Figure 4

PTEN regulates the impact of miR-221/222. SGC7901 cells were cultured and treated with pcDNA-PTEN, AS-miR-221/222, or pMSCV-miR-221/222, and subjected to MTT assay. Transfection with pcDNA-PTEN increased cell proliferation to a similar rate as cells transfected with AS-miR-221/222. Infection of pcDNA-PTEN-treated cells with pMSCV-miR-221/222 had no effect on proliferation (Figure 4A). Flow cytometry analysis showing that the fraction of cells in G1 phase following AS-miR-221/222 transfection was significantly higher than in the control. Transfection with pcDNA-PTEN resulted in statistically similar results as with AS-miR-221/222, and infection of pcDNA-PTEN transduced cells with pMSCV-miR-221/222 did not impact on cell cycle progression (Figure 4B). Annexin V staining and flow cytometry analysis showing that AS-miR-221/222 transfection induced significantly higher levels of apoptosis in SGC7901 cells than controls, and transfection with pcDNA-PTEN yielded similar results. Infection of pcDNA-PTEN-transfected cells with pMSCV-miR-221/222 did not impact on apoptosis (Figure 4C). Transwell assay showing that AS-miR-221/222 transfection decreased invasive ability compared to controls. Transfection with pcDNA-PTEN yielded similar results, and infection of pcDNA-PTEN-transfected cells with pMSCV-miR-221/222 did not impact on invasive ability. Data represents the number of migrated cells per field (Figure 4D). AS-miR-221/222 and pcDNA-PTEN transfection increased radiosensitivity, as determined by clonogenic assay following radiation exposure. Infection of pcDNA-PTEN-transfected cells with pMSCV-miR-221/222 did not impact clonogenic survival. Experiments were performed in triplicate. When applicable, data is represented as mean ± SE (Figure 4E).