Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis

Abstract

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3'-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.

Figure 1

(A) MiRNA reporter vectors used to analyze miRNA activity. The restriction sites for cloning the miRNA-binding sites are located in the 3′-UTR of the luciferase gene in the pmir-Report luciferase vector. Once cloned the miRNA-binding site vector is co-transfected with the pmir-Report β-gal vector as a control for transfection efficiency and a miRNA inhibitor molecule that is specific to the binding site or is mutated as compared with the binding site. (B) Enhanced expression of miRNA-regulated reporter by miRNA inhibitors. HeLa cells were plated at 50 000 cells/well in 24-well plates. Cells were transfected using lipofectamine 2000 in duplicate with pmir-REPORT β-gal, a luciferase reporter construct that contained one target site for either miR-23, miR-21, miR-15a, miR-16 or miR-19a and either inhibitors for these miRNA or a negative control (NC). HeLa cells were also transfected with a control luciferase reporter vector lacking a miRNA-binding site without an inhibitor to demonstrate the level of activity that can be achieved for an unmodified pmir-REPORT luciferase. Twenty-four hours post-transfection cells were assayed for luciferase and β-gal expression, and β-gal is used to normalize for differences in transfection efficiency.

Figure 2

Identification of miRNAs that alter cell proliferation in HeLa cells. In 96-well plates, 8000 HeLa cells were reverse transfected with miRNA inhibitors (5 pmol) in triplicates using Ambion siPORT Neo-FX. Seventy-two hours post-transfection, cells were fixed with 4% paraformaldehyde, permeablized with 0.1% TritonX-100 and stained with propidium iodide to look at total cell number. The plates were scanned using the TTP LabTech Acumen Explorer.

Figure 3

Screen for miRNA involved in cell viability in A549 cells. In 96-well plates, 8000 HeLa cells were reverse transfected with miRNA inhibitors (5 pmol) in triplicates using Ambion siPORT Neo-FX. Seventy-two hours post-transfection, cells were trypsinized and transferred to a non-tissue culture plate, so that cells would not adhere. Cells were stained using ViaCount Flex Reagent and analyzed for total cell number using the Guava PCA-96 (Personal Cell Analysis).

Figure 4

Effects of miRNA inhibitors on caspase activity in HeLa. In 96-well plates, 8000 HeLa cells were reverse transfected with miRNA inhibitors (5 pmol) in triplicates using Ambion siPORT Neo-FX. Seventy-two hours post-transfection, cells were analyzed for caspase and esterase activity as described in the Materials and Methods. Esterase activity was used to normalize for differences in cell number that may exist owing to the miRNA inhibitor causing defects in cell growth.

Figure 5

Effects of miRNA inhibitors on caspase activity compared with cell growth in HeLa cells. We graphed the data that were generated from Figures 2 and 4 together to compare hits that affected cell proliferation with those that affected apoptosis.