Purpose: Increasing evidence suggests that microRNAs (miRNAs) may be involved in the occurrence and progression of non-small cell lung cancer (NSCLC). In the present study, we used serum-starved A549 cells emulating tumor under a nutrient depletion stress in the microenvironment. Patients and methods: We first detected the expression level of miR-224 between tumor tissues and the adjacent normal tissues. We analyzed the expression levels of miR-224 and its predicted target phosphatase and tensin homolog (PTEN) using quantitative real-time PCR (qRT-PCR) in starved A549 cells. Following transfection with miR-224 mimic or inhibitor in starved A549 cells, MTT assay, Annexin V FITC/PI staining, and LC-3 immunofluore... More
Purpose: Increasing evidence suggests that microRNAs (miRNAs) may be involved in the occurrence and progression of non-small cell lung cancer (NSCLC). In the present study, we used serum-starved A549 cells emulating tumor under a nutrient depletion stress in the microenvironment. Patients and methods: We first detected the expression level of miR-224 between tumor tissues and the adjacent normal tissues. We analyzed the expression levels of miR-224 and its predicted target phosphatase and tensin homolog (PTEN) using quantitative real-time PCR (qRT-PCR) in starved A549 cells. Following transfection with miR-224 mimic or inhibitor in starved A549 cells, MTT assay, Annexin V FITC/PI staining, and LC-3 immunofluorence staining were performed to investigate the roles of miR-224 on proliferation, apoptosis, and autophagy. Next, the expression of apoptosis-related protein Bax and Bcl-2, autophagy-related proteins LC3, PI3K signaling, and target PTEN were measured using qRT-PCR and Western blot assays. The direct interaction between miR-224 and PTEN was validated with a dual luciferase assay. Results: We found that the expression level of miR-224 in tumor tissues was significantly higher when compared with the adjacent normal tissues. We discovered a reciprocal expression pattern between miR-224 and PTEN in starved A549 cells, and transfection with miR-224 mimic led to down-regulation of PTEN. A dual luciferase assay further confirmed the direct interaction between miR-224 and 3'UTR of PTEN. Transfection with miR-224 mimic in starved A549 cells resulted in enhanced cell proliferation, reduced apoptosis, and autophagy, accompanied by increased expression of anti-apoptotic protein Bcl-2, decreased expression of pro-apoptotic protein Bax, and autophagy-related protein LC3. Activation of PI3K was observed in miR-224 mimic transfected cells. The reverse effects by the miR-224 inhibitor in all experiments were observed. Conclusion: Taken together, we proved that miR-224 might play essential roles in cellular functions of nutrient-depleted A549 cells possibly through regulating the target PTEN and downstream signal PI3K, suggesting the potential of miR-224 to be a therapeutic target for NSCLC therapy.
