The microRNA-503 cluster, composed of six microRNAs (miR-424, miR503, miR-542, miR-450a-1, miR450a-2 and miR-450b), is located on the human X-chromosome at Xq26. This location is very near the gene encoding placenta-specific protein 1 (PLAC1) that we have shown is significantly over-expressed in several gynecologic cancers. We examined expression of the miR-503 cluster in a panel of endometrial endometrioid adenocarcinomas (EEAs) and found that all of the miRNAs are significantly under-expressed compared with benign tissue. Expression of the miRNAs is highly correlated suggesting that they are regulated from a single promoter. We have used DNA editing (CRISPR) of the miR-503 cluster promoter to down-regulate the entire cluster together. Additionally, known and validated targets of cluster members include a number of important oncogenes. Thus, suppression of miR-503 cluster members contributes to the establishment and maintenance of endometrial cancer. We further suggest that decreased miR-503 cluster expression is due to hyper-methylation of the promoter in tumor cells. Thus, we conclude that the miR-503 cluster is a potential therapeutic target in endometrial cancer

Cha, Elizabeth

English

Iowa Research Online

Honors Theses at the University of Iowa 
Fall 2018 
The mir-503 cluster is coordinately under-expressed in 
endometrial cancer 
Elizabeth Cha 
Follow this and additional works at: https://ir.uiowa.edu/honors_theses 
 Part of the Oncology Commons 
This honors thesis is available at Iowa Research Online: https://ir.uiowa.edu/honors_theses/253 
THE MIR-503 CLUSTER IS COORDINATELY UNDER-EXPRESSED IN ENDOMETRIAL CANCER 
by 
Elizabeth Cha 
A thesis submitted in partial fulfillment of the requirements 
for graduation with Honors in the Health and Human Physiology 
________________________________________________ 
Dr. Eric J. Devor 
Thesis Mentor 
Fall 2018 
All requirements for graduation with Honors in the 
Health and Human Physiology have been completed. 
________________________________________________ 
Dr. Lucas Carr 
Health and Human Physiology Honors Advisor 
This honors thesis is available at Iowa Research Online: https://ir.uiowa.edu/honors_theses/253 
The mir-503 cluster is coordinately under-expressed in endometrial cancer 
Elizabeth Cha,1 Eric J Devor,1,2 Akshaya Warrier,1 Marina D Miller,1 Jesus Gonzalez-
Bosquet,1,2Kimberly K Leslie1,2 
 
1Department of Obstetrics and Gynecology, University of Iowa Carver College of Medicine, 
Iowa City, IA 52246, USA; 2Holden Comprehensive Cancer Center, University of Iowa 
Hospitals and Clinics, Iowa City, IA 52246, USA
Abstract 
The microRNA-503 cluster, composed of 
six microRNAs (miR-424, miR503, miR-
542, miR-450a-1, miR450a-2 and miR-
450b), is located on the human X-
chromosome at Xq26. This location is very 
near the gene encoding placenta-specific 
protein 1 (PLAC1) that we have shown is 
significantly over-expressed in several 
gynecologic cancers. We examined 
expression of the miR-503 cluster in a panel 
of endometrial endometrioid 
adenocarcinomas (EEAs) and found that all 
of the miRNAs are significantly under-
expressed compared with benign tissue. 
Expression of the miRNAs is highly 
correlated suggesting that they are regulated 
from a single promoter. We have used DNA 
editing (CRISPR) of the miR-503 cluster 
promoter to down-regulate the entire cluster 
together. Additionally, known and validated 
targets of cluster members include a number 
of important oncogenes. Thus, suppression 
of miR-503 cluster members contributes to 
the establishment and maintenance of 
endometrial cancer. We further suggest that 
decreased miR-503 cluster expression is due 
to hyper-methylation of the promoter in 
tumor cells. Thus, we conclude that the 
miR-503 cluster is a potential therapeutic 
target in endometrial cancer. 
Introduction 
MicroRNA-503 cluster, composed of six 
microRNAs (miRs): miR-424, miR-503, 
miR-542, miR-450a-1, miR-450a-2 and 
miR-450b, is located on chromosome 
Xq26.3 in close proximity (26.5kb) to 
placenta-specific protein 1 (PLAC1) [1]. 
PLAC1 is normally exclusively expressed in 
the trophoblast cells of the placenta. 
However, PLAC1 has been shown to be 
expressed in a variety of cancers, including 
breast, prostate, ovarian and uterine. 
Expression of PLAC1 promotes 
invasiveness, proliferation, and migration, 
all of which are characteristics of cancer 
cells. Expression of PLAC1 in tumor cells 
increases with aggressiveness and advanced 
stage. PLAC1 may serve as a biomarker for 
endometrial cancers with the potential to be 
both a prognostic indicator and a therapeutic 
target [2]. The close proximity of the miR-
503 cluster to PLAC1 makes it an area of 
interest in endometrial cancer.  
Nearly 11,000 women in the US will die of 
endometrial cancer this year alone [3] and it 
is projected that by 2030, there will be a 
55% increase in the number of women with 
this cancer [4]. Endometrial cancer is the 
most common gynecologic cancer and is the 
6th most common cancer among women [5]. 
It is thus imperative to understand as much 
as possible about the genetics of endometrial 
cancer. Consequently, the miR-503 cluster 
and its chromosome region are the subject of 
this study. 
As noted, the mir-503 cluster is composed of 
six microRNAs but miR-450a-1 and miR-
450a-2 are identical so, for the purposes of 
this study, they are considered as a single 
locus: miR-450a. We report here that all five 
microRNAs in the 503 cluster are 
significantly down-regulated in endometrial 
cancer compared with benign uterine tissue 
and that their expression patterns are highly 
correlated. Further, we show that members 
of the cluster target a number of well-known 
oncogenes as well as DNA repair genes. Our 
hypothesis is that the entire cluster is 
transcribed as a single transcript and that the 
observed down-regulation in endometrial 
cancer is due to methylation of the tumor’s 
promoter. This model, in turn, suggests that 
targets of miR-503 cluster members are up-
regulated in endometrial tumors. The 
objective of our research is to detail the 
regulation of the mir-503 cluster and its 
relationship to oncogene and DNA repair 
gene targets as this could lead to developing 
it as a potential therapeutic target in 
endometrial cancer.   
Methods 
We created a tumor panel composed of 20 
endometrial endometrioid adenocarcinomas, 
the most common form of the cancer, and 4 
benign uterine tissues (table 1). These 24 
tissue samples were obtained with informed 
consent from the Gynecologic Tumor Bank 
which is part of the Women’s Health Tissue 
Repository maintained in the Department of 
Obstetrics and Gynecology [6]. 
 
Total cellular RNA was purified from these 
tissue specimens using the mirVana miRNA 
isolation kit following manufacturer’s 
(Thermo Fisher) recommendations. Equal 
mass amounts of the purified RNAs (250ng 
each) were then reverse transcribed using 
miRNA-specific primers and MultiScribe 
Reverse Transcriptase (Thermo Fisher). The 
resulting cDNAs were then amplified using 
miRNA-specific TaqMan expression assays 
(Thermo Fisher). All experiments were 
carried out in triplicate. 
Over-expression of each member of the 
miR-503 cluster in Ishikawa H Cells, a well-
validated endometrioid adenocarcinoma 
model cell line [7], was accomplished with 
miRNA-specific mimics (Thermo Fisher). 
Over-expression was confirmed using the 
same miRNA-specific assays as above. Each 
miRNA mimic transfection as well as the 
un-transfected control was carried out in 
triplicate. Total cellular RNA was purified 
as above and the RNA submitted for next 
generation RNA sequencing to identify 
additional gene targets. 
  
Previous identification of a large CpG island 
in the miR-503 cluster promoter and a recent 
study of miR-424 in glioma [8] suggested 
that expression is regulated by methylation 
(Figure 1). We exposed Ishikawa cells to a 
cytidine analog (5-aza-2’ deoxycytidine), a 
de-methylating agent, in order to assess the 
effect of de-methylation on miRNA 
expression and selected target gene 
expression. Cells were exposed to 1 µM 5-
aza-2’ deoxycytidine for 72 hours before 
TABLE 1: Tumor Panel used in study with clinical 
characteristics. Estrogen Receptor (ER) and 
Progesterone Receptor (PR) status were 
determined by Immunohistochemistry in the 
Department of Pathology. We determined the 
TP53 tumor suppressor mutation  through direct 
sequencing of tissue genomic DNAs  
harvesting. Control cells were grown in 
parallel. The treated and un-treated media 
was changed every 24 hours. Again, total 
cellular RNA was purified and all treatments 
and controls were maintained in triplicate. 
 
In all cases, miRNA and gene expression 
was measured as normalized fold change 
relative to controls. Expression values (Ct) 
for every RNA sample and assay are 
normalized as ΔCt using an endogenous 
control; RNU48 for miRNAs and 18S rRNA 
for mRNAs. Fold change is then determined 
by the standard ΔΔCt method in which ΔΔCt 
= ΔCtexperiment – ΔCtcontrol and fold change is 
computed as 2-ΔΔCt [9,10]. Significance is 
assessed on grouped ΔCt values via a two-
sided t-test with unequal variances with p < 
0.05 being taken as statistically significant 
[11].  
Correlations among normalized miRNA ΔCt 
values were computed as standard  Pearson 
Correlation and significance assigned from a 
standard look-up table at  df = 22. Again, p 
< 0.05 was taken as statistically significant. 
Results  
Coordinated under-expression of the miR-
503 cluster in Endometrioid 
Adenocarcinoma 
Results from miRNA-specific QPCR are 
seen in Table 2. Expression of miR-503 
cluster members in endometrioid 
adenocarcinoma relative to benign 
endometrium is presented in Figure 2. Each 
cluster member is significantly under-
expressed in the tumors compared with 
benign endometrium. The magnitude of 
under-expression ranges from around 5-fold 
in miR-503 to nearly 25-fold in miR-424. 
Using normalized expression values (ΔCt) 
for each miRNA in all 24 individuals in the 
panel, including the benign tissues, pairwise 
correlations among all five members of the 
cluster reveals a highly significant pattern of 
coordinated expression across the entire 
cluster (Figure 2A). Correlation coefficients 
range from 0.76 to 0.94 and all are 
statistically significant at p < 0.001. 
 
 
 
Table 2. Pearson correlation coefficients among the 
five miRNAs in the miR-503 cluster. All are 
significant to p < 0.0001 at df = 22 
Figure 1. The miR-503 cluster on X-Chromosome with 
identified promoter, CpG islands (methylation sites) and the 
transcription start site 
Figure 2. Relative expression differences (fold change) 
between endometrial tumors and benign uterine tissue. 
Fold change is calculated as 2-ΔΔCt where ΔΔCt is the 
difference between mean normalized expression in the 
tumors and mean normalized expression in the controls 
Figure 2a. Correlation between members of mir-503 with 
ΔCt values for each member for each of the 24 individuals 
on the panel  
Coordinate expression is confirmed in The 
Cancer Genome Atlas (TCGA) where 
expression of the five cluster members is 
also uniformly significant at p < 0.001. 
Correlation coefficients ranged from 0.56 to 
0.88. Comparing the pairwise correlation 
coefficients between our sample and TCGA 
is also statistically significant (r = 0.62,       
p < 0.05, df = 8). Thus, the pattern of 
expression in the two analyses are is the 
same. 
Catalog of experimentally validated miR-
503 cluster targets 
Using miRTarBase 7.0 a total of 55 unique 
experimentally validated targets were 
identified for members of the miR-503 
cluster (Table 3). Among these are 
numerous genes commonly regarded as 
oncogenes such as FGFR1, MYB, BCL2, 
PI3K, and MYCN. Additionally present are 
genes involved in DNA repair such as 
CHK1 and WEE1; cell cycle including 
several cyclins, RUNX2, and CDC25A; 
anti-apoptosis genes IGF1R and BIRC5 
(survivin). Also, chemotherapy response 
inhibitors such as TRAF5 (cisplatin), 
ZNF217 (paclitaxel), and ERBB3 (gefitinib) 
are also present.  
 
 
The targets currently validated for the miR-
503 cluster members represent a rich array 
of genes whose dysregulation in cancer 
would be advantageous for carcinogenesis, 
recurrence, and metastasis.  
Potential Target Identification 
The number of oncogenes, cell cycle, DNA 
repair, and drug resistance genes seen in the 
validated targets of the miR-503 cluster 
initiated the investigation into a number of 
other potentially target genes that may exist. 
Transient transfection with the miR-specific 
mimics at 10µM resulted in an average 
expression increase of 33,000-fold 
compared with mock transfected cells. We 
chose to set a False Discovery Rate (FDR) 
of 10% which placed the corrected 
acceptable significance level at p < 0.001. 
Loci whose expression was significantly 
influenced, either up or down, relative to 
untreated cells in response to miRNA mimic 
transfection (Figure 3).  As can be seen there 
are hundreds of potential additional targets 
for members of the miR-503 cluster and 
many of these are genes known to be 
involved in carcinogenesis.  
Table 3. miR-503 cluster member experimentally validated targets 
(miRTarBase 7.0). Targets classified in the COSMIC census 
(www.sanger.ac.uk/science/data/cancer-gene-census) as Tier 1 
oncogenes are shown in red. Targets validated for more than one 
member of the cluster are shown in Bold type.  
Figure 3. Heat Maps of the target genes from RNA sequencing data. The 
areas above the purple bars belong to the untreated cells and the areas 
above the orange belong to the cells treated with mimics.  Red is 
overexpressed and green represents genes that are under expressed. 
miR-424 miR-503 
miR-524 
miR-450a 
miR-450b 
Promoter De-methylation 
Changes in expression of miR-503 cluster 
members as a result of treating Ishikawa 
cells with Decitabine are shown in Figure 4. 
Though the fold changes are modest, three 
of the five cluster members did reach 
statistical significance. The pattern of 
alteration of expression due to de-
methylation in vitro is exactly the inverse of 
the expression pattern of the cluster 
members seen previously in Figure 2 from 
the primary tumor tissues. 
 
  
 
 
 
 
 
DISCUSSION 
The miR-503 miRNA cluster, located at 
Xq23.1 between HPRT and PLAC1, 
consists of six miRNAs (miR-424, miR-503, 
miR-542, miR-450a-1, miR-450a-2 and 
miR-450b). We have shown that this cluster 
is transcribed as a polycistron and that the 
entire cluster is significantly down-regulated 
in endometrial adenocarcinoma. Considering 
the cluster as a whole and assembling 
experimentally validated targets reveals a 
wide range of loci involved in 
carcinogenesis and the consequences of 
carcinogenesis which includes DNA repair, 
DNA damage response, cell cycle 
maintenance and chemotherapy response. 
Further, we have provided evidence 
consistent with suggestions from other 
cancers [12] that down-regulation of the 
cluster in endometrial adenocarcinomas is 
accomplished via methylation. Given all of 
this data, we suggest that the miR-503 
cluster, as a whole, functions as a tumor 
suppressor complex and is, therefore, a 
potentially important therapeutic target in 
endometrial adenocarcinoma. 
It is well documented that high PLAC1 
expression leads to more aggressive tumors 
and a poor prognosis [13]. Similarly, several 
studies have linked down-regulation of 
members of the miR-503 cluster to 
carcinogenesis and poor prognosis. Most of 
these studies have focused on miR-424 
and/or miR-503 [14, 15-18] but miR-542 
has also been implicated including a specific 
relationship with endometrial carcinoma 
[19]. While there are fewer studies involving 
the miR-450 family, their role in 
carcinogenesis has also been confirmed [20, 
21]. 
Future directions include continuing to 
archive and evaluate the genes revealed 
through RNA sequencing of over-expression 
of members of the miR-503 cluster. 
Finally, the fact that up-regulation of 
expression of miR-503 cluster members, 
possibly through use of cytidine analogs, 
will negatively affect targets whose actions 
are oncogenic, but such an effect will not 
apply to neighboring PLAC1 where up-
regulation is contrary to good outcomes. 
Thus, the focus in the future should be to 
find an appropriate balance between the two 
to further explore and exploit it.  
 
 
 
 
Figure 4. Fold Change from Decitabine treatment on 
Ishikawa cells vs. untreated cells.  
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The mir-503 cluster is coordinately under-expressed in endometrial cancer

https://ir.uiowa.edu/cgi/viewcontent.cgi?article=1382&amp;context=honors_theses

The mir-503 cluster is coordinately under-expressed in endometrial cancer

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