Mechanism of miR-30d-5p targeting ATG5 on proliferation,migration and autophagy of A549 cells
Objective To study the effects of miR-30d-5p on the proliferation,migration,and autophagy ability of lung ad-enocarcinoma cell line A549,providing new ideas for the subsequent diagnosis and treatment of lung adenocarcinoma.Method Screened differentially expressed miRNAs in lung adenocarcinoma from GEO database;Starbase database was used to query the expression of miR-30d-5p in lung adenocarcinoma;Kaplan-Meier website inquired about the influence of miR-30d-5p on the survival and prognosis of lung adenocarcinoma patients.Quantitative reverse transcription polymerase chain reaction(qRT PCR)was used to detect the expression and plasmid transfection rate of miR-30d-5p in cancer tissues and adjacent tissues of non-small cell lung cancer(NSCLC)patients,normal lung bronchial epithelial cells BEAS-2B,and lung adenocarcinoma cell lines.To explore the effects of miR-30d-5p on the proliferation and migration ability of lung ade-nocarcinoma A549 cells by using EdU cell proliferation assay,cell scratch assay,and Transwell migration assay;Predicting autophagy associated protein 5(ATG5)with potential binding to miR-30d-5p through bioinformatics databas-es;Double luciferase assay was used to verify whether miR-30d-5p has a targeted binding site with ATG5;Using stubRFP-sensGFP-LC3 lentivirus to detect the effect of miR-30d-5p on autophagy flow rate.Results The expression lev-els of miR-30d-5p in adjacent and cancerous tissues were 1.00±0.00 and 0.92±1.42,t=2.089,P=0.041.The expres-sion levels of miR-30d-5p in BEAS-2B,H1299,and A549 cells were 1.00±0.00,0.40±0.15 and 0.13±0.03,respec-tively,F=78.070,P=0.001.The EdU cell proliferation experiment results showed that the EdU positive cell rates in the miR-30d-5p mimetics/A549 group and con/A549 group were(23.00±4.00)％and(38.00±2.00)％,respectively,t=5.281,P=0.006.The results of cell scratch assay showed that the cell migration rate of miR-30d-5p mimics/A549 group(0.15±0.01)was lower than that of con/A549 group(0.29±0.01),t=21.830,P=0.001.Transwell assay showed that the number of cells passing through the microporous membrane of the miR-30d-5p mimics/A549 group(165.33±5.51)was less than that of the con/A549 group(410.67±9.50),t=7.482,P=0.002.The results of the du-al luciferase experiment showed that there was a targeted binding sequence between miR-30d-5p and ATG5.StubRFP-sensGFP-LC3 lentivirus infection results showed that the miR-30d-5p mimics/A549 group had an increased number of au-tophagosomes and an increased flow rate of autophagosomes to autophagolysosomes compared with the con/A549 group,the miR-30d-5p mimics/ATG5 group had reduced number of autophagosomes and reduced flow rate of autophagosomes to autophagolysosomes compared with the miR-30d-5p mimics/A549.Conclusion MiR-30d-5p inhibits the proliferation,migration and autophagy of lung adenocarcinoma A549 cells by targeting ATG5.
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