MiRNAs Expression And Function In Lung Cancer Sp(side Population) Cells

ObjectiveTo investigate the SP(side population) microRNA expression profiles and study their functional role in SP cells.Methods1. SP cells and non-SP cells were isolated using flow cytometry and Hoechst 33342 dye efflux assay from human lung adenocarcinoma A549 cell.2. SP an non-SP cells were subjected to cell cycle analysis.3. The microRNA expression profiles between SP cells and non-SP cells were investigated using miRNA array analysis.4. Real-time Quantitative PCR was performed to confirm the results of miRNA array analysis.5. Fow cytometry assay was used to analyzed RAS, Mlc-1 protein expression.6. Transfected SP and non-SP with antisense oligonucleotide using LipofectAMINE 2000 reagent (Invitrogen).7. Cell-proliferation assays were performed to analyze the proliferation potential of parental, empty vector and anti-miRNAs-transfected SP cells by using Cell-Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan).8. The parental, empty vector and anti-miRNAs-transfected SP cells were subjected to cell cycle and SP analysis in FACSCalibur.Results1. A549 cells contained a distinct fraction of SP cells (1.1%) which decreased significantly in the presence of verapamil.2. Larger proportion of SP cells were in G1 phase of the cell cycle as compared to non-SP cells.3. let-7, miR-29 and mir-31 were significantly downregulated in SP cells.4. RAS and Mcl-1 protein levels were significantly high in SP cells compared with non-SP cells.5. Anti-let-7e and anti-miR-29 transfection could enhance cell proliferation by accelerating G1/S phase transition; anti-miR-31 could inhibit cell proliferation by a cell-cycle arrest in G0/G1 phase. Co-transfection of anti-let-7e -miR-31 also could inhibit cell proliferation by a cell-cycle arrest in G0/G1 phase.6. Transfection of anti-let-7e could induce SP differentiation and decrease the number of SP cells. But transfection of anti-miR-31 could inhibit SP differentiation and increase the number of SP cells. Co-transfection of anti-let-7e -miR-31 also could inhibit SP differentiation and increase the number of SP cells.ConclusionThe reduced expression of miR-31 in SP is important in maintaining SP in undifferentiated state. Reduced miR-31 and let-7e maintain the balance between differentiation and quiescence of SP cells. In contrast, the reduction of anti-let-7e and anti-miR-29 can enhance cell proliferation by accelerating G1/S phase transition.