Paul Han
Biology 303
Dr. Ely
1 November, 2012
Potential MicroRNA-200b Therapeutics
Past studies have shown that almost all diseases show a difference in microRNA (miRNA
or miR) expression when compared to the genes of healthy tissues (Bader 2011). miRNA-200b
is part of the miRNA-200 family located on chromosome 1p36 and is expressed in a large variety
of cells including ovarian cancer cells, mammary stem cells, renal mesangial cells and
endothelial cells (Chan et al. 2012). Malignancies proven to be affected by miRNAs include
cancer, Alzheimer’s and Parkinson’s disease, obesity, diabetes, cardiovascular and autoimmune
disorders. miRNAs are small noncoding RNAs that bind to the 3’ untranslated region of specific
messenger RNAs (Feng et al. 2012). miRNAs can be anywhere from 19-22 nucleotides long.
During the post-transcriptional stage, miRNA can target and silence complementary mRNA
sequences so that the short lengths of mRNA are not translated into proteins or the proteins
become altered. miRNA expression can regulate a multitude of cellular functions not limited to
differentiation, proliferation, motility, apoptosis and survival (Chan et al. 2012). The significant
effect miRNAs have on gene expression and cellular control has opened the possibility of using
miRNA as potential therapeutic drugs. Discovered only 11 years ago, miRNAs have shown that
a level of molecular therapeutics has been neglected for many years. However, the idea of
miRNA therapeutics has developed rapidly over the recent years. Since then, potential miRNAs
such as tumor suppressor miRNAs have already been discovered and are in phase 2 of clinical
trials of drug testing (Bader 2011). Conversely, careful and thorough testing of miRNAs must be
done due to epigenetic processes that can easily lead to incorrect expressions of miRNA. This
event would cause even more detrimental effects on top of those already present.
Patients with peripheral blood diseases are more susceptible to chronic non-healing
wounds. Thus the reestablishment of blood flow by angiogenesis is an important element to
wound healing. Angiogenesis is the biological process of forming new blood vessels. Complete
restriction and loss of blood flow would normally call for an amputation procedure. However,
understanding the process of angiogenic wound healing could help advance therapeutic methods
that would avoid amputation. Endothelial miRNA-200b targets a cluster of proteins that
intercepts the vascular endothelial growth factor (VEGF). VEGF is important for the formation
and proliferation of endothelial cells. Thus, Chan et al. (2012) sought to determine if the
downregulation of miRNA-200b induced a greater angiogenic wound healing response.
Excisions were made on the dorsal skin of lab mice. miRNA-200b overexpressing lentivirus was
intradermally injected 1mm away from the wound location 3 days before the actual wound was
placed. Observations over a 14-day period showed that overexpression of miRNA-200b
significantly hindered angiogenesis during the wound healing process (Chan et al. 2012).
The above images were taken on days 0, 3, and 7 of the wound healing assay. The graph above
shows the significantly decreased blood flow due to the addition of miRNA-200b (Chan et al.
2012). The decrease of blood flow due to the addition of miR-200b validated the evident
impairment of wound angiogenesis. Next, the team performed a computer scan for potential
targets that regulated angiogenic response. They found that globin transcription factor binding
protein 2 (GATA2) has a single binding site for miRNA-200b while VEGF receptor 2
(VEGFR2) has 2 binding sites for miRNA-200b (Chan et al. 2012). GATA2 is a major
transcription factor involved in hematopoietic and endothelial development. Results from
Western blot and immunocytochemistry analyses revealed that silencing of endothelial GATA2
or VEGFR2 produced comparable, if not equal, effects of impairment on wound angiogenesis.
Since GATA2 controls the expression of VEGFR2, the silencing of GATA2 alone resulted in the
down-regulation of VEGFR2 expression (Chan et al. 2012). The down-regulation of miR-200b
caused a phenotype of the embryonic mesoderm, showing improved motility and invasiveness by
differentiating cells. This study provided evidence that miR-200b binds directly to the GATA2
mRNA in the 3' UTR repressing translation. The study also established that severe downregulation of miRNA-200b is required for a successful angiogenic response to a wound.
Angiogenesis, controlled by miRNA-200b, is a potential therapeutic method of wound
healing. However, angiogenesis is also an important step in tumor development. The formation
of new blood vessels supplies the overgrowth of cancer cells with nutrients. During metastasis
the cancer cells can enter the newly formed blood vessels and travel to different parts of the
body. Gastric cancer is one of the most prominent malignant cancers in the world (Hohenberger
et al. 2003). Unfortunately, the causes of gastric cancer development are still unclear. In their
study, Kurashige et al. (2012) sought to define the role of miRNA-200b in EMT. Epithelialmesenchymal transition (EMT) is characterized by the loss of adhesion between cells (Thiery
2003). This loss of adhesion between cells is caused by the down-regulation of the E-cadherin
protein. It was proposed that Zinc finger E-box binding homeobox 1 and 2 (ZEB1 & ZEB2)
directly inhibited the transcription of E-cadherin mRNA. To begin the experiment, frozen
gastric cancer tissue samples were obtained from 40 patients who underwent gastrectomy. Cells
were transfected with pre-miRNA-200b. Transfection is a non-viral method of introducing
nucleic acids, DNA or RNA, into a eukaryotic cell. This process usually requires the uptake of
material through the pores of the cell membrane or fusing the cell membrane with liposomes that
contain the material. 48 hours after transfection, the expression level of miRNA-200b was
quantified by reverse transcription – polymerase chain reaction (RT-PCR). Afterwards, the cells
were tested for invasiveness and proliferation. Cell invasiveness was assessed by using Matrigel
Invasion Chambers. Cell proliferation was assessed by the WST-8 assay. Kurashige et al.
(2012) discovered that there was no significant difference in the expression levels of miRNA200b between gastric cancer tissue and normal mucosa (the outer layer tissue lining the inside of
stomach). It was discovered that the up-regulation of miRNA-200b inhibited the expression of
ZEB2 because miRNA-200b binds to the 3’ untranslated region (UTR) of ZEB2 mRNA. Since
ZEB2 is a transcriptional repressor to E-cadherin, inhibited ZEB2 expression allows for Ecadherin production (Kurashige et al. 2012). The up-regulation of the E-cadherin protein allows
for increased cell to cell adhesion. Increasing cell-cell adhesion decreases the epithelial
mesenchymal transition (EMT). Decreased EMT inhibits cancer cell proliferation and motility.
Inhibited proliferation and motility of cancer cells decreases the risk of the gastric cancer cells
forming malignant tumors. In summary, there was a significant positive correlation between
miR-200b and ZEB2 expression. There was a significant negative correlation between miR200b and E-cadherin. The results of this study established foremost evidence confirming a
negative correlation between miRNA-200b expression and EMT in gastric cancer (Kurashige et
al. 2012). In light of therapeutic applications, these results propose the idea of using miR-200b as
oncogene or tumor-suppressor genes. Oncogenes are genes that have the potential to cause
cancer. Other studies have shown that other miRs are suppressors for other cancers such as
breast cancer (Tavazoie et al. 2008). A flowchart can be found on the final page of this paper to
help the informed reader understand the correlation between miR-200b, EMT, ZEB2 and gastric
cancer.
Lung cancer has become one of the world’s leading causes of carcinoma-related deaths
due to the difficulty of detection during the early stages of cancer development. By the time the
patients are diagnosed with lung cancer, advanced stages of cancer development have already
taken place (Jemal et al. 2008). About 70% - 80% of lung cancers are nonsmall cell lung cancer
(NSCLC). NSCLC include squamous cell carcinoma, large cell carcinoma, and adenocarcinoma.
Adeno-, meaning gland, denotes that adenocarcinoma is the development of cancer originating
from glandular and epithelial tissues. Docetaxel is a form of anti-mitotic chemotherapy and is
used to treat NSCLC. Docetaxel inhibits mitotic reproduction of cancer cells during the
transition from metaphase to anaphase by enhancing microtubule polymerization. Retarded or
unstructured microtubules activate the spindle assembly checkpoint (SAC) which leads to
apoptosis, also known as cell death (Yu 2002). Unfortunately, chemoresistance in cancer cells
makes docetaxel difficult to use as a clinical application. The chemoresistant phenotype in
cancer cells is observed in cells that have undergone selection by drug pressures. Recent studies
have shown that certain miRNAs are linked to the chemoresistant phenotype of various tumors.
However, to the researchers’ knowledge, there have not been any previous studies attempting to
find a relationship between miRNA dysregulation and docetaxel resistance in human lung
adenocarcinoma. The research team attempted to discover the role of miRNA-200b and E2F3 in
human lung adenocarcinoma cells. Microarray data collected by Feng et al. (2012) identified
miRNA-200b as the most down-regulated miRNA in docetaxel-resistant human lung
adenocarcinoma SPC-A1/DTX cells versus the parental SPC-A1 cells. Human lung
adenocarcinoma SPC-A1 and A549 cells were obtained from the American Type Culture
Collection. Docetaxel-resistant cancer cells were abbreviated as SPC-A1/DTX cells. To
determine if there was a relationship between miRNA-200b expression and the lung
adenocarcinoma chemoresistance to docetaxel, SPC-A1 cells were transfected with miRNA200b inhibitor. SPC-A1/DTX cells were transfected with miR-200b gene-expressing plasmid
and A549 cells were transfected with both. An assessment showed that miRNA-200b expression
had a close association with lung adenocarcinoma sensitivity to docetaxel (Feng et al. 2012). To
investigate the in vivo effects of miRNA-200b expression on the chemosensivity of lung
adenocarcinoma cells, SPC-A1/DTX cells, transfected with miR-200b plasmids and plasmids
with the negative control, were subcutaneously inserted into the mice. After one week, all mice
had tumor growths.
Figure A shows the negative effect miRNA-200b induces in lung adenocarcinoma tumor growth.
Figure B shows the tumor volume difference between the negative control and the cells treated
with miR-200b. Transcription factor E2F3 was picked as a favored target gene due to the 3
complementary sites of miRNA-200b in its 3’-UTR. Western blot analysis was conducted to see
if E2F3 had functional effects on SPC-A1 and SPC-A1/DTX cells. Feng et al. (2012) then
observed the effects of enforced miRNA-200b expression on E2F3 expression. They found that
the level of E2F3 expression was significantly decreased in cells with up-regulated expression of
miRNA-200b. The level of E2F3 expression was significantly increased in cells with miRNA200b inhibitors. Since miRNA-200b could directly target E2F3 mRNA, the possibility of
knocking down E2F3 using small interfering RNA (siRNA) was tested. E2F3 mRNA protein
and expression levels were both significantly down-regulated SPC-A1/DTX cells transfected
with siRNA. Feng et al. (2012) also discovered that E2F3 knockout inhibited the proliferation of
cancer cells. The team concluded that down-regulated expression of miRNA-200b is correlated
with the up-regulated expression of E2F3. Up-regulated expression of E2F3 decreases the
cancer cell’s chemosensitivity to docetaxel. This correlation explained the inability for docetaxel
to be used effectively in clinical applications against adenocarcinoma. Cancer patients with low
miRNA-200b expression showed shorter life spans than the patients with high miR-200b
expression. This study showed that miR's have the therapeutic potential to be reversers of
chemoresistance in cancers.
All three studies discussed in this literature review searched for the functional role of
miRNA in gene expression. Feng et al. (2012) and Chan et al. (2012) demonstrated a very good
experimental design. Although Feng’s research focused on lung adenocarcinoma
chemosensitivity and Chan’s research focused on wound angiogenesis, both teams saw that
miRNA-200b regulated the expression of other proteins. From this observation the researchers
sought to discover if other molecules such as GATA2 or E2F3 could have functional effects on
the subject being studied. It was quite interesting to note that Chan’s et al. research advocated
the inhibition of miRNA-200b expression in epithelial tissues while the other two studies
proposed up-regulation in miR-200b expression for positive health results. This aspect showed
that there many more relationships to be discovered between cancer cells and miRNA. Further
research of the miR-200 family can open new doors and opportunities for the development of
advanced gene therapy in the future.
Literature Cited
1.
Bader AG, Lammers P. The Therapeutic Potential of microRNAs. 2011. Innovations in
Pharmaceutical Technology March Issue: 52-55.
2.
Chan Y, Roy S, Khanna S, Cen CK. 2012. Downregulation of Endothelial MicroRNA200b Supports Cutaneous Wound Angiogenesis By Desilencing GATA Binding
Protein 2 and Vascular Endothelial Growth Factor Receptor 2. Arterioscler Thromb
Vasc Biol 32:1372-1382.
3.
Feng B, Wang R, Song HZ, Chen LB. 2012. MicroRNA-200b Reverses Chemoresistance
of Docetaxel-Resistant Human Lung Adenocarcinoma Cells by Targeting E2F3.
Cancer 118:3365-3376.
4.
Hohenberger P, Gretschel S. 2003. Gastric cancer. Lancet 362(9380):305–15.
5.
Kurashige R, Kamohara H, Watanabe M, Hiyoshi Y, Iwatsuki M, Tanaka Y, Kinoshita
K, Saito S, Baba Y, Baba H. 2012. MicroRNA-200b Regulates Cell Proliferation,
Invasion, and Migration by Directly Targeting ZEB2 in Gastric Carcinoma. Annals
of Surgical Oncology 19:S656-S664.
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Jemal A, Siegel R, Ward E, et al. 2008. Cancer Statistics. CA Cancer J Clin 58:71-96.
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Tavazoie SF, Alarcon C, Oskarsson T, et al. 2008. Endogenous human microRNAs that
suppress breast cancer metastasis. Nature 451(7175):147–52.
8.
Thiery JP. 2003. Epithelial–mesenchymal transitions in development and pathologies.
Curr Opin Cell Biol 15:740–6.
9.
Yu H. 2002. Regulation of APC-Cdc20 by the spindle checkpoint. Curr Opin Cell Biol.
14:706-714.
miRNA-200b
Up-regulated miRNA200b
Down-regulated miRNA200b
Inhibits repressor ZEB2
expression
Allows repressor ZEB2
expression
Allows for e-cadherin
production
Inhibits e-cadherin production
due to repressed transcription
by ZEB2
Increased cell to cell
adhesion using e-cadherin
Decreased cell to cell
adhesion using e-cadherin
EMT is down-regulated due
to cell-cell adhesion
EMT is up-regulated due to
lack of cell-cell adhesion
Cell invasion, proliferation
and motility is inhibited in
gastric cancer cell
Cell invasion, proliferation
and motility is increased in
gastric cancer cell
Cancer remains benign or
does not grow worse
Cancer is malignant and fatal.
Cancer can continue spreading.
A visual representation of the study done by Kurashige et al. (2012)