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inhibits stem cell characteristics of lung adenocarcinoma A549 cells through regulating miR-145 expression
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Abstract:
Objective:To investigate the mechanism of P53 regulating stem cell characteristics of lung adenocarcinoma A549 cells. Methods: Carcinoma tissues and relevant para-carcinoma tissues from 30 non-small cell lung carcinoma (NSCLC) patients who underwent surgical resection at No.85 Military Hospital from March 2014 to December 2015 were collected for this study; Levels of miR-145 in collected tissues were determined by Real-time PCR. Human P53 gene eukaryotic expression vector (pcDNA3.1-P53), mutant vector \[pcDNA3.1-P53R273H(CGT-CAT)\] and siRNA that trageting P53 (P53-siRNA) were constructed and transfected into A549 cells. The cells were divided into transfection group (vector or NC-siRNA) and experiment group (Flag-P53 or P53-siRNA). Western blotting was used to examine P53 protein over-expression and interference efficiency; Real-time PCR was used to determine the expressions of miR-145 and OCT4. Dual-luciferase reporter and Western blotting were used to confirm that miR-145 directly targeted OCT4 in A549 cells. Results: Compared with para-carcinoma tissues, miR-145 was obviously decreased in NSCLC samples (\[231±013\] vs \[3.51±0.27\], P<0.01), and P53 significantly promoted miR-145 expression in A549 cells (\[184±0.14\] vs \[1.00±0.00\], P<0.01); miR-145 mimics significantly inhibited the activity of pGAL3-OCT4-3′-UTR (P<0.01) and protein expression of OCT4 (P<0.05) in A549 cells. Additionally, miR-145 inhibitor transfection further increased OCT4 expression and reversed the inhibitory effect of P53 on stem cell characteristics of A549 cells.Conclusion: P53 down-regulats OCT4 expression by promoting miR-145 expression, and further suppresses the stem cell characteristics of A549 cells, which might be a new and safe treatment approach for NSCLC.
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