Figure 7.

La and HuR act cooperatively to mitigate miR-21–induced increase in cell proliferation and decrease in apoptosis. A, MCF7 cells were transfected with pCDNA3-HuR, pCDNA3-La, and pSUP-miR-21, individually (2 μg) or in combinations (1 μg each for La and HuR overexpressing constructs). pCDNA3 was used for mock transfection. Posttransfection, MCF7 cells were subjected to serum deprivation for 48 h as an apoptotic stimulus, followed by caspase 3/7 activation assay using a luminescent substrate. Relative light unit values of each sample, representing caspase 3/7 activity, are expressed as fold change from that of mock, taken as 1. ∗ represents significant difference (p-value ≤ 0.05) from mock-transfected cells and # represents significant difference (p-value ≤ 0.05) from miR-21–transfected cells. B, MCF7 cells, transfected with the same combination of DNAs as in A, were serum deprived for 48 h to induce apoptosis. Cells were stained with Annexin V-AlexaFluor 488 and Propidium iodide to detect apoptosis by fluorescent microscopy. Mean fluorescent intensity per cell of Annexin V and propidium iodide for 100 cells per treatment was determined and shown as fold change from mock, taken as 1. ∗, ∗∗, and ∗∗∗ represent significant difference (p-value ≤ 0.05, ≤0.01, and ≤0.001 respectively) from respective mock-transfected cells, and # and ## represents significant difference (p-value ≤ 0.05 and ≤0.01, respectively) from miR-21–transfected cells. C, MCF7 cells were transfected with the same combination of DNAs as in A and B. MTT assay was performed at 24 h, 48 h, and 72 h posttransfection. OD₅₉₅ readings are plotted for each sample as fold change from mock taken as 1. ∗ and ∗∗∗Represent significant difference (p-value ≤ 0.05 and ≤0.001 respectively) from respective mock-transfected cells, and # and ## represent significant difference (p-value ≤ 0.05 and ≤0.01, respectively) from miR-21–transfected cells. D, MCF7 cells were transfected with the same combination of DNAs as in A, B, and C, together with two combinations containing miR-21–expression plasmid (2 μg), La–expression plasmid (2 μg), and HuR siRNA (100 pmole), and miR-21–expression plasmid (2 μg), HuR-expression plasmid (2 μg), and La siRNA (100 pmole). Posttransfection, 1000 cells from each sample were seeded, and after 14 days, colony number was measured by crystal violet staining. Representative image of colony formation experiment is shown in left panel. Colony numbers from each sample are shown as fold change from mock, taken as 1 (right panel). ∗∗ represents significant difference (p-value ≤ 0.01) from mock-transfected cells, and # represents significant difference (p-value ≤ 0.05) from miR-21–transfected cells. HuR, human antigen R; La, lupus antigen.