microrna-29 expression in various cancer types · page 1 of 36 microrna-29 expression in various...

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MicroRNA-29 expression in

various cancer types

5.semester, efterår 2011

Studieretning: Molekylærbiologi og Medicinalbiologi

Af Mette Neiegaard Witt & Stine Herold Madsen

Vejleder: Annika Bagge

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Index Abstract ...................................................................................................................................... 4

Resume ....................................................................................................................................... 4

Introduction ................................................................................................................................ 5

Problem statement .................................................................................................................. 5

MicroRNAs ............................................................................................................................ 6

MicroRNA biogenesis ........................................................................................................ 6

Insulin-signaling pathway and type 2 diabetes mellitus......................................................... 8

The role of miR-29 in type 2 diabetes mellitus .................................................................... 10

The correlation of glucose concentrations and miR-29 .................................................... 12

miR-29 and the insulin-signaling pathway ....................................................................... 14

Cancer and the cell cycle ...................................................................................................... 14

Cyclins and cyclin dependent kinases .............................................................................. 15

Analysis.................................................................................................................................... 16

miR-29 and cancer in insulin-responsive tissues ................................................................. 16

Muscle, rhabdomyosarcoma ............................................................................................. 19

Pancreas, endocrine tumors .............................................................................................. 20

miR-29 and cancer in non-insulin-responsive tissues .......................................................... 20

Lymphocytes, B-cell chronic lymphocytic leukemia ....................................................... 20

Lymphocytes, AML.......................................................................................................... 21

Bile duct, cholangiocarcinoma ......................................................................................... 23

Is miR-29 a tumor suppressor, an oncomiR or both? ........................................................... 24

Summative discussion and conclusion..................................................................................... 27

The connection between miR-29a, 29b1, 29b2 and 29c. ..................................................... 27

Using miRNA in cancer treatment ....................................................................................... 27

Models vs. the human body.................................................................................................. 28

The role of miR-29 in cancer development .......................................................................... 29

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Perspective ............................................................................................................................... 29

Refferences .............................................................................................................................. 30

Appendix 1 ............................................................................................................................... 36

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Abstract

The recent discovery of microRNA’s has presented a new pool of regulators of diverse

pathways of the human body. It is suggested that miR-29a has negative effects on some of the

steps in the insulin-signaling pathway. This indicates that clinical regulation of miR-29a

could be part of type 2 diabetes mellitus treatment. With every clinical regulation of

pathways of the body risks of undesirable consequences may follow. These consequences

could be numerous but cancer is a frequent result of pathways being deregulated, and

therefore we conducted a literature study of the miR-29 family and its expression levels in

various cancer types. Several experiments showed that all three miR-29 paralogs were

expressed in increased or decreased levels in different cancer types compared to the

corresponding normal tissue. The studies showed that miR-29 was down-regulated in brain,

lung, liver, cervix, muscle and stomach cancers, and up-regulated in cholangiocytic, prostate,

pancreatic and lymphocytic cancers with possible exceptions. Thus no overall tendency of an

up- or a down-regulation of miR-29 in cancer was found. But it seems that that miR-29 do

play a role in cancer development though the role differs between cancer types. miR-29 could

thereby possibly be a part of cancer treatment if it can be regulated tissue specifically.

Resume

Den nye opdagelse af microRNA’er har frembragt en ny pulje af regulatorer i

menneskekroppens mange signalveje. Det er blevet foreslået, at miR-29a har en negativ

effekt på nogle af trinene i insulinsignalvejen. Dette indikerer, at en klinisk regulering af

miR-29a kunne blive en del af type 2 diabetes mellitus behandling. Med enhver klinisk

regulering af signalveje i kroppen følger risici for uønskede konsekvenser. Disse

konsekvenser kan være mange, men kræft er et typisk resultat af dysregulerede signalveje, og

derfor laver vi her et litteraturstudie over miR-29 familien og dens ekspression i forskellige

kræfttyper. Flere eksperimenter viste at alle tre miR-29 paraloger blev udryk i øgede eller

mindskede niveauer i forskellige kræfttyper sammenlignet med det korresponderende

normale væv. Studierne viste, at miR-29 var nedreguleret i hjerne-, lunge-, lever-,

livmoderhals-, muskel- og mavekræft og opreguleret i cholangiocyt-, prostata-, bugspytkirtel-

og lymfocyt-kræft med mulige undtagelser. Der er altså ingen overordnet op- eller

nedreguleringstendens af miR-29 i kræft. Men det ser ud til, at miR-20 alligevel spiller en

rolle i kræftudvikling selvom rollen varierer fra kræfttype til kræfttype. miR-29 kunne

dermed være en del af kræftbehandling, hvis den kan reguleres vævsspecifikt.

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Introduction

MicroRNAs (miRNAs) are a recently discovered pool of regulators in vertebrates,

invertebrates and plants (Laegos-Quintana et al., 2001; Lee, 1993; Chen, 2005). It has been

brought to our attention that the miRNA miR-29 might have a negative regulatory effect on

the insulin signalling pathway. This indicates a possibility of utilizing down-regulation of

miR-29 in diabetes mellitus type 2 (T2DM) treatments. Therefore we started out by

investigating literature regarding miR-29, the insulin signalling pathway and the connection

between miR-29 and T2DM. Even though a down-regulation of miR-29 might help people

suffering from T2DM, clinical regulation of pathways in the body could have undesired

effects. It is therefore important to consider the possible consequences of interfering with the

pathways before doing so. The consequences could have great diversity but we have chosen

to focus on the coherence of cancer and the expression of miR-29 because this field of

investigation is gaining more and more ground.

Problem statement

• Do expression levels of miR-29 play a role in cancer development?

Investigation of the expression levels of miR-29 in various cancers and the possibility of

using regulation of miR-29 in cancer treatment

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MicroRNAs

MicroRNAs (miRNAs) are single stranded non-coding RNA’s that do not translate into

proteins like regular RNAs do. They function either as a silencing molecule by suppressing

the translation of a target-mRNA, or as a direct degrader of a target-mRNA (Kolfschoten,

2009). The most important sequence for miRNA target binding is called the seed region and

is located between nucleotides 2-8 in the

5’end of the miRNA (figure 1).

Complementarities between the seed

sequence and the target sequence is required

for the miRNA to bind to the mRNA

(Lynam-Lennon, 2008). miRNAs are

divided into families according to identical

seed sequences (Ambros, 2003). The miR-

29 family is comprised by three paralogs; miR-29a, 29b and 29c. miR-29a and miR-29b1 are

located on chromosome 7q32 whereas miR-29b2 and miR-29c are located on chromosome

1q23 (Garzon, 2009). miR-29b1 and miR-29b2 sequences are identical but they are

distinguished as b1 and b2 due to the difference in locus. Some miRNAs are tissue specific

(Lagos-Quintiana, 2002) but miR-29 is present in many different tissues (Xiong et al., 2010;

Roldo et al., 2006; Zhao, 2010; Mott et al., 2007).

MicroRNA biogenesis

MicroRNA biogenesis describes the process of miRNA maturation. The first step in the

biogenesis is transcription of the gene encoding the miRNA. miRNAs are encoded in the

genome and are transcribed by RNA Polymerase II and the primary transcript is called pri-

miRNAs (Lynam-Lennon, 2009). Pri-miRNAs have a characteristic shape, with one or

several stem-loop structures (figure 2). They have a 5’-cap and a polyadenylated 3’ end like

mRNAs and they consist of a couple of thousands nucleotides (Kolfschoten et al., 2009). The

pri-miRNA is processed by a microprocessor complex consisting of two molecules; the

RNAse Drosha and the protein DGCR8. The Drosha-DGCR8-complex reduces the pri-

miRNA to a pre-miRNA of around 80 nucleotides, by excising the hairpins from the long pri-

miRNA. The pre-miRNA is translocated out of the nucleus and into the cytoplasm by the

transport protein exportin-5. A complex consisting of three units: an RNAse named Dicer, a

Figure 1: The sequences of the microRNA-29 family Families of miRNAs have very similar base sequences and identical seed sequences. The seed sequence is located between nucleotide 2 and 8 (Modified from He et al., 2007).

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RNA-binding protein and an endonuclease named Argonaut-2 (Ago-2) cleaves the loop from

the stem yielding a double stranded miRNA in the cytoplasm (Lynam-Lennon, 2009). The

duplex is now unwound by RNA helicase leaving only the mature miRNA consisting of 21-

23 nucleotides and Ago-2. The unwinding yields two strands, and either of them could be the

future mature miRNA, but the strand with the most stable 5’ end is degraded (Winter, 2011).

The mature miRNA is incorporated into the RNA-induced silencing complex (RISC) and the

miRNA guides the RISC complex to the miRNA target-sequence located in the 3’UTR of

their mRNA target (Lynam-Lennon, 2009; Winter, 2011). Binding of the miRNA-RISC

complex to the mRNA target leads to either degradation or translational repression of the

mRNA (Winter, 2011).

Figure 2: microRNA biogenesis The microRNA primary transcript (pri-miRNA) is processed by Drosha/DGCR8 yielding precursor miRNA (pre-miRNA) with a characteristic stem-loop structure. Exportin-5 translocates pre-miRNA from the nucleus into the cytoplasm. Dicer complex cleaves of the loop yielding to complementary strands. RNA helicase unwinds the double strand. The unwinding yields two strands, and either of them could be the future mature miRNA, but the strand with the most stable 5’ end is degraded. The single-stranded mature miRNA is incorporated into the RISC complex, followed by binding of the miRNA to the target mRNA. (Modified from http://www.bioscience.org/2009/v14/af/3412/fulltext.asp?bframe=figures.htm&doi=yes).

http://www.bioscience.org/2009/v14/af/3412/fulltext.asp?bframe=figures.htm&doi=yes�

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Because the miRNA most often is only partially complementary to their targets, the number

of potential targets is abundant (Shreenivasaiah, 2010). The field of miRNA-research is rather

new (Ambros, 2003), but several studies indicate that miRNAs are involved in regulating

many different pathways (Pekarsky, 2006; Garret, 2001; Wang, 2008; Garzon, 2009; Park et

al., 2009). Focus in this report is primarily on miR-29 and the correlation between miR-29

expression and cancer. Secondary focus is on the insulin signalling pathway and the

correlation of T2DM and miR-29, from where our motivation came.

Insulin-signaling pathway and type 2 diabetes mellitus

The body and in particular the brain and nervous system needs a constant supply of glucose

for optimal functioning. To maintain a constant glucose level in the blood, β-cells in the islets

of Langerhans in the pancreas produce and secrete the hormone insulin in response to

elevated glucose levels that follow food intake (Seeley, 2011; Hancock, 2010; Madsbad,

2007; Dirice, 2011). Insulin increases the uptake of glucose from the blood primarily into

muscle and adipose tissue by binding to the α-subunit of insulin receptors (IRs) on the surface

of the tissue cells. Binding to the α-subunit induces intracellular phosphorylation of the

transmembrane β-subunit, causing autophosphorylation of IR. These events lead to

translocation of intracellular vesicles containing the transmembrane glucose-transport protein

GLUT4 (figure 3). GLUT4 travels to the surface of the cell where it fuses with the plasma

membrane (Hancock, 2010; Skelly, 2006; Le Roith et al., 2003; Seeley, 2011).

Figure 3: The effect of insulin secretion on GLUT4 Insulin binds to the insulin receptor (IR) on the cell surface of muscle- and adipose tissue and induces a signaling pathway ultimately leading to translocation of glucose-transport protein GLUT4 from vesicles inside the cell to the cell membrane with which it fuses (modified from Hancock, 2010)

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The insulin-signaling pathway consists of many steps in between binding of insulin to IR and

translocation of GLUT4. miR-29 seems to regulate some of the steps and they are therefore

described in more details here and presented in figure 4. The binding of insulin to IR leads to

activation of insulin receptor substrates (IRS) which activates PI3-K. PI3-K has a regulatory

subunit called p85 and a catalytic subunit called p110 (Karlsson, 2007). When IRS binds to

p85 it leads to activation of p110 which catalyses phosphorylation of phosphatidylinositol

(3,4)-biphosphate (PIP2) yielding phosphatidylinositol (3,4,5)-triphosphate (PIP3) (Karlsson,

2007). The serine/threonine protein kinase Akt binds to PIP3 in the membrane and is then

phosphorylated and activated by either phosphoinositide dependent kinase 1 (PDPK1) at

Thr308 or rapamycin complex 2 (mTORC2) at Ser473 (Whitehead et al., 2000; Burén, 2003; Le

Roith et al., 2003). When Akt is activated it stimulates the translocation of GLUT 4 in the cell

(Karlsson, 2007; Whitehead et al., 2000; Lund, 2007).

Figure 4: Translocation of GLUT4 by the insulin signaling pathway The drawing shows some of the steps of the insulin-signalling pathway where binding of insulin ultimately leads to translocation of GLUT4. Insulin binds to insulin receptor (IR) thereby activating insulin receptor substrates (IRS) which binds to the p85 subunit of PI3-K. p85 then activates another subunit of PI3-K; p110, which leads to phosphorylation of phosphatidylinositol (3,4)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). Akt then binds to PIP3 and is thereby phosphorylated by phosphoinositide dependent kinase 1 (PDPK1) or rapamycin complex 2 (mTORC2) and this stimulates the translocation of GLUT4 to the cell surface (own drawing, 2011).

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Insulin plays another and just as important role besides increasing glucose uptake. Insulin

inhibits glyconeogenesis and also induces glycogenesis in the liver thereby storing more

glucose as glycogen. Glucose enters the liver through glucose transport protein 2 (GLUT2)

which is not insulin dependent but has a low affinity for glucose. The activity of GLUT2 is

therefore proportional to the concentration of glucose in the blood (Lund, 2007).

T2DM is characterized by β-cell dysfunction and decreased insulin response. Although there

are still disagreements on the field it seems as if the β-cell deterioration happens in early

stages of the disease before the reduction in insulin response (Kahn, 2003). This deterioration

reduces the performance of the β-cells thereby decreased insulin secretion which leads to

diminished ability of muscle and adipose tissue to take up glucose. Although not well

understood it seems as if a constant high blood glucose level ultimately leads to reduced

insulin response and also to negative alterations in insulin secretion (Kahn, 2003). Decreased

insulin secretion together with decreased uptake of glucose causes the glucose concentration

to rise, which again has a negative effect on glucose-uptake and insulin secretion, and this

cascade results in a vicious circle (figure 5).

Figure 5: The effects of β-cell dysfunction Constantly elevated glucose levels down-regulate glucose uptake in muscle and adipose tissue, and decrease insulin secretion, and both events lead to further elevation of glucose (Own drawing, 2011).

The role of miR-29 in type 2 diabetes mellitus

The first scientists to discover a difference in miR-29 expression in T2DM liver, muscle and

adipose tissue compared to non-diabetic tissue of the same type was He et al. (2007). By

Northern blot they found an up-regulation of miR-29a, 29b and 29c in skeletal muscle tissue

from the hyperglycemic, diabetic Goto-Kakizaki (GK) rats compared to the normoglycemic,

non-diabetic Wistar Kyoto (WKY) rats. An up-regulation of miR-29a, 29b and 29c was also

observed in adipose tissue and liver, but the difference in miR-29 expression was not as

profound as for skeletal muscle tissue (figure 6).

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Figure 6: Northern blot analysis of miR-29a, 29b and 29c expression in muscle, adipose tissue and liver from GK and WKY rats MiR-29a, 29b and 29c are up-regulated in muscle-, fat- and liver tissue samples from GK (G1, G2, G3, G4 and G5) compared to WKY (N1 and N2). U6 is used control, and here it validates the elevated miR-29 expression levels in the Nothern blot (He et al., 2007).

The science group of Herrera et al. (2009) compared miR-29 expression in liver and adipose

tissue from GK rats and Brown Norway (BN) rats. BN rats are normoglycemic just as the

WKY rats, but they have lower plasma glucose concentrations. miR-29a in the GK rats was

increased by a 1,51 fold in adipose tissue and miR-29c was increased by a 1,5b fold in liver.

These results came from microarrays but were not validated. Microarray is a way of

investigating the expression levels of many miRNAs at the same time. It is an indicator of

differential miRNA expression of different miRNAs. Different oligonucleotide probes are

spotted onto a chip and a miRNA sample is spread onto the chip. The microRNAs are labeled

with a fluorescence gene and the more miRNA that binds to a probes, the more it will light

up. Microarray must be validated because the oligonucleotide probes not only bind to the

mature miRNA but also pri-miRNA and pre-miRNA, and only the mature miRNA are of

interest. Hence, validation by either Northern blotting where the pri-, pre- and mature miRNA

are separated by size or by real-time RT-Q-PCR with primers for the mature miRNA are

necessary. real-time RT-Q-PCR is short for real time Reverse Transcriptation Quantitative

Poly Chain Reaction. real-time RT-Q-PCR is a method used for amplification and

quantification of small RNAs.

A study (Herrera, 2010) combined GK, BN and WKY rat models and found a 1,18 fold

increase of miR-29a expression in adipose tissue from GK compared to BN, which supports

the findings of Herrera et al. (2010). miR-29a expression was reduced by a 1,68 in BN

compared to WKY, and unexpectedly reduced by a 1,42 fold in GK compared to WKY

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(table 1) (Herrera, 2010). This contradicts the results from He et al. (2007) who found

elevated expression of miR-29 in GK compared to WKY.

GK/BN BN/WKY GK/WKY

Tissue miRNA Fold change p value Fold

change p value Fold change p value

Adipose miR-29a 1,8 2,8*10-1 -1,68 3,3*10-4 -1,42 4,0*10-2

Table 1: Comparison of miR-29a expression in GK, WKY and BN rats Expression of miR-29a is increased by a 1,8 fold in GK compared to BN and is decreased 1,42 fold compared to WKY. Expression of miR-29a is decreased by a 1,68 fold in BN compared to WKY. The results were validated by real-time RT-Q-PCR (Extract from Herrera, 2010).

Yet another group of scientists found that miR-29a levels were increased in diabetic liver

tissue, this time by using db/db mice liver compared to the control (Pandey et al., 2011). The

results of the experiment were validated by Northern blot and real-time RT-Q-PCR (Pandey

et al., 2011). Muscle and adipose tissue were not included in this trial. db/db mice are obese

and not lean as the GK rats (Srinivasan, 2007). Obesity induces stress in cells and can

therefore possibly alter expression levels of different transcripts. One could therefore argue

that GK rats constitute better models than db/db mice, but in this case the experiments

showed an up-regulated level of miR-29a in liver from both db/db nice and GK rats.

The correlation of glucose concentrations and miR-29

Expression of miR-29a and 29b is up-regulated in 3T3-L1 adipocytes under hyperglycemia

(25mM) and hyperinsulinemia (100nM) compared to physiologically normal conditions

involving a homeostatic glucose concentration of 5mM and no insulin (He et al., 2007). The

up-regulation was validated by Northern blot. Another group of scientist also showed

elevated levels of miR-29a in 3T3-L1 adipocytes when glucose concentrations rose from

5mM to 25mM (insulin was kept constant at 100nM) (Herrera, 2010). An interesting

observation was that miR-29 expression was elevated when glucose concentrations rose from

the 5mmol/L to 15mmol/L, but a further elevation of glucose concentration to 25mmol/L had

no significant effect on miR-29a expression (Herrera, 2010). real-time RT-Q-PCR was used

to validate this finding. It was also suggested that 3T3-L1 adipocytes transfected with miR-29

reduced glucose uptake by approximately 50%, although this result was not validated (He et

al., 2007).

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Unpublished data from A. Bagge et al. (2011) have shown that elevated levels of glucose in

human islets of Langerhans and in the β-cell cell line INS-1E increased the expression of

miR-29a (p<0,05 and p<0,001 respectively). Over-expression of miR-29a in INS-1E

decreased Glucose-Stimulated Insulin Secretion (GSIS) in both high glucose and low glucose

concentrations. RT-Q-PCR was used as validation method.

If elevated miR-29a expression decreases glucose uptake in 3T3-L1 adipocytes and reduces

GSIS in INS-1E, and elevated glucose concentrations increases miR-29a expression in 3T3-

L1 adipocytes and INS-1E, and elevated miR-29a expression in β-cells reduces insulin

secretion it might indicate a role of miR-29a in the vicious circle mentioned earlier (figure 5).

It is possible to imagine miR-29a also having an effect on glucose uptake in muscle tissue

although experiments on the connection between miR-29a and glucose uptake in muscle

tissue have not been conducted. We have chosen to include muscle tissue in figure 7 below

that shows a possible connection between elevated glucose concentrations, the de-regulation

it leads to and miR-29a.

Figure 7: The possible effect of miR-29a in the vicious circle of elevated glucose levels in β-cells, muscle and adipose tissue Elevated glucose levels down-regulate glucose uptake in muscle and adipose tissue, and decrease insulin secretion, and both events lead to further elevation of glucose. An increase in glucose concentration in β-cells, 3T3-L1 adipocytes (and possibly in muscle tissue as well) elevates miR-29a expression in these tissues which leads to a decrease in glucose uptake (not validated) and insulin secretion. Again both of these events lead to an increase in glucose levels and thereby also in miR-29a levels. (Own drawing, 2011)

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miR-29 and the insulin-signaling pathway

miR-29a targets p85α, the regulatory subunit of PI3K in a human hepatocellular cell line

called HepG2 (Pandey et al., 2011). When miR-29a targets p85α, p85αt cannot activate p110

and this leads to inhibited phosphorylation of PIP2 to PIP3. To activate translocation of

GLUT4 to the cell surface Akt must bind to PIP3. Since the amount of PIP3 is reduced

glucose uptake will be diminished due to the lacking translocation of GLUT4.

Comparison of the results from He et al (2007), Herrera et al. (2009) , Herrera (2010),

Pandey et al. (2011) and Bagge et al. (unpublished data, 2011) indicate that down-regulation

of miR-29 could be a possible treatment of T2DM. But regulation of the complicated systems

of the body might induce undesirable results, and it is therefore important to investigate

possible consequences of such interferences. We have chosen to take a closer look at the

coherence between miR-29 and cancer, as this a field attracting more and more attention. To

understand a possible role of miR-29 in cancer development it is convenient to know

something about the cell cycle of cancer for which reason it is described below.

Cancer and the cell cycle

The majority of human cells are in a resting or non-proliferating state; G0. Due to the fact that

the cell is not dividing in this state it is not a phase of the cell division cycle. The cell division

cycle is divided into four phases: G1, Synthesis (S), G2 and Mitosis (M). Cells stimulated by

growth factors enters G1 thereby beginning the cell cycle (figure 8). During S the

chromosomes are replicated and during M the cell divides into two identical daughter cells

(Campbell, 2008). Five checkpoints around the cycle regulate the transition from one phase

to the following. The checkpoints only allow the cycle to proceed if the previous phases have

progressed correctly. The checkpoints monitor the phases by detecting signals such as DNA

damage (Garret, 2001; Campbell, 2008; Vermeulen, 2003). One checkpoint; the restriction

point is often referred to as the “point of no return” because the cell is committed to enter the

cell cycle and proceed throughout the cycle if it passes this checkpoint (Murakami, 2001;

Garret, 2001). The cells need growth factor stimulation to pass the restriction point.

Afterwards growth factors are no longer required (Murakami, 2001; Garret, 2001). Only the

restriction point is of interest to this report and the four other checkpoints are therefore not

further described.

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Figure 8: The cell cycle and its checkpoints The cell cycle starts from G1 proceeding towards

the final phase of mitosis (M). The cell cycle is

divided into four phases: G1, S, G2 and M. Another

fifth phase, not part of the cell cycle is G0 which is

a non-proliferation phase. Besides the transition

states, different checkpoints around the cycle are

monitoring whether the phases have progressed

correctly. The restriction point (R), the G1/S DNA

damage checkpoint, the S Phase DNA checkpoint,

the G2 DNA damage checkpoint and the mitotic

checkpoint (Modified from Lynam-Lennon, 2008).

Normal cells that follow the general aging pattern of the cell cycle proliferate into a

monolayer and require attachment to a matrix to grow. Cancer cells do not have the same

requirements. Cancer cells proliferate without growth factors, do not require attachment to a

matrix and can proliferate to become more than a single layer such as a clot (Campbell,

2008). Cancer appears when one or more checkpoints of the cell cycle are deregulated

(Garret, 2001).

Cyclins and cyclin dependent kinases

In addition to the checkpoints the cell cycle is also controlled by cyclins and cyclin dependent

kinases (CDK’s) (Campbell, 2008). There are different types of cylins; A, B, D and E. The

different types of cyclins are present during different stages of the cycle and in varying levels.

The level of a specific cyclin tops just before a transition state (Vermeulen, 2003).

The enzymatic activity of the CDK’s is regulated through phosphorylation, binding to a

partner cyclin (Garret, 2001) and through binding to two CDK-inhibitors; Cip1/Kip1 and

INK4 (Murakami, 2001). Inactive CDK’s are located in the cytoplasm and the levels are

constant throughout the cycle (Collins, 1996). A connection between certain CDK’s and miR-

29 has been found, and will be described in section “Lymphocytes, AML”.

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Analysis

If down-regulation of miR-29 is a possible treatment to T2DM patients it may only be

necessary to down-regulate it in insulin-responsive tissues. Therefore the first part of the

analysis will concern these tissues except for adipose tissue as to our knowledge no

experiments have been made concerning this insulin-responsive tissue. Connections between

miR-29 and cancer in non-insulin responsive tissues have also been detected and this is

interesting in case miR-29 cannot be down-regulated tissue-specifically. Therefore articles

regarding non-insulin responsive tissues will be analyzed in the section “miR-29 and cancer

in non-insulin-responsive tissues” below.

The scientists of the many articles describe a few targets of mir-29 in their articles, and they

are in a directory of targets of miR-29 in appendix I. Some of them will be described in the

analysis where relevant, and the rest will only be presented in the directory.

miR-29 and cancer in insulin-responsive tissues

Liver, hepatocellular cancer

A study conducted by Xiong et al. (2010) has shown reduced levels of miR-29 in

HepatoCellular Carcinoma (HCC) cells compared to normal liver cells. The differential

expression of miR-29 was investigated by Northern blot

in four human hepatocellular carcinoma cell lines:

HepG2, QGY-7703, MHCC97H and SMMC-7721.

When looking at the Northern blot in figure 9 it could

seem as if miR-29 was not particularly down-regulated

in the HCC cell lines compared to normal liver cells, but

the U6 control line at the bottom shows weaker

expression for normal liver cells than the other samples,

indicating that the down-regulation postulate is right.

Xiong et al., (2010) validated the down-regulation by RT-Q-PCR. The control line should

always be equally expression in every tissue, and a smaller expression in one lane could

indicate that a smaller amount of RNA (including the control) was administered here.

Figure 9: miR-29 in HCC Northern blotting showed a reduction in miR-29 levels in normal liver tissue compared to HCC cell lines. (Modified from Xiong et al., 2010).

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A detection of a down-regulation of miR-29 in HCC cell lines says nothing about which

came first; the decreased mir-29 expression or the cancer? A group of scientists; Braconi et

al. (2011) showed that the decrease in miR-29a might be first on the scene. They found that

miR-29a up-regulates the gene MEG3 and down-regulates the DNA methyltransferases

DNMT-1 and DNMT-3B in HCC. A decrease of MEG3 was found in HCC cell lines and

down-regulation of DNMT in human primary HCC increases MEG3 which suggests that

miR-29a might target MEG3 through DNMT (figure 10). Braconi et al. (2011) validated all

the experiments by real-time RT-Q-PCR. All together these results indicate an increase in

miR-29a being a source in the development of HCC.

Figure 10: The connection between miR-29a and the possible development of HCC miR-29a down-regulation increases DNMT expression and reduces the expression of MEG3 which has been shown to correlate with the development of HHC (Own drawing 2011).

miR-29 also targets Bcl-2 and Mcl-1 in HCC, this time shown in HepG2 and HEK293T cells

(Xiong et al., 2010). Bcl-2 and Mcl-1 are two apoptosis regulating proteins. Bcl-2 is encoded

by the gene BCL-2 and Mcl-1 is encoded by the gene MCL-1. The targets were predicted in

silico and validated by Luciferase (Luc.) assay. Wild type MCL-1-3’UTR, BCL-2-3’UTR,

mutated MCL-1-3’UTR or mutated BCL-2-3’UTR was cloned into one plasmid each

downstream of a parental Luc. gene. HepG2- and HEK293T cells were transfected with one

of the plasmids and then with mature miR-29 (figure 11).

Figure 11: Luciferase assay on Mcl-1 genes: MCL-1 Wild type MCL-1-3’UTR or mutated MCL-1-3’UTR is cloned into a plasmid downstream of a parental Luciferase gene. Cancer cells are transfected with plasmids cloned with either wild type or mutant and then with mature miR-29 (Own drawing, 2011).

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If miR-29 targets Bcl-2 or Mcl-1 there will be a decrease in Luc. activity in cells transfected

with wild-type compared to the mutant. The mutated MCL-1-3’UTR and BCL-2-3’UTR is

formed by point mutation, differing in only a few nucleotides from the wild type. The less

mutated the sequence is, the less nucleotides differ between the wild type 3’UTR and the

mutated 3’UTR, and the more precise the examination of the respective target is. Xiong et al.

(2010) do not state the number of mutated nucleotides.

As shown in figure 12A1 from Xiong et al. (2010) the Luc. activity is significantly (p<0,001)

decrease in cells transfected with wt-BCL-2-3’UTR when compared to the Negative Control

(NC). Figure 12A2 shows only a small decrease in Luc. activity in cells transfected with

mutated wt-BCL-2-3’UTR compared to NC. The cells transfected with wt-BCL-2-3’UTR

shows a reduced Luc. activity compared to the mutated BCL-2-3’UTR. This shows that miR-

29 binds to the wild type and not to the mutated type, hence it has been shown that miR-29

targets Bcl-2. Figure 12B1 and B2 shows the same tendency for Mcl-1. The cells transfected

with wt-BCL-1-3’UTR creates a significantly (p<0,01) reduced Luc. activity compared to

mutated MCL-1-3’UTR, showing that miR-29 targets Mcl-1. Used as control in A1, A2, B1

and B2 Luc. assays were HepG2 and HEK293T cells transfected with a non-humane

nucleotide sequence (Xiong et al,. 2010).

Figure 12: miR-29 targets Bcl-2 and Mcl-1 (A1+A2) Luciferase (Luc.) assay was conducted on HepG2- and HEK293T cells (cancerous cells). Negative control (NC) was none-human nucleotides. Cells transfected with wild type BCL-2-3’UTR showed a diminished Luc. activity compared to the NC. Cells transfected with mutated BCL-2-3’UTR showed no significant (***= p<0,001) decrease in Luc. activity indicating that miR-29 targets Bcl-2 (Modified from Xiong et al., 2010). (B1+B2) Cell transfected with wild type MCL-1-3’UTR and mutated MCL-1-3’UTR. Wild type transfected cells show a significant decrease (**= p<0,01) in Luc. activity compared to mutated transfected cells, showing that miR-29 targets Mcl-1 (Modified from Xiong et al., 2010).

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Control cells must always undergo the same treatment as the cells of investigation to make

sure that it is not very event of transfection that causes the differences in miRNA expression.

Non-human nucleotide sequences are often used because it is assumed that they cannot target

anything in a human cell.

Mcl-1 exists in three different isoforms. Mcl-1 isoform 1 inhibits apoptosis and is thus MCL-

1 isoform 1 is an oncogene. Mcl-1 isoform 2 and 3 both induces apoptosis and are thus tumor

suppressor proteins (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=Show

DetailView&TermToSearch=4170). Xiong et al., (2010) showed a reduced level of miR-29

in HCC cells. A reduced level of miR-29 results in an increase in Mcl-1 and due to the cancer

development this Mcl-1 must be isoform 1. When miR-29 is down-regulated Mcl-1 isoform 1

is up-regulated resulting in a reduction of apoptosis repression. Repression of apoptosis

causes a diminished cell cycle arrest of the cells leading to uninhibited cell proliferation.

Muscle, rhabdomyosarcoma

Wang et al. (2008) showed a down-regulation of miR-29b2 and 29c in Rhabdomyosarcoma

(RMS) tumors versus normal human skeletal muscle, and also a down-regulation in RMS cell

lines (RH28, RH30, RH3, CW9019, SMS-CTR, RD2 and RH36) versus normal human

skeletal muscle cells. The down-regulations were validated by real-time RT-Q-PCR in both

trials. By Luc. assay they found that miR-29b2 targets Yin Yang 1 (YY1) in skeletal muscle

tissue and also that YY1 regulates mir-29b2 in a positive feedback mechanism. YY1 is a

regulatory protein of the NF-κB-pathway and it acts by inhibiting skeletal muscle cell

myogenesis (Wang et al., 2007). Activation of NF-κB is often amplified in RMS resulting in

too much reduction of miR-29, which causes an insufficient regulation of YY1 by miR-29.

This leads to a deregulation of YY1 resulting in RMS. Due to the targeting of YY1 by miR-

29, one might think that a down-regulation of miR-29 would result in an up-regulation of

YY1 which would repress cell proliferation and therefore unlikely result in cancer. But the

fact that a deregulation of YY1 causes RMS anyway indicates that any deregulation in a

pathway can induce undesirable consequences such as for instance cancer.

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Pancreas, endocrine tumors

miR-29 expression was also investigated pancreatic tissue and was shown to be up-regulated

in pancreatic endocrine tumors compared normal pancreas (Roldo et al., 2006). The increase

was shown by microarray and quantified using a computer program, but the result was not

validated. Consistent with this there is an elevated expression of miR-29a, 29b and 29c in

pancreatic cancer tissue specimens with respect to normal pancreatic tissue and in MiaPaCa-

2-, PANC-1- and BxPC-3-cell lines versus HPDE cells (Zhang, 2008). MiaPaCa-2, PANC-1

and BxPC-3 are epithelial-like human pancreatic carcinoma cell lines. HPDE cells are

immortalized human pancreatic duct epithelial cells. miR-29a and miR-29b is also elevated in

MiaPaCa-2 and PANC-1 cell lines compared to Mock controls (Lee et al., 2009). The

increase of miR-29 in pancreatic cell lines was found by microarray, but was not validated by

neither Zang (2008) nor Lee et al. (2009). Together these findings indicate a tendency of

elevated miR-29 expression in cancerous pancreatic tissue which is interesting for the

prospect of treating T2DM by down-regulating miR-29 expression in β-cells without

increasing the risk of developing pancreatic cancer.

miR-29 and cancer in non-insulin-responsive tissues

We have chosen to present the up- or down-regulation of miR-29 expression in three cancer

types in writing, and then present rest of the data found on the coherence between miR-29

expression and cancer in an accumulating figure in the end of the section (figure 15).

Lymphocytes, B-cell chronic lymphocytic leukemia

miR-29a and miR-29b is up-regulated in indolent and aggressive samples of B-cell Chronic

Lymphocytic Leukemia (CLL) compared to normal B-cells (Santanam et al., 2010). The up-

regulation was shown by real-time RT-Q-PCR. Over-expression of miR-29 in B-cells in

transfected mice lead to increased spleen-levels of the antigen CD5+ in 85% of the mice

(measured by real-time RT-PCR) and to an enlarged spleen over time (Santanam et al.,

2010). It has earlier been suggested that increased levels of the antigen CD5+ induces CLL

development (Bichi et al., 2002), and this supports the findings from Santanam et al. (2010)

of elevated miR-29a and miR-29b in CLL compared to normal B-cells. Together these events

indicate that an increase in miR-29 expression could induce CLL development. This is

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slightly supported by results from Santanam et al. (2010) showing that 4 of 20 miR-29

transfected mice developed frank leukemia and died.

However, contradicting findings have also been found. By Luc. assay it has been shown that

miR-29b targets tcl1 (Pekarsky, 2006), and that tcl1 over-expression might play an important

role in inducing the development of aggressive CLL (Pekarsky et al., 2008). The Luc. assay

did not involve a mutated target-3’UTR, and therefore it can be discussed whether the target-

result is valid. But if miR-29 targets tcl1, and tcl1 over-expression might play an important

role in inducing the development of aggressive CLL, a down-regulation of miR-29 could

induce CLL. But maybe the role of miR-29 in CLL is not that easy to categorize. Santanam et

al. (2010) surprisingly found that miR-29a expression was higher for indolent than for

aggressive CLL. All this suggest that any deregulation of miR-29 could induce CLL but that

a high up-regulation induces aggressive CLL and a smaller up-regulation, or maybe even a

down-regulation of miR-29 could induce indolent CLL.

A Western blot showed a decrease of CDK6 and DNMT-3A expression in B-cells from miR-

29 tranfected mice (Santanam et al., 2010). This correlates with the findings from Braconi et

al. ( 2011) where a miR-29a up-regulation in a HHC cell line causes a decrease in DNMT-3A

(and DNMT-1). The possible consequences of a decrease in CDK6 expression will be

discussed later in this section.

Lymphocytes, AML

miR-29a expression is elevated in Leukemia Stem Cells (LSC) collected from human primary

AML patients compared to normal Human Hematopoietic Stem cells (HSC) (Han et al.,

2010). Han et al. (2010) used different HSC’s: Lin-, CD34+, CD90+ and CD45Ra-, and

compared them to different LSC’s: Lin-, CD34+ and CD38+. The elevation was validated by

real-time PCR.

Another group of scientists have shown that miR-29b is down-regulated in primary Acute

Myolid Leukemia (AML) patient specimens compared to CD34+ cells from healthy donors

(Garzon, 2008). The down-regulation was shown by microarray and validated by real-time

RT-Q-PCR. The results contradict the findings from Han et al. (2010). Garzon (2008) did not

specify the names of the cells that were used for the miRNA expression profiling (Garzon,

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2008). The cells used by Garzon (2008) and Han et al. (2010) could differ from each other,

and this could be the reason for the different results.

Garzon (2009) showed that miR-29 targets cyclin dependent kinase 6 (CDK6) in ALM cells.

The target was validated by Luc. assay on K562 cells. K562 are human cancer cells from

AML and contains low levels of miR-29. The fact that miR-29 targets CDK6 is consistent

with the findings of Santanam et al. (2010) only they did validate their findings. Furthermore

Santanam et al. (2010) performed a Western blot using normal B-cells from miR-29-

transfected mice compared to wild type mice without miR-29. To understand how miR-29’s

targeting of CDK6 might induce cancer it is relevant to know how CDK6 functions in a

healthy cell.

CDK4 and CDK6 are active during G1 and their activity relies on cyclin D1. Cyclin D1 levels

top just before transition from G1 to S, and this results in an increase in cyclin D1/CDK4

complexes (cyclin D1/CDK4) and cyclin D1/CDK6 complexes (cyclin D1/CDK6) at this

transition state (Vermeulen, 2003). The transition from G1 to S requires activation of the

tumor suppressor protein Retinoblastoma (Rb). Rb is in a hypophosphorylated state bound to

the transcriptionfactor E2F in the beginning of G1. E2F is required for expression of genes

required for entry of S-phase (figure 13) (Giorgia, 2011).

Figure 13: Phosphorylation of Retinoblastoma Complexes of cyclin D1/CDK4, cyclin D1/CKD6 and cyclin E/CDK2 phosphorylate the tumor suppressor protein Retinoblastoma (Rb) thereby releasing the transcription factor E2F. E2F promotes cell cycle progression, gene transcription and the cell proceeds to S-phase. Two cyclin dependent inhibitors: INK4 and CIP/KIP, regulate the cycle by inhibiting the CDKs and indirectly regulate the phosphorylation of Rb (Modified from Giorgia, 2011).

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Cyclin D1/CDK4(6) phosphorylates Rb yielding a free E2F, and allowing cyclin E

expression. To be completely phosphorylated Rb requires enzymatic activity from cyclin

E/CDK2 complex. Expression of cyclin E results in formation of cyclin E/CDK2, and so the

Rb is fully phosphorylated (Sherr, 1996; Giorgia, 2011).

Mutations in the gene encoding cyclin D1 can cause undesirably elevated levels of cyclin D

which leads to increased levels of cyclin D1/CDK4(6) complexes resulting in an elevation of

phosphorylated Rb and thereby a deregulation of the checkpoint. Mutations in the gene

encoding cyclin D1accours in Mantle Cell Lymphoma (MCL) (Zhao, 2010). miR-29 levels in

MCL is decreased in comparison to non-cancerous CD19+ lymphocyte control cells (Zhao,

2010). This was shown by microarray and validated by real-time RT-Q-PCR where CD19+

peripheral blood lymphocytes and lymph nodes were used as controls. miR-29 is also

negatively correlated with CDK6 in MCL (Zhao, 2010). Due to the high expression of cyclin

D1 in MCL and the high expression of CDK6 transcripts caused by low miR-29 expression

there is a lot of cyclin D1/CDK6 complexes in MCL. These events result in uninhibited

phosphorylation of Rb and thereby uninhibited cell proliferation that could lead to MCL

developing.

Bile duct, cholangiocarcinoma

miR-29b is diminished in malignant cholangiocarcinoma cells (KMCH) with respect to non-

malignant H69 cholangiocytes (Mott et al., 2007). H69 cholangiocytes are epithelial cells of

the bile ducts. The finding was validated by real-time RT-Q-PCR (figure 14A). Consistent

with Xiong et al. (2010), Mott et al. ( 2007) found Mcl-1 to be a miR-29 target, this time by

Luc. assay on HeLa cells and KMCH cells (figure 14B) (Mott et al., 2007). HeLa is a human

cervical cancer cell line. A parental Luc. and a wt-MCL-1-‘3UTR was cloned into a plasmid.

The controls were cells transfected with either parental Luc. and single-nucleotide-mutated-

Mcl-1-‘3UTR or parental Luc. alone. miR-29b is down-regulated in KMCH and HeLa cells

and targets Mcl-1 in the same cells. This means that Mcl-1 is most likely increased in KMCH

and this reduces apoptosis and therefore induces risk of cancer development.

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Figure 14: miR-29 expression in malignant cholangiocarcinoma cells (KMCH) and Mcl-1 targeting (A) real-time RT-Q-PCR showed a decrease in miR-29 expression in KMCH cells compared to H69 cells. (B) Luciferase (Luc.) assay showed that miR-29b targets Mcl-1. Parental Luc. gene (pLuc.) was transfected into HeLa cells and KMCH cells. The control was HeLa/KMCH cells cotransfected with pLuc. mutated 3’UTR Mcl-1. Cells transfected with pLuc-Mcl-1 3’UTR and miR-29 (p29b) was down-regulated versus pLuc. and pLuc-Mutant 3’UTR (Modified from Mott et al., 2007).

Is miR-29 a tumor suppressor, an oncomiR or both?

Targets such as CDK6 and Mcl-1 have been described in several articles regarding miR-29

and cancer. CDK6 is a key-player of cell cycle regulation and is a promoting factor of the

cycle, and is thus a proto-oncogene. In the context of targeting CDK6, miR-29 is therefore a

tumor suppressor miRNA. miR-29 has been shown to target Mcl-1 in HHC and AML (only

miR-29b), and miR-29/b is down-regulated in both cancer types. We therefore conclude that

the target of miR-29 in AML and HHC must be Mcl-1 isoform 1, and not isoform 2 or 3. One

could imagine that miR-29 could also target Mcl-1 isoform 2 and 3, and thereby be an

oncomiR instead of a tumor suppressor miR.

In general miR-29 can be an oncomiR or a tumor suppressor miR dependent on the target.

Four cascades can be proposed:

Target Regulation of miR-29 Induces cancer development Oncogene Up No Tumor suppressor Up Yes Oncogene Down Yes Tumor suppressor Down No

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Besides CDK6 and Mcl-1 we also encountered an article concerning miR-29 and another

tumor suppressor protein; p53. Park et al. (2009) found that miR-29 is positively correlated

with p53, and that miR-29 induces apoptosis in p53-positive HeLa cells compared to HeLa

cells with p53 knocked down suggesting that the induction of apoptosis by miR-29 happens

through p53. p53 regulates the G1/S-checkpoint (figure 8) (Vermeulen, 2003) and is activated

when the checkpoint receives a signal of DNA damage (Garret, 2001). Activation followed

by up-regulating of p53 increases expression of genes that induces cell arrest and apoptosis

(Garret, 2001). When not activated p53 levels are low in the cells due to MDM2-protein

binding leading to ubiquitin-degradation of p53. Ubiquitin is a cellular trash-machine that

guides proteins to degradation when they are no longer needed (Garret, 2001; Murakami,

2001). If p53 is not properly activated when DNA damage is detected it will not induce cell

arrest and apoptosis, and an uninhibited proliferation can occur.

By Luc. assay Park et al. (2009) found that miR-29 directly targets p85α and Cell Division

Cycle Protein 42 (CDC42) in HeLa cells. Through p85α miR-29 might reduce the

phosphorylation of Akt which normally inactivates MDM2 (Park et al., 2009). When MDM2

levels are not properly inactivated the levels become too high causing excessive binding to

p53 and thereby a degradation of p53.

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Figure 15: Overview of up- or down-regulation of miR-29 in cancers of various tissues

* = Validated

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Summative discussion and conclusion

Whether miR-29 is up- or down-regulated in cancer tissues compared to normal tissue is

highly dependent on the tissue type. miR-29 seems to be down-regulated in brain, lung, liver,

cervix, muscle and stomach cancers, and up-regulated in cholangiocytes, prostate, pancreas

and some lymphocytes with possible exceptions.

Up-regulation of miR-29a, 29b and 29c in pancreatic cancers are especially interesting in

relation to the possibility of using a repression of miR-29a to treat patients suffering from

T2DM. But it is important to notice that the findings were not validated! If these findings

become validated and miR-29a repression could be done tissue-specifically, then it might

serve as part of T2DM-treatment. Some miRNAs are tissue-specific, but miR-29 is clearly

not. In case a miR-29a repression occurs in all tissues and not only in pancreas one would

possibly expose the T2DM patients to an increased risk of developing cancer if miR-29a

expression was clinically repressed.

The connection between miR-29a, 29b1, 29b2 and 29c.

miR-29a and 29b1 is expressed on the same gene. The same accounts for miR-29b2 and 29c.

Li et al. (2011) found decreased levels of miR-29a and 29c in cervical cancer. The result was

not validated, and one would expect to see reduced levels of miR-29b1 and 29b2 too, if miR-

29a and 29c are both decreased. As for experiments conducted with reference to brain

(Silber, 2008) and cholangiocytes (Mott et al., 2007) differences in expression was detected

only for miR-29b, whereas experiments conducted with reference to lung (Yanaihara, 2006)

detected miR-29b2 expression differentiation (not validated). miR-29a or 29c should in this

case follow the increase or decrease that miR-29b exhibits in brain and cholangiocytes,

whereas only miR-29c should follow the increase or decrease miR-29b2 exhibits.

Using miRNA in cancer treatment

The detection of an either elevated or reduced level of a certain miRNA in a certain type of

cancer can be utilized in patient-treatment in at least three ways; prognosis assessment,

regulation of the particular miRNA in that cancer tissue type and for classification of the

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cancer. There have been examples of miRNA-levels rightfully placing tumors in the

appropriate category (Subramanian et al., 2007). The right categorization plays an important

role in choosing the optimal treatment.

A down- or up-regulation of the particular elevated or reduced miRNA could also be part of

cancer treatment. But as discussed earlier it is of utmost importance to make sure that the

clinical regulation of the miRNA occurs only in the tissue-type chosen as target of treatment.

Regulation of miRNAs could be done either by inducing a “fake target” of the miRNA to

which it will bind or by repressing the miRNA. TGF-β, YY1 and c-Myc are different genes

found to target and repress miR-29 (Winbanks, 2011; Wang, 2008; Mott, 2010). Two

miRNAs; miR-29a and 29b have been shown to induce apoptosis in cholangiocarcinoma and

AML cells (Mott et al. 2007; Garzon, 2009). Furthermore it has been shown that miR-29b

has a tumor suppressive effect in vivo on nude AML mice (Garzon, 2009; Xiong et al.,

2010). This indicates that induction of miR-29 is a possible treatment cholangiocarcinoma

and AML.

The detection of either down- or up-regulation of a certain miRNA can also be utilized in the

prognosis of cancer patients. The expression profiling of miRNA in a certain cancer type in

different stages of the cancer development compared to corresponding normal tissue can be a

guide in choosing the optimal treatment. Some scientists have investigated indolent versions

of a cancer type compared to the aggressive version (Pekarsky, 2006; Santanam et al., 2010;

Muniyappa 2009; Calin, 2007) instead of comparing cancer and non-cancer. The focus is

thereby the miRNA-induced or -repressed pathogenesis effect of the cancer. If regulation of

one or more miRNAs could shift the cancer from being aggressive to being indolent it gives

hope that the cancer might be beaten by additional cancer-treatments that alone would not

have killed the cancer.

Models vs. the human body

It is important to keep in mind that it is seldom possible to conduct experiments under

physiologically relevant or clinically relevant conditions. Cell lines are often utilized for

experiments, though they are not a perfect analogue to the human body because they are not

part of the overall unit that the body constitutes. But they have the advantage of being easy to

achieve, easy to grow in a petri dish and easy to transfect. A cell line used for experiments is

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chosen from many cell cultures, and it is chosen because it is judged to be the best analogue

to the tissue it resembles. Some scientists utilize specimens from human patients for their

experiments, which makes the experiment more physiological relevant than if cell lines were

used.

All the experiments described in this report are conducted on cell lines, specimens or mice,

which means that the results are only indications of what might occur in a real live human

being. And additionally it has been suggested that gender and starvation periods could alter

miRNA expression in rat liver (Cheung et al., 2009). It is possible that these parameters and

maybe other additional ones could also alter miRNA levels in humans. The experiments

might only give results that concern the particular model the experiment was conducted on,

and not general results.

The role of miR-29 in cancer development

There is no overall tendency of miR-29 being up- or down-regulated in cancer. miR-29

expression levels are highly dependent on the cancer tissue type. But it seems probable that

miR-29 do play a role in the development of cancer, although no absolute conclusions can be

made before clinical trials on humans are conducted.

Perspective

It would be interesting to investigate the role of other miRNA’s and their targets in cancer

development. Also there has been conducted several trials concerning miR-29 and other

diseases such as for instance sclerosis. This could also be interesting to further investigate.

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Appendix 1

Directory of targets of miR-29

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microrna-29 expression in various cancer types · page 1 of 36 microrna-29 expression in various...

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