The overall goal of this methodology is to describe how a transcription factor of interest regulates the microRNA transcriptome. This method can help answer key questions on whether transcription factors that modulate transcription of protein coding genes are also involved in transcription of microRNAs. The main advantage of this methodology is that it integrates data mining, laboratory data, and bioinformatic analysis to provide a comprehensive description of microRNA genes transcriptions that is dependent on the activity of transcription factor of interest.
Though this method can provide insight into the interaction of microRNA gene transcription and activity of the transcription factor status, it is important to remember that this is a descriptive method and confirmation of such an association should be done using additional assays. To begin, open a genome browser, such as the University of California Santa Cruz genome browser, in order to extract chromatin immunoprecipitation sequencing data. Once on the site, go to the top menu, select Tools and open the Table Browser.
Enter the specifications, as shown here, into their respective boxes, and then select Get Output to extract the table. Save the output as a text file. Then, import the file into a spreadsheet.
Once in the spreadsheet program, sort the name column and filter the results for a transcription factor of interest, such as STAT2. Then, copy the list of microRNA promoters based on their H3K4me3 epigenetic signature and paste the list into a txt file. Then, use the supplemental code written in SQL and provided in the supplementary data files to map the collected data and determine the median binding affinity.
First, add 1.5 million HEK293 cells to a 10 centimeter dish and add DMEM supplemented with 10%FBS. Culture the cells for 24 hours until they reach about 50%confluency. Next, replace the media.
Then, add a transvection agent combined with five micrograms of a green fluorescence protein Lentivirus containing the targeted shRNA and five micrograms of packaging vectors in accordance with the manufacturers instructions. As a control, transvect an identical plate of cells using scrambled shRNA and the packaging vectors. Incubate the transvection mix on the cells for 16 hours and then change the media to 10 milliliters of fresh DMEM media supplemented with 10%FBS.
48 hours post-transvection, remove the media from the cells. Then, centrifuge for five minutes in order to collect infectious supernatant. Filter the supernatant through a 0.45 micron syringe filter to remove any floating cells.
Next, add the media into an ultracentrifigal filter device with a threshold of 100 kilodaltons. Centrifuge the media to concentrate the Lentivirus until the volume is less than 250 microliters. Transfer the remaining liquid into a cryotube and store the concentrated virus at minus 80 degrees Celsius until it is needed.
When ready to continue, remove the frozen Lentivirus from the minus 80 freezer and bring the vial to room temperature. Transfer 100 microliters of viral supernatant to a fresh 1.5 milliliter microfuge tube and add 900 microliters of medium without serum. Next, add 10 nanograms per milliliter hexadimethrine bromide to the one milliliter virus suspension.
Mix the tube gently, and let the mixture stand for five minutes. Harvest five million cells for each planned transduction and then gently re-suspend the cell pellet in 0.5 milliliters of media containing virus. Transfer the cells to a 12-well plate and incubate them for four to 24 hours.
Then, add an additional 0.5 milliliters of medium containing 20%FBS. At 48 to 72 hours post-transduction, harvest the cells and stain them with propidium iodide according to the manufacturer's instructions. After staining, protect the cells from light and use facts to estimate transvection efficiency by measuring the rates of GFP positive, propidium iodide negative cells.
Then, collect the sorted GFP positive cell population and extract their proteins. Finally, use western blotting to determine levels of the targeted transcription factor before and after infecting the cells with the designated shRNA. After the transvection of CLL cells with STAT3 shRNA, the levels of STAT3 mRNA and STAT3 protein both significantly decreased.
In addition, a microRNA array of the CLL cells depicted 23 microRNAs whose expression differed significantly between CLL cells that were trasnfected with STAT3 shRNA and CLL cells that were transvected with an empty vector. The results of the microRNA array were validated for seven of the genes, shown here, using quantitative RT-PCR. Following this procedure, other methods like chromatin immunoprecipitation and electromobility shift assay and a difference assay can be performed in order to validate that this transcription factor binds and activates the promoter of a specific mirror gene.
After watching this video, you should have a good understanding of how to integrate bio informatics analysis, publicly available data and silencing a transcription factor using shRNA approach to find how this transcription factor regulates the expression of microRNA genes.
