited.So the treatment is more dependent on chemotherapy,radiotherapy,targeted therapy and biological immunotherapy.Although molecular targeted drugs are more effective in lung cancer patients,chemotherapy is still the main medical treatment of lung adenocarcinoma.Drug tolerance is the key factor leading to chemotherapy failure.Studying the molecular mechanism of chemotherapy resistance and the potential impact on the patient,the mechanisms of the occurrence of chemotherapy resistance,detection and reversal methods of the chemotherapy resistance can provide new ideas for the clinical treatment of lung adenocarcinoma and may have important implications.Aurora-A kinase is a member of the serine/threonine family of proteins involved in the regulation of cell mitosis in many processes,such as centrosome mutagenesis and isolation,spindle assembly and maintenance,chromosome pairing and so on.The expression and activation of Aurora-A mainly occur in the course of G2 to M phase,and the subcellular localization of Aurora-A changes during the cell cycle.Aurora-A gene expression and protein levels increase abnormally in lung adenocarcinoma,liver cancer,colon cancer,breast cancer and other malignant tumors,our laboratory has confirmed that Aurora-A kinase can regulate NF-kB/miRNA-21/PTEN signal transduction pathway to promote primary hepatocarcinoma cells to develop drug resistance.In early basic research,we established the human lung adenocarcinoma drug resistant cell lines SPCA1/DTX by gradually increasing the concentration of docetaxel.The established SPCA1/DTX can tolerate a certain concentration of docetaxel,and the expression of Aurora-A in SPCA1/DTX-resistant strains was significantly higher than that in SPCA1 parental strains.We speculated that the expression of Aurora-A may also play an important role in drug-resistant lung adenocarcinoma.The expression level of Aurora-A is regulated by many factors.The abnormal expression of epigenetic regulation is one of the causes of uncontrolled expression.MicroRNA(miRNA)inhibits target gene translation at post-transcriptional level,achieving negative expression of genes,regulates different molecular signaling pathways,and participates in the regulation of cell growth,differentiation,apoptosis,metastasis and the malignant phenotype of tumor cells.We predicted that miRNA-885-3p might be a potential upstream regulatory gene for Aurora-A.MiR-885-3p,which is a 3'-terminal splice of miR-885,locates on the short arm of chromosome 3.Studies have shown that it is involved in lung adenocarcinoma,colon cancer,squamous cell carcinoma and other biological behavior,in which miR-885-3p can regulate the proliferation of squamous cell carcinoma and apoptosis,and may affect the chemosensitivity of squamous cell carcinoma to platinum.In the SPCA1/DTX resistant strains,miRNA-885-3p was down-regulated compared with the parental strains.We speculated that miRNA-885-3p could affect the chemosensitivity of lung adenocarcinoma by regulating Aurora-A expression.Therefore,the study group constructed a model of lung adenocarcinoma dr?g-resistant strains and explored the mechanism of Aurora-A and miR-885-3p involving in the regulation of the docetaxel resistance of human lung adenocarcinoma cells from the process of cell proliferation,apoptosis,cell cycle,etc.This study can help to reveal the possible mechanism of chemoresistance in lung adenocarcinoma and provide a new access to the clinical treatment of lung adenocarcinoma.Materials and Methods1.MTT assay was used to determine the sensitivity of Human lung adenocarcinoma SPC-A1 cells and docetaxel-resistant SPC-A1/DTX cells to docetaxel;Clone formation assay was used to determine the difference of proliferation;Aurora-A and miR-885-3p expression between the two groups was analyzed by Real time PCR.2.Aurora-A gene was transfected into SPC-A1/DTX and H1299/DTX cells by shRNA/Aurora or overexpression plasmid.The sensitivity of SPC-A1 cells to docetaxel was determined by MTT assay.The cell proliferation was evaluated by clone formation assay.Cell cycle and apoptotic rate were analyzed by flow cytometry.Western blot was used to detect the proliferation and apoptosis-related protein.3.Using two different miRNA target gene online analysis software:?TargetScanHuman 6.0(http://www.targetscan.org/),? Microrna.org(http://www.microrna.org/microrna/home.do).Bioinformatics analysis of miR-885-3p target genes was performed and the results were analyzed by double luciferase Reporter gene experimental validation.4.The miR-885-3p was selected as the upstream regulatory gene of Aurora-A.The miR-885-3p-mimics and miR-885-3p-inhibitor were transferrd into Human lung adenocarcinoma SPC-A1?SPC-A1/DTX?H1299?H1299/DTX cells,respectively,regulating the expression of miR-885-3p.MTT assay was used to determine the sensitivity of the cells to docetaxel.The cell proliferation were analyzed by clone formation assay.Cell cycle and apoptosis rates were detected by flow cytometry.Western blot was used to detect the proliferation and apoptosis-related proteins.5.The miR-885-3p mimics and inhibitor were transfected into human lung adenocarcinoma cells SPC-A1/DTX,SPC-A1,H1299/DTX,H1299 cells,respectively.Then each group of cells were transfected with Aurora-A short hairpin RNA(shRNA/Aurora)or overexpression of Aurora-A plasmid in the same time.The sensitivity of the cells to docetaxel was detected through MTT assay,Clone formation assay was used to determine the proliferation ability of the cells,and the apoptotic rate and cell cycle was detected by flow cytometry.The western blot was used to detect the proliferation and apoptosis-related proteins.6.The miR-885-3p mimics and inhibitor were transfected into human lung adenocarcinoma cells SPC-A1/DTX,SPC-A1,H1299/DTX,H1299 cells,respectively.Aurora-A gene was transfected into SPC-A1/DTX and H1299/DTX cells by shRNA/Aurora or overexpression plasmid.Cell migration ability was detected by cell scratch test and transwell assay,and EMT-related protein was detected by western blot.Results1.Compared with the parental SPC-A1 cells,SPC-A1/DTX cells exhibited higher dr?g resistance to docetaxel than those of parental SPC-A1 cells(p<0.05).The level of Aurora-A in SPC-A1/DTX cells significantly increased compared to SPC-A1 cells parental strain,miR-885-3p expression level was significantly reduced.2.After Aurora-A was artificially down-regulated(p<0.05),the sensitivity of the cells to docetaxel was increased(p<0.05),and the proliferation ability of the cells was decreased in SPC-A1/DTX and H1299/DTX cells(p<0.05),the early cell apoptosis was significantly increased(p<0.05)and the cell cycle was blocked to G2/M phase(p<0.05).The expression of apoptotic related protein a-caspase3,Bax increased,Bcl2 expression decreased,and NF-kB expression decreased(p<0.05),The.The sensitivity of the cells to docetaxel was decreased and the proliferation ability of the SPC-Aland H1299 cells in vitro was increased(p<0.05)when the expression of Aurora-A was up-regulated(p<0.05).3.The up-regulation of miR-885-3p expression in the two strains of drug-resistant cells significantly increased the sensitivity of docetaxel(p<0.05)and the cell proliferation ability was significantly decreased in vitro(p<0.05).The expression of apoptosis-related protein A-caspase-3,Bax,Bcl2 and NF-kB decreased,whereas in SPC-A1 and H1299/DTX cells,the sensitivity of the cells to docetaxel was decreased and the proliferation of SPC-A1 cells was increased in vitro(p<0.05)when the expression of miR-885-3p was down-regulated.4.The expression of miR-885-3p was negatively correlated with the expression level of Aurora-A,SPC-A1/DTX and H1299(p<0.05).MiR-885-3p is an upstream regulatory gene of Aurora-A due to luciferase reporter assay.In SPCA1/DTX and H1299/DTX cells,the expression of miR-885-3p was abolished and the expression of Aurora-A was increased abnormally.Increasing the expression of miR-885-3p could reverse the influence of Aurora-A on the sensitivity of chemotherapy,the proliferationof cells,the early apoptosis rate,the cell cycle and the expression of apoptosis-related proteins.5.In the SPC-A1/DTX and H1299/DTX cells,when the expression of miR-885-3p was abolished or the expression of Aurora-A increased,the migration of drug-resistant cells increased,E-cadherin decreased,Vimentin increased,EMT level was significantly increased(p<0.05).The up-regulation of miR-885-3p expression or the down-regulation of Aurora-A expression decreased the cell migration and metastasis ability(p<0.05),the expression of EMT-related protein E-cadherin increased,Vimentin decreased and the EMT level decreased.Conclusions1.Overexpression of Aurora-A can induce docetaxel-resistant chemotherapy in lung adenocarcinoma cells in vitro and in vivo significantly,accompanied by cell proliferation hyperresponsiveness,apoptosis reduction,and significant changes in cell cycle distribution.2.MiR-885-3p can increase the docetaxel chemosensitivity in lung adenocarcinoma cells in vitro and in vivo,accompanied by cell proliferation arrest,increased apoptosis,and significant changes in cell cycle distribution.3.The sensitizing effect of MiR-885-3p to chemotherapy of lung adenocarcinoma cells may be related to the negative regulation of Aurora-A,promoting apoptosis and inducing the expression of apoptosis-related proteins.4.The change of EMT caused by miR-885-3p expression may be an important way to regulate the chemosensitivity of drug-resistant lung adenocarcinoma cells.5.The expression of miR-885-3p/Aurora-A in human lung adenocarcinoma and its relationship with chemosensitivity were first probed,and the expression of miR-885-3p in lung adenocarcinoma may be one of the reasons of cell EMT changes.It is suggested that miR-885-3p may be a new molecular target for reversing the chemoresistance of lung adenocarcinoma.