A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

Our protocol addresses the effects of regulatory RNA-binding proteins on microRNA biogenesis and maturation, straight from cell cultures. The method we describe can be performed with common cell culture reagents and is relatively fast. This method can be important to study the phenotypic effects of microRNAs and their targets in different types of eukaryotic cells.

Normal or disease-like cells can be used. It is important to choose cell lines amenable to transfection, perform adequate transfection and induction controls in order to observe the changes in target affinity through luciferase activity. To begin, maintain the cells in DMEM, high glucose supplement in 100 millimeters Petri dishes and incubate the cells at 37 degrees Celsius in a humidified, controlled atmosphere incubator with 5%carbon dioxide.

To detach adherent cells, incubate it with trypsin-EDTA solution at 37 degrees Celsius for three to five minutes. To perform transfections, mix 200 nanograms of DNA and 1.25 microliters of transfection reagent in 100 microliters of the culture medium and add the transfection mixture to the cells. Then, incubate the cells with the transfection mixtures for four hours at 37 degrees Celsius.

Then replace the medium with medium containing 10%FBS and incubate for another 24 hours before adding the antibiotics for selection. To transfect pFLAG-HuR and MT-pFLAG into HeLa, select cells by increasing the concentration of Geneticin from 100 microgram per milliliter to 1000 microgram per milliliter, generating stable cell lines. Then, confirm overexpression with quantitative PCR using specific primers for HuR mRNA.

Transfect the plasmids in HeLa-Cre cells with 40 nanograms pTK-Renilla, then pRD miR 17-92, generating HeLa-Cre miR 17-92, and selecting with one microgram per milliliter puromycin. Then, transfect HeLa-Cre miR 17-92 with pTK-Renilla and pmirGLO RAB1B 3'UTR or pmirGLO scrambled 1B 3'UTR separately. Next, select using penicillin and streptomycin, resulting in generation of luc RAB1B and luc scrambled.

Next, transfect the cells with pFLAG-HuR or MT-PFLAG as them demonstrated previously. Proceed to cell culture with HeLa-Cre miR 17-92 luc, HeLa-Cre miR 17-92 scrambled, HeLa-Cre miR 17-92 HuR, and HeLa-Cre miR 1792 HuR lock until approximately 80%of confluency is reached. Then, induce with one microliter of doxycycline for 30 minutes at 37 degrees Celsius and also keep all the cells without doxycycline as controls for the same period.

To quantify and compare different luminescence intensities, use the dual luciferase reporter assay kit. Before beginning the assay, thaw luciferase assay solutions and leave them at room temperature. Next, prepare the mix Stop and Glo by mixing 200 microliters of the substrate in 10 milliliters of luciferase assay buffer two.

Then mix 10 milliliters of luciferase assay buffer two into the amber vile of luciferase assay substrate two to prepare the mix, LAR II, and shake well. Afterward, transfer the solutions to 15 milliliter centrifuge tubes previously identified and protected from light. Next, perform the luminescence readings using equipment such as Synergy and measure expressed luciferase as relative light units, and then export the results to a spreadsheet for further statistical analysis.

Perform normalization of firefly luciferase activity by the control Renilla and plot that as relative light units in a graphic while repeating for groups with and without doxycycline induction. Overexpression of HuR upon transfection of pFLAG-HuR was confirmed in HeLa, BCPAP, and HEK293T cell line, and it was observed that HuR overexpression in these cells stimulates the expression of miR19A. A strong reduction in luciferase activity was observed with RAB1B 3'UTR upon increased miR19A and miR19B expression from pRD miR 17-92, in comparison to HeLa-Cre cells.

The transfection of pFLAG-HuR in HeLa cells did not change the luciferase activity. However, overexpression of HuR, coupled with doxycycline supplementation stimulating expression of the miR 17-92 cluster, further reduced luciferase activity. After confirming the role of the RNA-binding protein in microRNA maturation, cell kinetics and cell cycle analysis will be important to understand the major changes promoted by the protein in microRNAs.