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Wnt/beta-catenin signaling activates microRNA-181


expression in hepatocellular carcinoma

Cesar Ceja, Jenny Gong, Anyi Magaa, and Brian Parkins
California State University Fullerton, Department of Biological Science

Student 1 (Anyi Magaa): Introduction
Hepatocellular carcinoma (HCC) most commonly known as cancer of the liver is the
fifth most common cancer and the third leading cause of cancer deaths worldwide [4]. The
disease is more common in parts of sub-Saharan Africa and Southeast Asia [9]. Majority of
patients with liver cancer will die within one year of being diagnosed. In most cases, the cause of
liver cancer is usually scarring of the liver known as cirrhosis [10]. Cirrhosis may be caused by
alcohol abuse, autoimmune disease of the liver, hepatitis B or C infection, and iron overload in
the body known as hemochromatosis.
MicroRNAs (miRNA) are small ( ~22 nt), non-coding functional RNAs whose function
is the regulation of gene expression at the post-transcriptional level [4]. A miRNA expressing
gene is transcribed by an RNA polymerase II generating a primary-miRNA (pri-mRNA)
sequence [1]. This pri-mRNA undergoes post transcriptional processing by two members of the
RNase III enzyme family, Drosha and and Dicer to form a mature-miRNA. Only after the
mature-miRNA is incorporated into an RNA-induced silencing complex (RISC) can it become
involved in gene silencing. Mature-miRNA have complementary base pairing to its intended
target genes mRNA. Upon locating its target mRNA the miRNA undergoes gene silencing via
mRNA degradation, cleavage, or repression of protein translation. The function of mature-
miRNAs, as post-transcriptional regulators of gene expression, can be observed in various
biological processes such as apoptosis, stem cell differentiation, hematopoiesis, carcinogenesis
and more. MicroRNAs play an important role in maintenance of tissue identity and maintaining
target gene expression at an optimal level. Once the target mRNA has a specific function in the
cell the miRNA becomes mutated and overexpressing that miRNA can be detrimental to that cell
line.
The microRNA-181 (miR-181) is composed of four family members: miR-181a, miR-
181b, miR-181c, and miR-181d [4]. The miR-181s have high expression in hepatic cancer stem
cells (HepCSCs) where the Wnt/-catenin signaling pathway had been activated. The Wnt
ligand is a gene expression regulator and is instrumental in the Wnt/-catenin pathway [5]. The
pathway is activated (turned on) when a wnt ligand attaches to a cell membrane and inhibited
(turned off) when the wnt ligand is not attached. In the off state glycogen synthase kinase (GSK-
3), a Wnt antagonist, phosphorylates -catenin effectively marking it for proteosomal
degradation by -TrCP. In the on-state the Wnt ligand binds to Frizzled receptors, leading to the
activation of the Dishevelled (DvI) proteins and preventing the degradation of -catenin by
displacing the GSK-3. -catenin can now accumulate in the cytoplasm and translocate into the
nucleus to function as a co-transcriptional activator of LEF/TCF transcription factors. Altogether
the activation of the Wnt/-catenin pathway activates expression of miR-181s in HCC cells.

Student 2 (Jenny Gong): Establishing Experiments.
In order to confirm that the Wnt/Beta-catenin signaling was related to the expression of
miRNA-181s in liver cancer cells the researchers were trying to establish if there was a relation
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between miRNA 181 expression and the Wnt/Beta-catenin signaling? The first experiment was
Beta-catenin was present in the HCC cancer cells? This was done with the use of Western blot
and the control, Beta-actin, was used in order to compare the expression levels of Beta-catenin in
the cancer cells. A step further with the use of qRT-PCR in order to see if there was miRNA -181
also present in the liver cells. The final and the most important establishing experiment that was
used a qRT-PCR was what would the level of miRNA-181 expression be with a Wnt antagonist?
This experiment was done where they took three wild-type mice liver tissue miRNA-181, which
was used as a control and two miRNA-181s from mices liver cells that had the knockdown of
the Klotho transmembrane protein [6]. Klotho is a Wnt antagonist, in where in the Wnt pathway
the Beta-catenin is activated which leads to the transcription of the miRNA.

The results of the establishing experiment correlate that the Wnt/Beta-catenin signaling
has relation with the miRNA-181 family members. In miRNA-181a with the Klotho knockout
had the highest amount of expression when compared to the wild type and miRNA-181 b, c, and
d had similar but a slightly higher expression in the Klotho knockout liver tissues then of the
wild type liver tissues. Overall the results of the experiment was that the mice of Klotho
knockout the expression in the miRNA-181 had relatively higher levels in expression when
compared to the expression levels of the wild type mice. This indicates that the expression of
miRNA-181s increases when there was an activation of Beta-catenin in the HCC cells. All four
of the miRNA-181s are related the Beta-catenin expression in the cancer cells lines.
This is considered an establishing experiment because they wanted to confirm that the
Wnt/Beta-catenin signaling and the level of miRNA-181 expression are related to each other in
the HCC cancer cell lines. So they first established at there was B-catenin in the different types
of cancer cells and they compared the expression levels to Beta-actin a loading control. The
cancer cells HuH1 and HuH7 have around 50% of the expression levels of Beta-catenin
compared to Beta-actin. Then they ran an experiment that identifies that miRNA-181s were in
the different cancer cell expression. The miRNA-181s expression in the cancer cell Hep3B was
the highest and the other cancer cells had relative amount of expression. Finally in the last
establishing experiment they confirmed that with the knockout of the Wnt antagonist, Klotho,
there was a jump of expression of miRNA-181s. So this shows the relationship between
Wnt/Beta-catenin pathway and the mi-RNA-181s are correlated with each other.
This experiment is the start foundation of the paper because of these results are what
confirms the hypothesis which was Wnt/Beta-catenin pathway affects the level of expression of
miRNA-181s in HCC cells. These first three experiments were the like the start of an ongoing
process of learning about the miRNA-181 and its relations to the HCC cells. These experiments
are important because HuH1 and HuH7 cancer cells they are used for later experiments, which
will further test if the Wnt/Beta-catenin pathway was, involved in the expression levels of
miRNA-181s. The experiments also lead to the exploration of more experiments to come even
after this paper when the questions are completed.
Student 3 (Cesar Ceja): Hypothesis driven Experiments.
Ji et al.s experiment demonstrated how Wnt/-catenin signaling activated microRNA-
181 in hepatocellular carcinoma (HCC). The researchers investigated the relationship between
Wnt/-catenin and miR-181 to observe if Wnt/-catenin signal activation induced miR-181
expression. Multiple experiments were performed to support the relationship between Wnt/-
catenin signaling pathway and miR-181s expression.
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The Wnt/-catenin pathway followed the premise that the Wnt signaling allowed for -
catenin levels to increase and in turn elevated miR-181 expression. As established by the author,
-catenin levels increase as the miR-181 expression increases. Experimentally, the author
prepared known HCC cells and used a western blot to establish that those HCC cells have the -
catenin protein. The same cell lineage was subjected to qRT-PCR that monitored the levels of
miR-181 (a-d). The western blot and the qRT-PCR revealed a direct correlation between the -
catenin levels and the miR-181 expression. To confirm the results, miR-181 expression was
compared in wild type mice and Klotho KO mice. Klotho is a Wnt antagonist and it does not
allow the mice to suppress the Wnt activation as in the wild type. The relative miR-181
expression in the Klotho KO was consistent with the western blot and qRT-PCR results. This
assay directed focus on the effect of Wnt signaling and miR-181 expression.
Since the experiments were hypothesis driven, a correlation between Wnt signaling and
miR-181 expression must be established. An experiment chosen by the author must permit the
activation of the Wnt pathway and also allow for quantification of the miR-181 expression.
Because of this, the experiment chosen was 4 qRT-PCR that followed these conditions: 1. HuH7
vs. HuH7 Wnt10BR8, 2. Normal Media vs. Conditioned Media, 3. NaCl vs. LiCl (HuH1), and 4.
NaCl vs. LiCl (HuH7).
Under the first condition, qRT-PCR involved HuH7 (a HCC cell) and HuH7 Wnt10BR8
(a Wnt overproduction cell) along with the relative miR-181 expression levels were measured.
The Wnt overproduction cell had up to an eight-fold increase in miR-181 expression. The same
media from the Wnt overproduction cell held many Wnt signaling proteins and was used as the
conditioned media versus normal media in the second qRT-PCR test. Once again, there was an
increase of two-fold in miR-181 expression with the conditioned media. LiCl is a wnt/-catenin
activator and functions by inhibition of GSK3-. The inhibition prevented GSK3- to
phosphorylate -catenin so it would accumulate within the cell. The last two qRT-PCR involved
LiCl and NaCl affect upon HCC cells HuH1 and HuH7. The HuH1 cell received 40mM
concentrations of NaCl and LiCl, while HuH7 received 20mM concentrations of NaCl and LiCl.
The concentrations of NaCl and LiCl correlated with miR-181 expression. The HuH1 cell had a
three-fold increase exposed to the LiCl treatment. The HuH7 cell received half the concentration
of NaCl and LiCl treatments and had a 1.75 fold increase in miR-181 expression. LiCl acting as
a GSK3- deactivator allowed for -catenin accumulation respective to its concentration. This
data reiterates what was established by the expression of miR-181 is associated with -catenin
levels. As a whole, the qRT-PCR experiments indicated that the Wnt/ -catenin signaling
activation induced the level of miR-181 expression.
Ji et al. confirmed the Wnt/ -catenin activation of miR-181 expression from an
additional qRT-PCR experiment. The test involved HCC cells, HuH1 and HuH7, and the relative
miR-181 expression under four treatments: control, -catenin, wt-Tcf4 (wild type), and dn-Tcf4
(dominant negative). The negative control was the treatment of HCC cells transfected with an
empty vector. In addition to the control a -catenin vector was transfected and allowed
constitutively activated -catenin that served as a positive control. Wild type Tcf4, a -catenin
co-transcriptional activator, showed the natural expression miR-181 found in nature, while the
dominant negative Tcf4 is a mutant without Tcf4. The dn-Tcf4 is not able to recruit -catenin for
expression of miR-181. It was important to establish that both the control and the dn-Tcf4 were
expected to behave similarly, while both -catenin and wt-Tcf4 are expected to have increased
miR-181 levels relative to the control and dn-Tcf4 because of their ability to produce -catenin
and Tcf4. Both HuH1 and HuH7 cell results were as expected. Both -catenin and wt-Tcf4 levels
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had a two-fold increase from the control vector that indicated that Wnt/ -catenin signaling
activation induced the level of miR-181 expression. This was significant to the study because it
answered the researchers hypothesis that Wnt/-catenin signaling transcriptionally activates
microRNA-181s in Hepatocellular carcinoma (HCC). Finally, rather than reaching a conclusion,
it was needed to confirm that Wnt was the cause of the increased miR-181 expression and not
another factor. For this reason, another assay was performed that established the inhibition of
Wnt and its effects on miR-181.

Alternative Experiment (Cesar Ceja & Anyi Magaa)

Figure 1. The microarray scheme used to measure the miR-181 in non-transfected HuH7 and
transfected HuH7 Wnt 10B R8 This method uses fluorescent probes that can be allow the
relative miR-181 expression to be quantified (Left). The quantified results in graph form of
microarray experiment. There is at least a 4 fold increase in the HuH7 Wnt 10B R8, the Wnt
transfected over-expressing cell, when compared to the non-transfected cell (right).

Ji et al. experiments were hypothesis driven, a correlation between Wnt signaling and
miR-181 expression must be established. Ji et al. establishes the relationship between Wnt
signaling and the miR-181 expression using 4 different qRT-PCR on Hepatocellular Carcinoma
(HCC) cells with the following conditions: 1. HuH7 vs. HuH7 Wnt10BR8, 2. Normal Media vs.
Conditioned Media, 3. NaCl vs. LiCl (HuH1), and 4. NaCl vs. LiCl (HuH7). Instead of qRT-
PCR microarray is advocated as the best alternative. The microarray, much like the qRT-PCR, is
another RNA experiment capable of comparing miR-181 expression in HCC cells observing
multiple conditions using different colored fluorescent tags and quantifying it. For clarity, the
alternate experiment will focus on the more important condition, HuH7 (a HCC cell) and HuH7
Wnt10BR8 (a Wnt overproduction cell). The HuH7 is a non-transfected HCC cell while the
HuH7 Wnt10BR8 is a Wnt transfected HCC cell that allows constitutive Wnt expression. The
microarray, as an alternate experiment, observes RNA expression, the data can be quantified, and
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the same HCC cells are observed, we propose that it is a valid alternative to qRT-PCR and can
be used interchangeably with the figures published by Ji et al.
The microarray process begins by obtaining and purifying RNA (miR-181) from both
HuH7 and HuH7 Wnt10BR8 cells (figure 1-left). The miR-181 undergoes reverse transcriptase
to cDNA where fluorescent labeling hybridization occurs by complementary base pairing of the
cDNA and the fluorescent label. This process requires prior knowledge of the target RNA
nucleotide sequencing in order for fluorescent probes to hybridize to cDNA. Afterwards, the
microarray chip is analyzed and the data is quantified and tabulated (figure 1-right).
To validate the hypothesis the Wnt transfected HuH7 Wnt 10B R8 must show a higher
relative expression of miR-181 than the non-transfected HuH7 cell. That will suggest that Wnt
signaling activation is the cause of miR-181 expression increase. The tabulated results (figure 1-
right) show that the relative expression of the Wnt transfected HuH7 is at least 4 fold higher than
the non-transfected HuH7.The results turned out as expected and the hypothesis that Wnt/-
catenin signaling transcriptionally activates microRNA-181s in Hepatocellular carcinoma (HCC)
is validated.

Student 4 (Brian Parkins): Hypothesis driven Experiments.
Upon learning that Wnt/-catenin activation led to all four miR-181s being expressed, the
authors wanted to explore the possibility this signaling pathway could be inhibited, which could
lead to repression of the miRNAs. One possible avenue is by degrading -catenin before it
enters the nucleus, a process the authors chose to pursue. Adenomatosis polyposis coli (APC) is
a protein that has been found to promote -catenin degradation, in addition to blocking Wnt
target genes [7]. The HT29 cell line was isolated from a colorectal adenocarcinoma, and has
been used in many studies exploring tumorigenesis. The particular HT-29 used in this study,
HT29-APC, includes an APC gene controlled by a zinc-activated metallothionein promoter in
the presence of zinc, the APC gene is made available for transcription, allowing for control of
this protein through a simple on-off switch mechanism [2]. For a negative control they used
HT29-GAL cells, which contain a simple -galactosidase gene. When both cell cultures had
been incubated with zinc chloride (ZnCl
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) for 0, 24 or 48 hours, qRT-PCR was used to measure
all four miR-181s expression levels. In HT29-APC cells, the levels of these miRNA were
significantly reduced, while levels in the control HT29-GAL cells remained relatively
unchanged.
Following this success, a new strategy was used to characterize the relationship between
Wnt/-catenin signaling inhibition and miR-181 expression levels. This test would utilize
siRNA, small interfering RNA that reduce the expression of specific genes, in this case -
catenin. siRNA is a double-stranded RNA script of 20-25 base pairs, with two overhanging
nucleotides and phosphorylated 5 and hydroxylated 3 ends. In eukaryotes, siRNA is an
important part of the RNA interference (RNAi) pathway, which uses RNA to inhibit gene
expression. The siRNA is split into two single-stranded RNAs, referred to as the passenger
strand and guide strand. The guide strand is selected by an Argonaute protein embedded in the
RNA-induced silencing complex (RISC), based on the thermodynamic stability of the 5 end [8],
while the passenger strand is degraded as a substrate for the RISC [9]. In animal cells, the RISC
is necessary and sufficient for the binding of miRNA to the target mRNA, which prevents
transcription of the mRNA while simultaneously promoting degradation by deadenylation. As a
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result, the target gene is silenced. To verify the efficacy of the -catenin siRNA obtained for this
study, HuH1 and HuH7 cell lines were transfected with either the -catenin siRNA or an siRNA
targeting an unrelated gene as a control via liposome transfection. Following Western blotting
analysis, a marked decrease in -catenin was found in cells transfected with the -catenin siRNA
versus those transfected with the control siRNA. qRT-PCR was performed with these cells to
explore CTNNB1 expression, the gene responsible for -catenin production, and found a
significant, nearly 75% decrease in CTNNB1 expression in -catenin siRNA transfected cells
versus control cells.
Having established the efficacy of the -catenin siRNA, the final test involved measuring
the expression levels of miR-181 expression in the presence of -catenin siRNA versus control
siRNA. Again, HuH1 and HuH7 cells transfected with -catenin siRNA and control siRNA
were analyzed by qRT-PCR. As expected, the reduction in -catenin resulted in significant
reduction of all four miR-181s when compared to levels with control siRNA. This not only
confirmed that -catenin regulates the expression of miR-181s, but also demonstrated a positive
correlation between the expression levels of these microRNAs and the amount of -catenin
present.


Alternative Experiment (Brian Parkins)



Figure 2 Western blot analysis (left) was used to verify the integrity of -catenin siRNA in HuH1 and HuH7 cells versus a
control siRNA which targeted an unrelated gene. A significant decrease in expression levels of -catenin were seen following
application of -catenin siRNA versus control siRNA in both cell lines. -actin was used as a loading control. Northern blot
analysis was used to purify all four mature miR-181s in both the HuH1 and HuH7 cell lines when exposed to the control siRNA
and -catenin siRNA. Quantification (right) was performed using fluorescein-labeled miRNA probes, and indicated a nearly
30% decrease in miR-181 levels.

Replication of the study exploring the effect inhibition of -catenin would have on miR-
181 expressions can be done in multiple ways. The use of siRNA is a simple and efficient
method of determining this relationship, but there are some alternatives. Transfection of shRNA
targeting -catenin allows for siRNA to be made naturally within the cells [10]. The shRNA is
transcribed within the nucleus, and the RNA product, pri-miRNA, is processed by Drosha, an
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RNase III enzyme. Cleavage of this pri-miRNA by Drosha results in a stem-loop structure
known as pre-miRNA, which is further processed by Dicer, another RNase III enzyme, into the
siRNA product [11][12]. While similarly effective, an increased risk is seen in this method due
to the necessity of a mammalian expression vector, which hijacks the cells protein synthesis
mechanism and thus poses a danger to researchers if not properly handled.
The use of either the siRNA or the shRNA method requires it first be verified for
integrity and function, especially in the case of shRNA. This is generally best performed using
Western blot analysis to determine the expression levels of the targeted protein. A Western blot
analysis performed in the context of this experiment would require two controls a loading
control for the gel, as well as a control siRNA. For a loading control, -actin is probably the
most widely used. The control shRNA or siRNA should target an unrelated mRNA, and serves
as verification that introduction of any siRNA does not affect -catenin expression. Successful
-catenin siRNA would show decreased levels of -catenin in cell cultures used, but these same
cell cultures must be used for further investigation of the -catenin/miR-181 relationship.
The Northern blot analysis would begin by loading samples into polyacrylamide gel
containing urea to denature secondary RNA structures [13]. Staining with a fluorescent stain
such as SYBR gold could then be used to visualize the miRNA either prior to or following
blotting. An alternative method would use fluorescent labeled probes, such as 5-fluorescein
labeled miRCURY LNA miR-181 probes. Quantification in either case would be performed
using an imager such as the Typhoon 8600.


Student 1 (Anyi Magaa): Authors findings and Conclusions
The overall molecular paradigm studied in the article is that Wnt/-catenin signaling
pathway directly regulates miR-181s expression in HCC [14]. The author drew inspiration from
research in molecular profiling of HCC revealing that Wnt/-catenin signaling is crucial in
deregulated genes. Also, hepatic cancer stem cells (HepCSCs) that were isolated from HCC cells
were found to be highly expressive of miR-181s. The earlier work allowed the author to
speculate that the Wnt/-catenin signaling pathway transcriptionally activates microRNA-181s in
HCC cells. Through experimentation the author confirmed the Wnt/-catenin signaling pathways
involvement in regulating miR-181s expression in HCC cells. This was accomplished by: 1.
proving, in vitro using HCC cell lines and in vivo using livers of mice, that miR-181 expression
is positively correlated with -catenin level, 2. activation or inhibition of Wnt/-catenin signaling
positively correlates with miR-181 expression, 3. Tcf4/-catenin complex and the TCF/LEF
binding elements are located within the promoter region of miR-181a-2/miR-181b-2 transcript
indicating further that the Wnt/-catenin signaling pathway is a direct target in HCC. Overall, the
authors concluded that there is a positive feedback between miR-181s and the Wnt/-catenin
signaling pathway and that it needs to be further studied in order to find a solution for HCC.




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