MicroRNA181a-inhibitor Reverses Epithelial To Mesenchymal Transition By Targeting PTEN In Drug-resistant Lung Adenocarcinoma Cells

Part ⅠThe resistance properties of the A549/PTX and A549/DDP with the treatment of the first-line chemotherapy drug (paclitaxel/cisplatin) associated with the EMT processObjective1. The development of resistance is a natural biological process with the first line of chemotherapy drugs to treat lung adenocarcinoma.2. Evaluation of lung adenocarcinoma cancer drug resistance.3. To establish a multidrug resistance cell line of human lung adenocarcinoma.4. Research the characteristics of lung resistant adenocarcinoma cell lines. Preliminarily, the relationship between EMT and the ability of invasion and migration.Methods1. The first-line chemotherapy drugs (paclitaxel and cisplatin) to treat lung adenocarcinoma cells induced resistance.Gradually increase the drug concentration treatment of lung adenocarcinoma A549 cells and lasted longer about 12 months. Each chemotherapy cycle of survival cells were about 70% with pancreatic enzyme digest cultivate together with the increased concentrations of paclitaxel and cisplatin.Until reach resistance target chemotherapy concentration:paclitaxel (0.23μmol/ml) and cisplatin (3.3 μmol/ml).Such cells were cultured in high concentrations of drugs till after the state stability about 3 months.2. MTT method to evaluate the lung adenocarcinoma cells the proliferation ability.(1) PTX (Paclitaxel) different concentrations of (0μmol·-1,0.2μmol·-1,0.4μmol·-1, O.8μmol·-1,1.6μmol·-1,3.2μmol·-1) in 24h,48h,72 h at different times of human lung adenocarcinoma A549 cancer cells and A549/PTX proliferation inhibition..(2) The different concentrations of cisplatin DDP (0μmol·-1,4μmol·-1 8μmol·-1, 16μmol·-1,32μmol·-1,64μmol·-1) effect on lung adenocarcinoma A549 and A549/DDP at 24h,48h,72 h. In order to make clear its resistant propertie, dose-effect relationship and resistant index were adopted.3. wound healing, migration and invasion assays were performed to assess the migratory potential of A549/PTX and A549/DDP cells in relation to A549 cells(1) Wound healing was observed under an inverted microscope and imaged at the appropriate fields to calculate healing percentages.The results show that significantly increased numbers of cells migrated across the "wound" in a scratch assay. Using Transwell Chambers for cell migration experiment and reorganization of the basement membrane experiments to detect lung adenocarcinoma A549 cancer cells and A549/PTX and A549/DDP in vitro migration and invasion ability.4.Microscopy at ×200 magnification was used to assess the sensitive cells and drug-resistant lung adenocarcinoma cell (A549, A549/PTX and A549/DDP) morphological characteristics:cell polarity, shape and arrangement, etc.5. EMT-related proteins including:E-cadherin, B-catenin, Vimentin, MMP-2 and MMP-9 were assessed in terms of expression levels. EMT-related transcription factors (Snail, Slug, Twist and ZEB1) were measured in A549, A549/PTX and A549/DDP cells using Western blot analysis.6. The expression changes were confirmed at the mRNA level by qRT-PCR. Expression was standardized to the expression of GAPDH and normalized to 1.0 in the parental cellsResults1. Establish the paclitaxel resistance (A549/PTX) and cisplatin resistance (A549/ DDP) lung adenocarcinoma cancer cells(1) The procedure was performed repeatedly until the cells showed resistance to the inhibitory activities of 0.23μmol/ml paclitaxel and 3.3μmol/ml cisplatin.(2) The resulting cells (A549/PTX and A549/DDP cells) were cultured for an additional 3 months in RPMI 1640 medium containing 0.23μmol/ml paclitaxel or 3.3μmol/ml cisplatin.2. Chemoresistance was demonstrated with Paclitaxel-resistant A549/PTX cells and cisplatin resistant A549/DDP cells exhibit(1)A549 and A549/PTX cells were treated with paclitaxel (PTX) at increasing concentrations (0.2,0.4,0.8,1.6 and 3.2μmol/1) for 24,48 and 72 h, A MTT assay was used to assess viability.(2) A549 and A549/DDP cells were treated with cisplatin (DDP) at various oncentrations (4,8,16,32 and 64μmol·l-1) for 24,48 and 72 h, and viability was assessed by MTT assay. Results are expressed as the percentage of control levels at each time point.3. The resistant cells of A549/PTX and A549/DDP had more the potential of invasion and migration than the parental A549 cells increases(1) wound healing, migration and invasion assays were performed to assess the migratory potential of A549/PTX and A549/DDP cells in relation to A549 cells(2) The migration and invasion potential of A549/PTX, A549/DDP and parental A549 cells were compared after 24 h of incubation using Transwell Boyden chamber assays (×200 magnification). Representative images of migrating and invading cells are shown.(3) the Transwell Chambers method, according to the results of A549/PTX, A549/DDP cells relative to the ability of parental migration and invasion in vitro enhanced significantly statistically significant.4. A549/DDP and A549/PTX cells showed molecular and morphological changes that were consistent with EMT(1) microscopy at ×200 magnification was used to assess cell morphology. The A549 cells (parental cells) had an epithelioid, rounded cobblestone appearance and there was limited formation of pseudopodia. A549/PTX and A549/DDP cells exhibited a spindle-shaped morphology and an increased formation of pseudopodia, indicating a loss of cell polarity.(2) E-cadherin, B-catenin, Vimentin, MMP-2 and MMP-9 which were EMT-related proteins, were assessed in terms of expression levels. EMT-related transcription factors (Snail, Slug, Twist and ZEB1) were measured in A549/PTX and A549/DDP cells using Western blot analysis.(3) The expression changes were confirmed at the mRNA level by qRT-PCR. Expression was standardized to the expression of GAPDH and normalized to 1.0 in the parental cells (compared with the parental A549 cellsConclusion1. Long-term first-line chemotherapy drugs (paclitaxel and cisplatin) may induce human lung adenocarcinoma cells A549 resistant;2. Drug-resistant cell (A549/PTX and A549/DDP) gain more the ability invasion and migration ability than parental A549 cell;3. A549/DDP and A549/PTX cells showed molecular and morphological changes that were consistent with EMTPart Ⅱ MiRNA-181a-inhibitor reverse EMT and weaken the ability of invasion and metastasis human lung adenocarcinoma cellObjective1. To understand the underlying mechanisms of drug-resistant cells and induction of EMT-like properties, miRNA-181a was identified among A549/PTX, A549/DDP and A549 cells.2. To explore the potential role of the significantly upregulated miRNA in regulating metastasis and EMT in lung adenocarcinoma cells.3. MiRNA-181a-inhibitor may reverse drug resistance.Methods1. QRT-PCR was applied to compare the content of miRNA-181a in A549/PTX, A549/DDP to A549 cells.2. To explore the potential role of miR-181a in regulating metastasis and EMT in lung adenocarcinoma cells, we used miR-181a mimic and inhibitor to modulate miR-181 expression.3. Determined by MTT method to evaluate the miRNA-181a-inhibitor treatment A549 /PTX and A549/DDP resistant lung adenocarcinoma cellsResults1. miR-181a was the most significantly dysregulatedin A549/PTX and A549/DDP resistant lung adenocarcinoma cells than parental A549 cells.QRT-PCR analysis verified that miR-181 a was up-regulated 16-fold in A549/PTX compared with A549 cells. Furthermore, up-regulated 12-fold of miR-181a was found in A549/DDP than parental A549 cells. Therefore, we hypothesized that miR-181a may represent a primary regulator in lung adenocarcinoma cells.2. over-expression of miR-181a promotes the acquisition of EMT phenotype in parental A549 cells and that inhibition of miR-181 a expression reverses the EMT phenotype in A549/PTX or A549/DDP cells.(1) A549 cells transfected with miR-181a mimic:a more mesenchymal appearance;both the protein and mRNA levels, miR-181a mimic caused a reduction in the expression of the epithelial adhesion molecules (E-cadherin and B-cadherin) and an increase in the expression of the mesenchymal markers (Vimentin, MMP-2, MMP-9, Snail, ZEB1 and Slug), which was similar to the pattern observed for A549/PTX and A549/DDP cells.(2) Transfection of A549/PTX cells with miR-181a inhibitor may reverse drug resistance, as indicated by MTT. These cells also showed a modest but clearly visible change in morphological characteristics,changing from mesenchymal-like spindle-cell shape to epithelial-like shapes,with opposite effects on the expression of epithelial and mesenchymal markers.(3) Similar results were observed for A549/DDP cells like A549/PTX.3. MiR-181a regulates the protein expression of PTEN by directly targeting 3’UTR of PTEN(1) Using miRTarBase software to predict miR-181a target genes, which report to PTEN playing an important role in the pathogenesis and drug resistance.(2) The protein levels of PTEN were reduced in the paclitaxel-or cisplatin-resistant A549 cells compared with the parental A549 cells(3) The protein expression of PTEN is increased when miR-181a inhibitor is transfected into A549/PTX and A549/DDP cells4. Inhibition of miR-181a expression reverses the EMT phenotype in A549/PTX or A549/DDP cells.(1)A549/PTX cells transfected with miR-181a-inhibitor and control inhibitor were treated with paclitaxel at increasing concentrations (0.2,0.4,0.8,1.6 and 3.2 umol/1) for 48 h, and viability was assessed by MTT assay.(2) A549/DDP cells transfected with miR-181a-inhibitor and control inhibitor were treated with paclitaxel at increasing concentrations (4,8,16,32 and 64μmol/l) for 48 h, and viability was assessed by MTT assay. Results are expressed as the relative number of control levels at each point in time.Conclusion1. MiR-181a responds to chemotherapy in lung adenocarcinoma cells by changing the levels of both itself and its target, PTEN.2. Down-regulation of miR-181a could successfully sensitize cancer cells to chemotherapy.3. Therapeutic strategies could be developed based on the predictive levels of miR-181a.4. MiR-181a repression could potentially be combined with chemotherapy to prolong drug sensitivity in lung adenocarcinoma, and potentially other types of cancer.