Investigation of a link between microRNAs, cell senescence and Cancer

Background and Rationale

MicroRNAs (miRNAs) are small, single-stranded RNA molecules roughly 21-25 nucleotide long which negatively regulate gene expression. They have been shown to control cell growth, differentiation, and apoptosis. MiRNAs work in one of two ways to regulate gene expression, if the miRNA is perfectly, or near perfectly, complementary to the 3’-untranslated region (3’UTR) of the mRNA the RNA interference (RNAi) pathway is induced and the mRNA is cleaved by ribonucleases found in RISC (RNA-induced silencing complex) causing the mRNA to be degraded. The second mechanism of regulation occurs when the miRNA is only partially complimentary to the 3’-UTR of the mRNA. This method does not involve the degradation of the mRNA but represses the expression of the gene post-transcriptionally, and therefore prevents the mRNA transcript being translated, again using RISC. Through this method the level of the target protein decreases but the level of mRNA remains unchanged, with the first method both the level of mRNA and the level of protein decreases.

MiRNAs have been shown to function as both tumour suppressors and oncogenes, as they are able to act as both activators and inhibitors depending on the type of gene the miRNA is acting upon. If the miRNA acts as an inhibitor on an oncogene it is then acting as a tumour suppressor, conversely if the miRNA acts as an inhibitor on a tumour suppressor gene, it is inhibiting an inhibitor and therefore causing it to become an oncogene. Therefore this suggests a strong link between miRNAs and tumour formation. Lu et al (2005) observed a down regulation of certain miRNAs in tumours compared to normal tissue, this observation highlights the link between miRNAs and tumour formation and shows a potential for miRNA profiling in cancer diagnosis. He et al (2006) further proved this link after studying B cell lymphomas and a cluster of miRNAs, the miR-17-92 polycistron. It was observed that primary or mature miRNAs derived from this polycistron are substantially increased in B cell lymphomas compared to normal tissues. It was observed that when the expression of the miR-17-92 cluster was enforced, it acted with c-myc expression (a proto-oncogene which encodes a transcription factor that regulates cell proliferation, growth and apoptosis) and tumour development was accelerated in mouse B cell lymphoma models. He et al came to the conclusion that these studies indicate that miRNAs can modulate tumour formation, and that the miR-17-92 cluster is implicated as a potential human oncogene.

Example of how miRNAs act as tumour suppressors and oncogenes, taken from Caldas and Brenton (2005), Sizing up miRNAs as cancer genes.

Esquela-Kerscher and Slack produced a review in Nature (2006) detailing oncomirs, miRNAs with a role in cancer. In this review it was described that about 50% of annotated human miRNAs are located in regions of the genome, known as fragile sites, which are associated with cancer, again indicating that miRNAs might have a crucial function in tumourigenesis. Calin et al (2002) were one of the first groups to indicate miRNAs acted as tumour suppressors by studying patients diagnosed with B-cell chronic lymphocytic leukaemia (B-CLL), a common form of adult leukaemia. It was observed that in more than half these patients there were deletions or down-regulations of two clustered miRNA genes, miR-15a and miR-16-1.

P53 is a gene which encodes a transcription factor, that is involved in multiple cellular processes and regulates the cell cycle. It acts as a sensor for many cancer-associated stress signals, including DNA damage, telomere depletion and oncogene stress, and therefore functions as a tumour suppressor. P53 has been described as the guardian of the genome as it conserves stability by working to prevent mutations. P53 is able to detect DNA damage and work to repair it if possible by initiating survival pathways. If the damage to the DNA is too severe p53 is able to stop the cell cycle and move the cell into programmed cell death by inducing the apoptotic pathways. This shows that although p53 is mainly a transcriptional activator it also has the ability to work as an inhibitor, for example inhibiting apoptosis for a cell to survive. Mutations in p53 are associated with rapid tumour progression, with p53 being mutated in roughly 50% of cancers.

The p53 tumour suppressor network, taken from He et al (2007), The guardian’s little helper: MicroRNAs in the p53 tumour suppressor network.

Recent studies have highlighted miR-34 as a microRNA component of the p53 network. He et al (2007, a microRNA component of the p53 tumour suppressor network) is a key study in the establishment of MicroRNAs in the p53 tumour suppressor network. Wild type cells were compared with p53-deficient cells for miRNA expression, and it was found that expression of the miR-34 family of miRNA was related to p53 status. This allowed for the identification of miR-34s as an important p53 transcriptional target, which is able to regulate cell proliferation and apoptosis. The miR-34 family of miRNAs are directly induced by p53 in response to oncogenetic stress and DNA damage (illustrated in the figure above); this induction then blocks other functions such of that of BCL2 and the apoptotic molecules and Cyclin-Dependent Kinase 6 (CDK6) which is involved in cell cycle transit. MiR-34 reiterates the effects of p53 by inducing growth arrest and apoptosis by inhibiting the expression of pro-proliferation and anti-apoptotic genes. Extensive studies have been carried out to ascertain the regulation of miR-34 family by p53; both exogenous and physiological stresses are capable of miR-34 expression in a p53-dependent manner both in vivo and in vitro.

The following figure is adapted from He et al (2007, MicroRNAs join the p53 network – another piece in the tumour suppression puzzle) and shows the relationship between p53 and miR-34 and their roles in regulating cell proliferation and cell death.

MiR-34 has also been reported to be deleted in some human cancers. MiR-34a is located at 1p36, a region of frequent heterozygous deletion in many tumour types, as shown by Versteeg, R et al (1995). Minimal deletions containing miR-34b and miR-34c have also been shown in lung and breast cancer, evidence of which was observed by Calin et al (2004).

Normal human cells (i.e. not stem cells) don’t have telomerase activity, and so with every division telomeres at the end of chromosomes become smaller and smaller until they get to a stage where they are too small for the cell to divide anymore, at this point the cell is still viable but no longer dividing and has reached the stage of senescence. This uses the activation of critical tumour suppressor genes, namely p53. This process happens as the cell ages, the older the cell becomes the shorter telomeres become, and is recognised as an anti-cancer defence. The older a cell is the more susceptible the DNA is to damage, so the cell shuts down to stop the damage being passed on and therefore preventing tumour progression. Kumamoto, K et al (2008) observed that Nutlin-3a, an MDM2 inhibitor, is able to activate p53 to induce apoptosis in many types of cancer cells. Nutlin-3a is able to induce senescence by both activating and inhibiting a number of p53 dependent genes, including activating miR-34a, miR-34b and miR-34c and down-regulating inhibitor of growth 2 (ING2). Studying normal human fibroblasts and using chromatin immunoprecipitation, electrophoretic shift assays and monitoring Luciferase activity, Kumamoto and colleagues came to the conclusion that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, as well as concluding that p53-mediated miR-34a; miR-34b and miR-34c up-regulation and ING2 down-regulation may be involved in the senescence pathway.

Metalloproteinases are a group of enzymes which have protease activity, allowing them to cleave protein. Metalloproteinases work on the cell surface where they were thought to cut a path for the cancer cell to move through the extracellular matrix, but these proteases have very complex functions and it is not fully understood what function they perform.

Zmpste24 is a different sort of metalloproteinase which works inside the cell, as first observed by Carlos Lopez-Otin (2002). Zmpste24 is involved in the process of the maturation of nuclear lamin A which provides structural support and transcriptional regulation of the nuclear lamina, and therefore is an essential part of the nuclear lamina. Zmpste24 deficient mice exhibit accelerated aging and have severe nuclear structure abnormalities leading to a shortened life-span, as shown by Lopez-Otin et al (2005, Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling). A similar phenomenon is observed in humans, with Zmpste24 knock out humans suffering from Hutchinson Gilford Progeria Syndrome (HGPS), a condition that causes sufferers to exhibit accelerated aging. Lopez-Otin and colleagues decided to investigate the molecular mechanism that underlies these diseases. It was demonstrated that the Zmpste24 deficiency induces a stress signalling pathway that was discovered through an up-regulation of p53 target genes and a senescence phenotype at the cellular level and accelerated aging for the organism. It was also demonstrated that this phenotype could be rescued by re-introducing Zmpste24 in Zmpste24-/-, lamina+/- mice, and partially rescued in mice deficient in both lamin A and Zmpste24. These observations allowed Lopez-Otin to come to the conclusion that there is a checkpoint response which is activated by nuclear abnormalities caused by prelamin A accumulation, which also supports the theory that hyperactivation of p53 may cause accelerated aging.

The gap in the science that has been identified is whether Zmpste24 is a target of miRNAs, specifically the miR-34 family.

Hypothesis

Therefore from reading the literature I will be testing the hypothesis that the following pathway is correct:

Design and Methodology

During the research project I will be undertaking at least 5 experiments to test my hypothesis and will investigate the roles miR-34 and Zmpste-24 have in senescence and cancer. The experiments will be as follows:
1. Effect of miR-34 on endogenous Zmpste24 mRNA and protein

2. - Construct Luciferase reporter with 3’-UTR region of Zmpste-24 (containing putative miR-34)

- Transfect miR-34 and scrambled (mutated) control into vectors. Does miR-34 suppress reporter activity?

3. Mutate putative miR-34 site. Does this wipe out miR-34 suppression in the reporter?

4. Is there a relationship between miR-34 and Zmpste-24 in cancer cell lines

5. How does this relationship relate to p53 status?

A western blot will be used to investigate the effect of miR-34 on endogenous Zmpste24 protein in experiment to determine the concentration of Zmpste24 protein within the cell. This method is used as it allows a clear and easy comparison of the different concentrations of protein when different amounts of miR-34 are available.

In experiment 2 the reporter gene, Luciferase, will be transfected into cells and the activity will be measured. When Luciferase is active, i.e. the reporter gene has been turned on, light will be emitted. An extract of cells will be incubated in a luminometer which is able to measure the level of light. Luciferase is used over other reporter genes, for example GFP (green fluorescent protein) as it is a convenient assay system which can be easily manipulated and is widely used for these types of experiments. This method will also be used over other methods as the Biomedical Research Laboratory has a luminometer.

In each of these experiments I will be using normal, non-senescent human fibroblasts; most likely MRC-5 cells and I will be using the Targetscan program to determine the miR34 site.

Data Summaries and Analyses

Western blotting uses a gel which can be stained and then photographed to allow the different concentrations of protein to be visualised, when there is a bigger concentration of protein the band will be darker than a lesser concentration, the blot will be similar to:

Which has been adapted from Eulalio, A et al (2008) who used a western blot to show that GW182 interaction with argonaute is essential for miRNA mediated translational repression and mRNA decay.

Raw data from each of the experiments will be held in excel tables, these will then be used to construct histograms and line graphs as appropriate. The raw data will also be used in a standard T-Test which will allow me to statistically test whether my observations are significant and therefore whether my hypothesis is correct. Other statistical tests may be applied as appropriate, depending on the nature of the data generated. An example of how I will use data obtained from my experiments is shown using dummy data for experiment 2, below.

Concentration of miR-34 (µg)	Relative Light Units (x1000)
0	700
0.1	623
0.2	587
0.3	504
0.4	466
0.5	379
0.6	297
0.7	243
0.8	198
0.9	105
1	62

Mean of dummy data: 378.5

Standard Deviation of dummy data: 0.22

Planning Schedule

Experiment	Semester	Week(s)
1	1	2 - 4
2	1	4 - 8
3	1	8 - 12
4	2	1 - 4
5	2	4 - 7

As my research project is comprised of many smaller experiments instead of one long one they will be carried out over the two semesters, and therefore parts of my final project report and presentation will not be completed at a certain time, but on going as I complete each experiment. The components of my report that will be carried out continuously and the other components which have a more set time scale are shown in the table below.

Component	Date intended to carry out task
Literature Search	SEM1 WK 1-6
Testing Methodology	SEM1 WK 2-4
Preliminary Studies	ONGOING AS PER EXPERIMENT TIMETABLE
Data Collection	ONGOING THROUGHOUT YEAR
Preparation of Progress Report	SEM1 WK 4-7
Data Analyses (Expt 1 - 3)	ONGOING THROUGH SEMESTER 1
Data Analyses (Expt 4- 5)	ONGOING THROUGH SEMESTER 2
Preparation of first draft of written report	EASTER BREAK - SEM2 WK 1
Drafting written report	SEM2 WK 2-5
Preparation of oral presentation	SEM2 WK 5-9

Component	Submission Deadline
1st blog assessment	SEM1 WK 1-2
2nd blog assessment	SEM1 WK 11-12
3rd blog assessment	SEM2 WK 3-4
Submit report	SEM2 WK 9
Presentation	SEM2 WK 9

I have produced a Gantt chart to illustrate more clearly the time periods over which each component of the project will be carried out. The Gantt chart is present in days, with day 0 being Monday SEM1 WK1 (21/9/09). I have designed my Gantt chart so it does not include weekends or holidays, therefore a week is presented as 5 days. Using this method the project is scheduled to end on day 100, which is Monday SEM 2 WK 9 (08/03/10), as this is the deadline for both the completed report and the presentation.

The project may potentially be disrupted if the cloning in experiment two does not work correctly. Successfully cloning a vector which has been transfected with new genes is a very rare event, and quite often the genes will not be transfected properly and therefore no reporter activity will be present. If this happens this part of the experiment needs to be repeated until an adequate amount of cells show successful transfection.

Relevance

This project is relevant to the wider field of cancer biology as if the results confirm my hypothesis it will give a wider understanding of the process of senescence, more specifically senescence as an anti-cancer defence. This potentially could allow for drugs to be developed which mimic miR-34 function, inhibiting Zmpste24 function causing the cell to become senescent.