| Objective: Lung cancer is currently one of the most common solid tumor in the world with high fatality rate.Cisplatin as the basic clinical chemotherapeutic drugs of lung cancer,the resulting resistance is one of the main reasons of ineffective treatments.Therefore,the new therapeutic targets to reverse or reduce the drug resistance demand to search,and finally to reduce the incidence of side effects and improve the patients survival rates.Exosome,as nanovesicles of intercellular signal transmission,play an important role in tumorigenesis,growth,invasion,metastasis and drug resistance.This experiment focus on the possible drug resistance mechanism through the research of the biological behaviour of the human lung adenocarcinoma cell line A549 and the cisplatin resistant cell line A549/DDP,and the cisplatin resistant influence of exosomes derived from A549/DDP to homologous cell A549.The aim is to discover new possible drug resistance target for improving the curative effect of lung cancer.Methods:1 Comparision of cisplatin resisitance,cell cycle and migration ability between human lung adenocarcinoma cisplatin resistant strain A549/DDP and human lung adenocarcinoma wild strain A5491.1 Lung adenocarcinoma cisplatin resistant cell line A549/DDP was purchased and continued to induce to stably proliferate in vitro with 1640 medium suppled with 10% FBS containing cisplatin(DDP)at the final concentration of 2 μg/ml.The IC50 value of A549 and A549/DDP cell lines to cisplatin were determined by the MTS experiment.After A549 and A549/DDP cell lines being treated with cisplatin at the final concentration of 6 μg/ml for 48 hours,apoptosis was detected by flow cytometry using Annexin V-PE and 7-AAD staining.1.2 Cell cycle was examined by flow cytometry with PI staining and p27 expression was detected by Western Blot.1.3 The migration rates were detected by Wound healing assay and Transwell assay,respectively.EMT related proteins,including N-Cadherin,E-Cadherin and Vimentin,were detected by Western Blot.2 The effect of D-exo derived from A549/DDP cell line on cisplatin resistance and migration ability of homologous cells A5492.1 A549/DDP and A549 cells were cultured in vitro with 1640 medium containing 10% FBS without exosomes for 72 hours.Supernatant was collected for centrifugation.Precipitation was obtained after a series of differential centrifugation and dissolved with PBS.Exosome concentration was determined by nanodrop.Exosome morphology was observed by Transmission electron using phosphotungstic acid staining.2.2 D-exo from A549/DDP cell line labeled by fluorescent dye Dil was cocultured with A549 cells overnight to observe the uptake of labeled exosomes in the fluorescence microscope.2.3 A549 cells were pretreated with A-exo derived from A549 cells or D-exo(final concentration of 50 μg/ml)derived from A549/DDP cells respectively for 72 hours,IC50 value of A549 cells to cisplatin were detected by MTS assay.After A549 cells being pretreated as above,6μg/ml of cisplatin was added to them and treated for another 48 hours.Cell apoptosis were detected by flow cytometry using Annexin V-PE and 7-AAD staining to observe the impact of exosomes on A549 cells drug resistance.2.4 After pretreatment as 2.3,Wound healing assay and Transwell assay were used to evaluate the impact of exosomes on migration ability of A549 cell line.3 miRNAs in the exosomes related with cisplatin resistance of A549/DDP cell line3.1 miRNAs expressions of A549 and A549/DDP cell lines,including miR-221-3p,miR-221-5p,miR-222-3p,miR-222-5p,miR-21-3p,miR-21-5p,were examined by Real-time PCR to screen the research object.3.2 The expressions of miR-222-3p of the two cell lines and exosomes from the two cell lines were detected by Real-time PCR.3.3 After A549 cells being pretreated with PBS,A-exo and D-exo(final concentration of 50 μg/ml)respectively for 72 hours,the expressions of miR-222-3p of A549 cells were examined by Real-time PCR to observe the influence of exosomes derived from A549/DDP cell line on homologous cell line A549.3.4 Micro RNAs related proteins(PTEN,AKT,APE1)expressions of the two cell lines were determined by Western Blot.After A549 cells being pretreated with PBS,A-exo and D-exo(final concentration of 50 μg/ml)respectively for 72 hours,the expressions of PTEN and AKT1 were determined by Western Blot.Results: 1 The differences between A549 and A549/DDP cells on cisplatin resistance,cell cycle and cell migration ability.1.1 The establishment of A549/DDP cell line MTS assay showed that IC50 value of A549 or A549/DDP cells to cisplatin was 8.37±1.82(μg/ml)or 63.71±16.87(μg/ml),respectively.The Resistance Index was 7.56±0.59.The statistical difference between two cell lines was significant(P<0.01)(Fig.1).Apoptosis results showed that the apoptosis rates of A549 cells treated with PBS and 6 μg/ml of cisplatin for 48 h were 6.37%±1.67% and 38.44%±3.99%.The apoptosis rates A549/DDP cells treated with PBS and 6 μg/ml of cisplatin were 6.84%±0.19%,20.86%±2.36% respectively.There were statistically significant difference between A549 cell line and A549/DDP cell line pretreated with 6 μg/ml of cisplatin(P<0.01)(Fig.2).1.2 A549/DDP cells slowed the growth and prolonged the cell cycle With comparison with A549 cells,the cell number of G0/G1 phase of A549/DDP cells elevated significantly(P<0.01),S phase decreased significantly(P<0.01)and G2/M phase increased(P<0.05)according to the results of cell cycle analysis(Fig.3).Western Blot results showed that the cell cycle inhibition protein P27 of A549/DDP cells increased significantly,compared with the A549 cells,which had a significant difference(P<0.05)(Fig.4).1.3 The migration ability of A549/DDP cells enhanced Wound healing assay showed that the lateral migration rates of A549 and A549/DDP cells were 23.34%±6.86% and 44.44%±6.41%,which had a significant difference(P<0.05)(Fig.5).Transwell assay revealed that the migration cell numbers of A549 and A549/DDP cells were 26±7 and 198±22,which had a significant difference(P<0.01).The OD values of A549 and A549/DDP cells were 0.35±0.24 and 1.42±0.14,which had a significant difference(P<0.01)(Fig.6).The expression of N-Cadherin of A549/DDP cells was elevated compared with A549 cells(P<0.05).While the expressions of E-Cadherin and Vimentin of A549/DDP cells had no statistical change(Fig.7).2 D-exo derived from A549/DDP cells promote cisplation and migration ability of A549 cells2.1 Exosome extraction and identification Exosomes were obtained after a series of differential centrifugation.A large number of scattered vesicles were observed with the diameter of 40-100 nanometer in the Transmission electron(Fig.8).2.2 A549 can uptake exosomes A lot of red fluorescence were observed in the A549 cells in the fluorescence microscope after being cocultured with D-exo from A549/DDP cells for 24h(Fig.9).2.3 D-exo can enhance the resistance of A549 cells to cisplatin After pretreatment with PBS,A-exo and D-exo,IC50 value of A549 cells to cisplatin assayed by MTS were 8.37±1.82(μg/ml),14.44±1.14(μg/ml)and 39.44±8.46(μg/ml).There were statistically significant higher IC50 values for A549 pretreated with A-exo or D-exo(P<0.05 or P<0.01)(Fig.10).Apoptosis results showed that the apoptosis rates of A549 cells,pretreated with PBS,A-exo or D-exo and then with 6 μg/ml of cisplatin,were 6.37%±1.67% and 38.44%±3.99% respectively.D-exo can promote the resistance of A549 cells to cisplatin significantly(P<0.05)(Fig.11).2.4 D-exo can promote the migration ability of A549 cells Wound healing assay showed that the migration rates of A549 cells pretreated with PBS,A-exo and D-exo were 25.68%±10.74%,33.57%±3.28% and 55.0%±1.69%.D-exo can promote the lateral migration ability of A549 cells significantly compared with PBS and A-exo(P<0.05)(Fig.12).Transwell assay showed that the migration cell numbers of A549 cells pretreated with PBS,A-exo and D-exo were 27±8,67±14 and 139±13.The OD values of A549 cells pretreated with PBS,A-exo and D-exo were 0.35±0.24,0.81±0.07 and 1.19±0.08.Exosomes can enhance tha longitudinal migration ability(P<0.05)and the effect of D-exo was more prominent(P<0.05)(Fig.13).3 D-exo could enhance the resistance of A549 cells to cisplatin via miR-222-3p3.1 The expression of miR-222-3p in A549/DDP cells upregulated significantly The expression of miR-222-3p in A549/DDP cells was apparently upregulated by 10.79±1.72 folds(P<0.01)compared with A549 cells.While the expressions of miR-221-3p,miR-221-5p,miR-21-5p were also upregulated by 2.47±0.63,3.62±0.16,2.21±0.06 folds respectively(P<0.05)(Fig.14).We choose miR-222-3p as our target miRNA for further study.3.2 D-exo carry high levels of miR-222-3p The expression of A-exo and D-exo were 2.28±0.34 and 3.29±0.39 folds(P<0.05)compared with the homologous cells respectively(Fig.15),which indicated that exosomes carry high levels of miR-222-3p.3.3 D-exo could enhance the resistance of A549 cells to cisplatin by transfer of miR-222-3p The expressions of miR-222-3p in the A549 cells pretreated with A-exo or D-exo were 3.84±2.81(P<0.05)and 9.44±3.71 folds(P<0.01),compared with PBS control(Fig.16).3.4 PTEN/AKT signaling pathway may participate in the resistance of A549 cells to cisplatinThe expression of PTEN of A549/DDP cells was downregulated significantly(P<0.01)and p-AKT1 was upregulated(P<0.01).The expression of APE1 between the two cell lines had no significant difference(Fig.17).Compared with PBS and A-exo stimulating groups,the expression of PTEN or p-AKT1 in A549 cells treated with D-exo were significantly reduced(all P<0.01)(Fig.18).Conclusion: 1 Proliferation of A549/DDP cell line slowed down,and the ability of migration enhanced.2 Both the migration ability and the resistance to cisplatin of A549 cell line enhanced when A549 cells were treated with exosomes from A549/DDP cell line.3 A549/DDP cell line expressed miR-222-3p with a high level,the promoting A549 cell resistance to cisplatin could be transferred via miR-222-3p rich exosomes from A549/DDP.4 PTEN/AKT could participated in the enhancement of A549 cell secondary resistance to cisplatin induced by miR-222-3p rich exosomes from A549/DDP. |
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