Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

This method will help understand and perform lab techniques necessary to identify the activity of two microRNA in lung cancer cells in vitro. Lung cancer is the leading cause of cancer-related death worldwide. Here, we present techniques spanning from basic lab methodologies to more complex protocols, such as gene expression analysis and antibody protein microRNA, specifically used for identifying the effect of microRNA's actions.

The following methods will help us answer key questions regarding the effect of two microRNAs, microRNA 143 and microRNA 506, on regulating the cell cycle in lung cancer cells. We present different lab techniques, like cell transfection, RNA extraction, quantitative real time PCR and microRNA analysis. We hope our presentation will contribute on successfully completing these analyses.

First, seat the cultured cells in an appropriate flask sufficient for RNA and protein extraction, to perform gene expression using qPCR or micro-array analysis. Then incubate overnight with media supplemented with 10 percent FBS and 1 percent penicillin streptomycin. The next day, prepare the microRNA sample for transfection by diluting microRNA 143 and microRNA 506 in media at a concentration of 100 nanomolar each.

Take out the flask from the incubator and remove the media. Wash with 1x PBS. All the untreated controls will contain only incomplete media with cells.

Incubate the cells with transfection media for six hours in an incubator. After six hours, remove the media and replace with four milliliters of fresh, complete media. Harvest the cells after 24 hours and 48 hours by trypsinization.

Next, wash twice with 1x PBS in each tube and remove supernatant. In order to perform RNA extraction at a later time, freeze the cells at minus 80 degrees Celsius. RNA extraction was performed using an RNA kit, following manufacturer's instructions.

First, remove the tubes from minus 80 degrees Celsius and allow to thaw. Add 300 microliters of lysis buffer and pipette up and down to break cell membrane. Add equal volume of 100 percent ultra-pure ethanol.

Then mix well and place in separated column. Centrifuge and remove the flow-through. Add 400 microliters of wash and buffer and centrifuge to remove the buffer.

Add 5 microliters of DNAse 1 with 75 microliters VNA digestion buffer in each sample, and incubate for 15 minutes. Then wash the sample with 400 microliter RNA prep buffer. Next, wash twice with RNA wash buffer.

Now, add nuclease-free water to the column, centrifuge and then collect the RNA. Measure the RNA concentration. In this stage, two micrograms of RNA sample can be sent for the RNA sequencing.

Prepare the cDNA according to the protocol described in the text section, using thermocycler. Prepare forward and reverse primer solutions with DNAse RNAse-free water to a concentration of 10 micromolar. Prepare a master mix for each gene to be detected according to the number of samples.

For each cDNA sample, reaction quantity is according to the table described in the text. Place each sample to their respective wells. For qPCR, it is always good to prepare an Excel document with 96 well plate layout, in order to keep track of the samples.

For each sample and analyzed gene, perform their reaction in triplicates, or at least duplicates. Seal the plastic plate with transparent, optically clear plastic sealer. Avoid touching the thin, plastic layer with fingers, as this can produce false signal.

Quickly spin the plate to mix all of the reagents to the bottom of their wells. Next, run the sample plate using a quantitative, real-time PCR machine. Get the CT values generated by the qPCR machine, and analyze for relative gene expression.

Transfect the cells as described in the earlier section of this video. Harvest cells from each sample in separate, 15 milliliter sterile tubes for trypsinization. Next, wash the cells twice with 1x PBS, by centrifugation at 751G for five minutes, and then remove the supernatant.

Re-suspend and break the palette by adding 200 microliter ice-cold 1x PBS through a pipette. Add two milliliters of 70 percent ice-cold ethanol, drop-wise to the tube, while vortexing the tube gently to fix the cells. Incubate tubes for 30 minutes at room temperature and place them in four degrees Celsius for one hour.

Remove the tubes from four degrees Celsius and centrifuge. Add 2ml ice-cold PBS and vortex, and then remove the supernatant by centrifugation. Add 500 microliters of 1x PBS containing propidium iodide and ribonuclease A with concentration 50 and 200 micrograms per milliliter, respectively, in each sample, and incubate for 30 minutes at room temperature.

Transfer samples into appropriate tubes, protecting from light, and run on flow cytometer. This experiment is to identify all the cell cycle pathway proteins'expression changed by the microRNA treatment. Collect protein samples according to the procedure described in the transfection section, and quantify with BCA assay.

Remove the slides from minus four degrees Celsius one hour before experiment to bring to room temperature and to remove moisture. Take 70 microgram of protein samples and prepare sample slides according to the manufacturer's instructions, which is also described step-by-step in the text section. Here to note, proper washing of slides with ultra-pure deionized water is another important step, as this will help to reduce the background of the slides.

Additionally, it is very critical in this experiment not to dry up the sample slides until the final step. Finally, scan the slide using micro-array machine and analyze the data. The qPCR analysis of the microRNA-treated samples took place to determine the gene expression of CDK1, CDK4 and CDK6.

These genes regulate the cell cycle of normal and cancer cells, and have been targeted as a means for inhibiting tumor progression. The data demonstrated the downregulatory effect of these genes due to the treatment with microRNA 143 and 506. Cell cycle distribution data from flow cytometry demonstrated a population increase in the G0 and G1 phase, and a population decrease in the S phase due to the effect of combinatorial treatment of microRNA 143 and 506.

The cell cycle's specific micro-array analysis detected protein expression changes of approximately 60 genes. The represented heat map indicated downregulation at the protein level of multiple genes necessary for the progression of the cell cycle and proliferation, such as CDK2, Helen 1 and 3, RE2F1. In contrast, proteins commonly recognized by their grown inhibitory effect were upregulated.

Although the results are semi-quantitative and further analysis is required, the micro-array analysis provides a quick overview of the pathway's activity. We believe, after watching this video, you will be able to perform fundamental, as well complex, lab experimentation that will assist you on the identification of microRNA activity in cells.