Transcript Poster 4 - REU Program - Iowa State University

Research Experience in Molecular Biotechnology & Genomics
Summer 2010
Center for Integrated Animal Genomics
1
Wright ,
Kanwarpal S. Bakshi, Elane C.
1Department
Caixia
1
Yang ,
Josephat
1
Njoka ,Jason
W.
1
Ross
of Animal Science, Iowa State University, Ames, Iowa 50011
Gene Expression in microRNA-21 Inhibited GranulosaCells in the Pig
Abstract
Materials and Methods
Cumulus oocyte complexes (COC) were collected and cultured in oocyte
maturation media. After collection of cumulus cells from metaphase II
(MII) oocytes, RNA was extracted and checked for purity using the
NanoDrop or Agilent Bioanalyzer RNA nano chip. The GeneChip® 3' IVT
Express Kit was used in order to determine the levels of gene expression in
three type of cumulus cells: miR-21 inhibited, control, and negative
control. A pool of 360 ng was used for each treatment to determine the
gene expression in each of these cells. Finally, Q-PCR was used in order to
analyze the gene expression of four genes: PPP1CB, TGFB1, CCL2, and
BMPR1B. Three replications were done for the negative control cells,
while four replications were done for the control and miR-21 inhibited
cells. Reverse transcriptase was used to create cDNA, and then primers
specific for teach transcript were designed to amplify cDNA and analyze
gene expression.
Improved reproductive efficiency in agriculturally
important species such as pigs is especially vital in
today's world. Because maturing oocytes are
transcriptionallyinactive following germinal vesicle
breakdown, any changes to mRNA or protein
abundance are controlled through interactions with
the
surrounding
cumulus
cells
or
posttranscriptional gene regulation (PTGR)
(Prather et al. 2009). MicroRNAs have been found
to have a heavy influence in the regulation of
mRNAs and proteins in oocytes through PTGR.
One miRNA of interest, microRNA-21, is thought
to repress the expression of oocyte specific proteins
that are involved apoptosis, such as PDCD4.
Inhibition of miR-21 during porcine oocyte
maturation increased gene expression for several
hundred
genes
as
determined
through
AffymetrixGeneChip
analysis.
Using
the
microRNA.org, database it was determined that
miR-21 is predicted to interact with up to 36 of
those genes. Further analysis of four genes;
PPP1CB, TGFB1, CCL2, and BMPR1B, was
conducted using quantitative real time PCR. The
data from the RT-PCR were inconsistent with the
GeneChip analysis requiring further investigation
into the potential regulation of these candidate
genes via miRNA-21.
Primers:
BMPR1B For- 5’- AAACGAGGTCGACATACCACCCAA -3’
BMPR1B Rev- 5’- TCCTGTTCAAGCTCTCATCCACGCA -3’
CCL2 For- 5’- AGTCACCAGCAAGTGTCCTAA -3’
CCL2 Rev- 5’- GCTTCAAGGCTTCGGAGTTTGGTT -3’
PPP1CB For- 5’- TGTGCAGATGACTGAAGCAGAGGT -3’
PPP1CB Rev- 5’- AAGATAGTTGGCCTCTGGTGGGAA -3’
TGFB1 For- 5’- CGATAGGTGGAAAGCGGCAACCAA -3’
TGFB1 Rev- 5’- AGCTCCGACGTGTTGAACAGCATA -3’
Conclusion
Table 1. Expression levels of the genes PPP1CB, TGFB1, CCL2, and BMPR1B,
along with alignment scores. The numbers in the first three columns indicate the
fluorescence intensity from the gene chips. The miR-21/Cont and miR-21/NC
indicate the fold increase in gene expression when comparing the miR-21 inhibited
cells to those control and negative control, respectively. The alignment score
indicates the theoretical binding capacity of miR-21 to the mRNA transcript.
3 (b) Relative Gene Expression of CCL2
8
7
6
5
4
3
2
1
0
16
14
12
10
8
6
4
2
0
Relative Expression
Relative Expression
3 (a) Relative Gene Expression of TGFB1
Control
miR21 Inhibitor
Negative Control
Control
miR21 Inhibitor
Figure 1. Binding of MiR-21 to 3’-UTR of PDCD4.
3 (c) Relative Gene Expression of PPP1CB
18
16
14
12
10
8
6
4
2
0
3 (d) Relative Gene Expression of BMPR1B
12
Relative Expression
Relative Expression
Our objective is to identify mRNA molecules
whose expression level is affected by the
presence of miRNA-21. In addition, we want to
determine how these genes relate to oocyte
maturation and early embryo development. Our
hypothesis is that cells inhibited by miR-21 will
have increased expression of specific transcripts
compared to non-inhibited control cells.
4 (d)
• Results from the pig gene chip analysis demonstrated that miR-21 resulted
in the differential expression of a number of genes. Alignment scores from
microRNA.org indicated 36 genes with an alignment score ≥ 140.
• From the list of 36 genes, 35 of them were down-regulated, and while only
one of them was up-regulated. The four genes that we choose were all
down-regulated by at least 3 fold when compared to the control cells (Table
1, Figure 2).
• Results from the real time PCR indicated opposite findings: the relative
gene expression of three of the transcript was decreased in the miRNA-21
inhibited cells compared to controls while one transcript remained
unchanged.
Results and Discussion
Negative Control
Objective
4 (c)
Figure 2. A picture of several cumulus
oocyte complexes. The oocyte is
shown as the dark mass in the center
of the complex, while the cumulus,
or granulosa cells, are shown
surrounding the oocyte.
Introduction
•Oocytematuration is an intricate process that is
influenced by many factors.
• Because transcription stops following GVBD
until the four-cell stage the amount mRNA and
protein in the oocyteuntil the maternal-zygotic
transition is significantly influenced by interactions
between the cumulus cells and PTGR.
•MicroRNAs are small endogenous RNA
fragments approximately 22 nucleotides long
(Bartel 2004).
• They function in down-regulating many genes
through through degradation, inhibition of
initiation,
elongation,
and
termination,
deadenylation, and promotion of ribosome
detachment (Lewis and Steel 2010).
• MiR-21 has been known for its expression in
many cancer lines, contributing to their ability to
rapidly proliferate and resistance to apoptosis.
• MiR-21 has had a post transcriptional regulatory
impact on programmed cell death 4 (PDCD4),
which is a novel tumor repressor.
Figure 4 (a)-(d). Real Time PCR amplification plots of (a) TGFB1, (b) CCL2, (c)
PPP1CB, and (d) BMPR1B. Data from each shows that the miR-21 inhibited cells
have a greater cycle threshold, which indicates that their level of gene expression
is reduced compared to the control cells.
4 (a)
4 (b)
10
8
6
4
0
Control
miR21 InhibitorNegative Control
Acknowledgements
I would like to thank Dr. Jason Ross, Elane C. Wright, Dr. Ciaxia Yang, and
JosephatNjoka for all of their help and support during this summer. I would also like
to thank Dr. Max Rothschild, Justin Rice, and Ann Shuey for their guidance
throughout this program, and the National Science Foundation (NSF) for giving me
this opportunity.
References
2
Negative Control
The results from the pig gene chip analysis and Q-PCR were inconsistent.
Affymetrix analysis demonstrated an increase in gene expression in the miR21 inhibited cells while the Q-PCR amplification demonstrated that there
was a decrease in gene expression in the miR-21 inhibited cells. This
confliction indicates that we cannot make a clear conclusion regarding
miRNA-21 regulation of these candidate genes, although the results from QPCR are much more precise and represent the biological variation present in
each treatment. One plausible reason for the decrease in the amount of gene
expression from the Q-PCR results could be the fact that we only took the
data from one time point, instead of a variety of different times. At this time
point, the amount of mRNA could have been reduced as a result of a
negative feedback mechanism. A time point assay should be done in order to
determine the relative expression of genes at different times in response to
miRNA-21 inhibition.
Control
miR21 Inhibitor
Figure 3 (a)-(d). Relative gene expression of (a) TGFB1, (b) CCL2, (c) PPP1CB, and
(d) BMPR1B shown for control, negative control, and miR-21 inhibited cumulus cells.
Results are from Q-PCR.
Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. (2004). Cell, 116, 281–297.
Prather, RS., Ross, JW., Isom, SC., & Green, JA. (2009). Transcriptional, post-transcriptional and epigenetic
control of porcine oocyte maturation and embryogenesis. Soc ReprodFertil Suppl., 66, 165–176.
Lewis MA, Steel KP. MicroRNAs in mouse development and disease. Semin Cell Dev Biol (2010),
doi:10.1016/j.semcdb.2010.02.004
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371