Chapter 5: Expression of MMP-9 mRNA following PMA induced Differentiation

As we learned in the previous chapter, chemical agents may be used to induce differentiation in HL-60 cells. Like stem cells, HL-60 cells are capable of becoming a different cell type upon the proper inductive stimulus. HL-60 cells are multipotent and can be induced to differentiate. Think of differentiation as moving from general to specific function. Technology enables us to view and document the changes in a cell as it differentiates.

In the last chapter we observed the differences in cell morphology when HL-60 cells are treated with either DMSO (dimethylsulfoxide) or PMA (phorbol 12-myristate 13-acetate). In this exercise we will examine the changes that occur in HL-60 cellular products, specifically RNA, when this differentiation occurs. Remember the central dogma of molecular biology, DNA transcribes to RNA, RNA translates to protein. Because we know this, we can design experiments to compare the differences in chemically induced differentiation. When HL-60 cells are treated with DMSO, the cells change from predominate promyelocytes, to granulocytes and when treated with PMA change to monocytes. As the function of these cells changes, the cellular products made by these cells change as well. These changes may be followed by monitoring the products, or more accurately, the messenger RNA. RNA transcripts lead to the production of proteins (central dogma), therefore it can be assumed that if there is accumulation of a transcript, then the cells also produce the corresponding protein, unless translation is inhibited.

In this lab exercise, we want to look at mRNA transcript production in cells that have been treated with either PMA or DMSO. This allows us to examine changes in gene expression following external stimuli exposure to the two ligands (due to signal transduction mechanisms induced by the PMA and DMSO ligands). We will isolate mRNA, and then perform a reverse transcription reaction to convert those mRNA transcripts into the more stable cDNA, since RNA is so easily degraded and unstable. We will then perform a PCR reaction to amplify genes of interest that may be present in our cDNA library. We will determine whether PCR amplification of our genes of interest occurred by running our samples on an agarose gel containing ethidium bromide. The ethidium bromide is an intercalating DNA dye that allows us to view the individual bands of DNA when exposed to UV light. Remember, agarose gels are used to separate DNA molecules based on their size and charge.

The two genes that we will be trying to amplify with the PCR in this lab are MMP-9 and ß-actin. ß-actin is one of the most abundant proteins found in eukaryotic cells (Xu, et al, 2010). It is a highly conserved and highly distributed cytoskeletal protein and is involved in many cellular processes that are common amongst all cell types, such as cell motility, growth, and cytokinesis (Xu, et al, 2010; Hofmann, 2009). ß-actin is referred to as a “housekeeping” gene because it is a highly distributed protein in many cell types and, because of this, it is great to use as a positive control when analyzing gene expression (Xu, et al, 2010). We will perform a PCR using primers that amplify a region of ß-actin cDNA for each experimental group. Since this “housekeeping” gene is highly distributed and conserved amongst eukaryotes, we should be able to successfully amplify ß-actin cDNA in both the PMA and DMSO treatment groups. Presence of amplified ß-actin cDNA on our agarose gel allows us to verify that our RNA was successfully isolated from the treated HL-60 cells and that the RNA successfully underwent reverse transcription to make cDNA.

We will also perform a PCR using primers that amplify a region of MMP-9 cDNA, which is a gene that is not expressed by undifferentiated HL-60 cells. MMP-9 is an enzyme that monocytes use to digest extracellular matrix proteins, to promote their motility in between cells as they move towards sites of an immune response. Presence of amplified MMP-9 PCR product would confirm that exposure of HL-60 cells to an experimental treatment induces transcription of MMP-9. Absence of amplified MMP-9 PCR product would suggest that transcription of MMP-9 is not induced by that sample’s experimental treatment. Based on our knowledge of HL-60 cells and the PMA signal transduction pathway, the only treatment that should yield amplification of MMP-9 cDNA should be the PMA treated group. This is because when our HL-60 cells are treated with PMA, they undergo differentiation into monocytes through the signal transduction process, which stimulates the upregulation of MMP-9 transcript production. DMSO causes the cells to differentiate into granulocytes which do NOT have an increase in MMP-9 transcription, so no MMP-9 amplification should occur from the DMSO treated group’s cDNA.

In order to look for production of mRNA transcripts of b-actin and MMP-9, we will utilize Reverse Transcription, and PCR (RT-PCR). First, we will isolate total RNA from our cells. RNA is very unstable due to the fact that it is single stranded, and its free hydroxyl (OH) group on the 2' carbon. The OH group makes RNA susceptible to hydrolysis, and its single stranded nature makes it very susceptible to ribonucleases (RNases). RNases are enzymes that digest RNA, and are present everywhere; they're on our lab bench, our hands, our pipette tips, they're ubiquitious! Thus, it is very important to use gloved hands and sterile equipment throughout this experiment. It also important to keep our samples on ice when they're not being used.

Because of RNA's instability, we also must convert RNA into cDNA using reverse transcription. The enzyme Reverse transcriptase will add nucleotides on to the ends of random decamers that we will add to our RNA. Random decamers are short primers (small DNA sequences) that will bind to various places on the strand of RNA. This will yield a strand of cDNA complementary to our RNA. This will then be amplified over and over, and strands will anneal to each other to become double stranded cDNA.

Next, we will perform PCR to amplify our genes of interest. The cDNA will then be mixed with a polymerase enzyme, dNTPs, and primers so that a particular region of the cDNA (which represents our original mRNA) may be amplified. A primer is a short, single-stranded oligonucleotide that is sequence-specific for a desired region of interest (i.e. a gene). A forward primer and a reverse primer are needed so that a region of interest will be specifically amplified. We will use two sets of primers: one specific to MMP-9, and one specific to β-actin. Once the cDNA is mixed with the various reagents, it is placed in a thermal cycler that will automatically control the steps of PCR.

The steps of PCR involve:

1. Denaturing the cDNA

2. Annealing the forward and reverse primers

3. Extending the primers to generate a product.

The first step involves heating the reaction to 95°C, which denatures the DNA by causing the two strands to “unzip”. The second step cools the reaction down to around 55°C(this temperature will vary based on the specificities of our primer set), enabling the primers to bind to the corresponding sequence on the DNA template. The third step involves heating the reaction up to 72°C, the temperature at which a polymerase enzyme will elongate the strand by adding on nucleotides to the end of the primers, complementary to the template strand. These three steps are repeated 30-35 times. As each cycle is completed, there is an exponential increase in the number of specific products, which may then be run on an agarose gel to be visualized. (Note: the polymerase enzyme must be heat-stable, or else the denaturing step would also denature the enzyme; a common polymerase used is Taq polymerase. Also note: an initial 2-5minutes of denaturing occur before the 3 cycling steps. This occurs at 95°C. After the 30-35 cycles of the three steps above, a prolonged elongation step occurs at 72°C, usually for 5 minutes.

Agarose gel electrophoresis: To view our RT-PCR products, we will view them on an agarose gel. Gel electrophoresis is a method used to separate molecules based on size and charge. Agarose gels are used to separate nucleic acids (DNA/RNA). We will insert our amplified PCR products into the wells of a gel made of agarose. Agarose comes in powder form. It can then be mixed with a buffer, boiled, and then poured into a mold, and allowed to cool. Once cooled, it forms a gelatin like product in which we can insert our amplified PCR products. The gel forms a matrix through which our PCR products can move. Our primers amplify a specific size of DNA. The larger this section is (the more nucleotide base-pairs it contains), the slower it will move down the gel, while smaller sections will move much more quickly down the gel. We'll place the gel in buffer, load our samples into the wells of the gel, and run an electrical current through the gel. (Note: samples will be mixed with loading dye, which contains glycerol. Glycerol will make our samples more dense than the buffer and inhibit them from leaking out of the well and into the buffer.)

A negative electrode is placed at the top of the gel, and a positive electrode is placed at the bottom of the gel. Due to the phosphate group of the DNA, it has an overall negative charge, so when we apply this electrical current, DNA will be attracted to the positive electrode at the end of the gel. The gel will also contain a dye that fluoresces in the presence of UV light (in our case, we'll be using Syber Safe). This allows us to visualize our PCR products on the gel (the brighter the band, the more PCR product, if analyzing at the same size as generally larger PCR products will be brighter due to more dye binding to it than smaller PCR products). A base-pair ladder will be inserted into the very first lane of our gel. Base-pair ladders contain DNA fragments of known base-pair lengths and allow us to compare our band to these bands of known sizes in the base-pair ladder. If we know that our PCR product should be 400 base-pairs, then we look on the base-pair ladder to see where 400 base-pairs will be on the gel. Then look at the lane we placed our sample in and see if we have a band adjacent to the 400bp band on the base-pair ladder.

Summary: One of the protein products of these cells is β-actin, which is produced by both granulocytes and monocytes. Monocytes, however, produce a protein not produced by granulocytes, matrix metalloproteinase-9, or MMP-9. MMP-9 is an enzyme that the monocytes use to digest matrix proteins. Remember, in this chapter we are performing a set of experiments to observe cellular differentiation at the transcriptional level (production of mRNA). We can see whether these genes are being transcribed by comparing the mRNA found in HL-60 cells treated with DMSO or PMA. In this series of experiments, we will 1) extract mRNA from HL-60 cells that have either been exposed to DMSO or to PMA; 2) convert the mRNA to cDNA using the reverse transcription reaction (RT reaction); 3) use PCR to amplify certain regions of the cDNA present, in order to see if the PCR products formed contain the expected gene products (β-actin and/or MMP-9 for untreated, DMSO, and PMA treated HL-60 cells).

Why do we need to isolate RNA, not DNA?

The same genomic DNA (gDNA) is present in all cells of eukaryotic organisms. The function of each particular cell is controlled by gene expression in response to a variety of genetic control mechanisms or signals. These instructions are transcribed from specific genes in the genomic DNA as messenger RNA (mRNA). The instructions are then translated into a polypeptide product in the cellular cytoplasm. The protein carries out the specific function. Regulation of these products can be executed at many different levels; therefore, gene expression should be investigated at the RNA level (transcriptional level) and at the protein level (translational level). In this way, we can analyze what genes are being expresssed at the time of RNA isolation!

Additional Information:

PCR Video

Electrophoresis Video

1. Annealing: pairing of DNA or RNA by hydrogen bonding to form a complementary double-stranded sequence

2. Denaturing: process of splitting the hydrogen bonds between complementary strands of DNA to form single strands

3. Extending: addition of nucleic acid bases to the oligonucleotide primer by Taq polymerase

4. Granulocytes: any of several white blood cells with granules in their cytoplasm that help in digestion of bacteria

5. Monocytes: the largest white blood cell, able to move outside of the vasculature in pursuit of invading pathogens

6. Multipotent: stem cells that are only capable of giving rise to a cells in a certain family of cell types (in the way that blood stem cells give rise to all blood cell types)

7. Oligonucleotide: a short single-stranded nucleic acid of fewer than 20 bases

8. Primer: small single-stranded nucleic acid molecule required for the initiation of DNA replication