Download (A). Egr1 gene expression in B16 cells

Effect of Decreasing Hsp27 on Egr1 Gene Expression in B16 cells
Marissa Weinfeld, Department of Biology, York College
Introduction
Methods
Prostate cancer is the most common form of cancer found in
men in the United States as well as the second largest cause of
cancer-related death in men signifying the importance of
research in this major public health problem (Jemal, 2005).
Design Hsp27 and scrambled
control siRNA plasmid
•
Elevated expression of heat shock protein 27 (Hsp27) has been
found in patients with prostate cancer and is associated with
castration-resistant prostate cancer (CRPC) progression. CRPC
progression is a process where cancer cells obtain the ability to
survive and proliferate in the absence of testicular androgens.
•
•
SiRNA
Well 1
Well 2
Well 3
Hsp27
Untransfected
Transfected
Transfected/ DMSO
scHsp27
Untransfected
Transfected
Transfected/ DMSO
Table 1. A six well plate was used to transfect B16 cells with Hsp27 and scHsp27.
Hsp27 insert primer sequence
GTACCTCGTCCCTGGATGTCAACCACTTTCAA
CCACTTTCAAGAGAAGTGGTTGACATCCAGG
GACGAAAAGCTTTTCCAAAAAGTCCCTGGAT
GTCAACCACTTCTCTTGAAAGTGGTTGACATC
CAGGGACGAG
Ligated insert at Hind III and ACC651
Transformed bacteria and selected for KAN
resistance to isolate plasmid
Primer
Sequence 5’ to 3’
Fragment size
Egr1For
Egr1Rev
Egr1.3For
Egr1.3Rev
TAATAGCAGCAGCAGCACCAGC
GTCGTTTGGCTGGGATAACTCG
CCACCATGGACAACTACCCC
TCATAGGGTTGTTCGCTCGG
220
205
Table 2. Primer sequences for Egr1 and their expected fragment size.
A more recent study shows Hsp27 initiatives the movement of
human prostate cancer cells out of the prostate gland to distant
organs (Voll, 2014).
Conclusions
Transfection
•
•
Early growth response protein 1 (Egr1) levels are constitutively
high and closely linked to cancer development and progression
(Giteny, 2009).
Results from a previous study indicate that the activation of Egr1
is an early event in the development of prostate cancer and the
absence of the Egr1 gene results in significantly delayed tumor
development. It is also suggested that Egr1 induced genes that
accelerate tumor progression either by mitogenesis ,
angiogenesis or invasion (Svaren, 2000).
Created plasmid construct
Transfected B16 cells with 1 ug plasmid
using the LyoVec reagent for 1 hour
Cells were treated with DMSO dose for
4 hours
Figure 1. Showed that the Egr1 primer set 1 (Table 2) did not
produce expected bands.
Figure 2. Performed a DNase treatment on RNA prior to PCR and
no bands were present.
RNA isolation and
quantification
Figure 3. A faint band was present in the untreated B16 cells.
• Trizol reagent was used to isolate total RNA
• 0.5 ug of RNA was used for Reverse
transcriptase
• RT was amplified with Egr1 primers (Table 2).
• Universal primers (Ambion)
Figure 4. Non specific interactions appeared in lane 3. A band
was present for Egr1 gene expression in human DNA around 190.
While primers were able to amplify in DNA little to no bands were
observed in RNA isolates.
The popular anticancer drug in chemotherapy 5-Fluorouracil (5FU) inhibited the phosphorylation of Hsp27, which decreased the
induction of Egr1 (Zhao, 2008).
Future Studies
Reisolation of RNA to measure the effect of Hsp27 and Egr1
gene expression
Hsp27 has been shown to induce NF-kB which induces expression
of Egr1 (Shiota, 2013).
References
Biron-Pain, K., Grosset, A., Poirier, F., Gaboury, L., and Pierre, Y. 2013. Expresion and functions of galectin-7 in human and murine
melanomas. PLOS ONE 8:1-10.
Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R.C., Ghafoor, A., Feuer, E.J., and Thun, M.J. 2005. Cancer Statistics, 2005. CA
Cancer J Clin 55:10-30.
Shiota, M., Bishop, J., Nip, K., Zardan, A., Takeuchi, A., Cordonnier, T., Beraldi, E., Bazov, J., Fazil, L., Chi, K., Gleave, M., and Zoubeidi, A.
2013. Hsp27 regulates epithelial mesenchymal transition, metastasis, and circulating tumor cells in prostate cancer. The Journal of
Cancer Research 70:3109-3119
Svaren, J., Ehrig, T., Abdulkadir, S., Ehrengruber, M., Watson, M., and Milbrandt J. 2000. Egr1 Targets genes in prostate carcinoma cells
identified by mircroarray analysis. The Journal of Biological Chemistry 275: 38524-38531.
Voll, E.A., Ogden. I.M., Pavese, J.M., Huang, X., Xu, Li., Jovanovic, B.D., and Bergan, R.C. 2014. Heat shock protein 27 regulates human
prostate cancer cell motility and metastatic progression. Oncotarget 9:2648-2663.
Zhao, H.Y. Ooyama A., Yamamoto, M., Ikeda, R., Haraguchi, M., Tabata, S., Furukawa, T., Che, X., Zhang, S., Oka, T., Fukushima, M.,
Nakagawa, M., Ono, M., Kuwano, M., Akiyama, S. 2008. Molecular basis for the induction of an angiogenesis inhibitor,
thrombospondin-1, by 5-Fluorouracil. American Association for Cancer Research 68:7035-7041.
The melanoma B16 cell line was used in previous experiments
expressing Hsp27 and Egr1 (Biron-Pain, 2013).
Objective and Hypothesis
The objective of this study was to determine if decreasing Hsp27
alters Egr1 gene expression in B16 cells.
The hypothesis was that the knockdown of Hsp27 would lead to a
reduction in Egr1 expression.
Acknowledgements
I would like to thank Dr. Kaltreider for his patience, work and support on this senior thesis project.
Results
Human DNA
ScHsp27
Untransfected
Hsp27/DMSO
Hsp27
ScHsp27/DMSO
ScHsp27
Untransfected
ScHsp27
Hsp27/DMSO
Untransfected
ScHsp27/DMSO
ScHsp27/DMSO
ScHsp27
ScHsp27
Untransfected
Hsp27/DMSO
Hsp27
Hsp27
ScHsp27
Hsp27/DMSO
Hsp27
Untransfected
Untransfected
Hsp27
Hsp27
Hsp27/DMSO
Hsp27/DMSO
Untransfected
ScHsp27
ScHsp27
ScHsp27/DMSO
ScHsp27/DMSO
Hsp27
ScHsp27
Untransfected
Hsp27
Untransfected
ScHsp27
Untransfected
Untransfected
Hsp27
Hsp27
Hsp27/DMSO
Hsp27/DMSO
Untransfected
ScHsp27
ScHsp27
ScHsp27/DMSO
ScHsp27/DMSO
Hsp27
Hsp27
Untransfected
Hsp27
300
200
200
100
200
200
100
A
100
100
B
300
200
200
100
100
Fig.3. Egr1 expression in the control B16 cells. Total RNA was
Fig. 1. 18S expression in B16 cells (A). Egr1 gene expression in B16
cells (B). Total RNA was isolated from the cells and the reverse
transcription reaction was performed using the High Capacity cDNA
Reverse Transcriptase Kits Protocol (Applied bio systems). The cDNA
was then amplified by PCR using the Egr1 primer (Table 1.)PCR
produced was electrophoresed on a 2% agarose gel and PCR
fragments were visualized by SYBR green.
300
Fig. 2. PCR reaction of RNA isolates. Total RNA was
isolated from the B16 cells. The RNA was treated with the
DNase treatment and was electrophoresed on a 2% agarose
gel. PCR fragments were visualized by SYBR green.
isolated from the cells and the reverse transcription reaction was
performed using the High Capacity cDNA Reverse Transcriptase Kits
Protocol (Applied bio systems). The cDNA was then amplified by PCR
using the Egr1 primer (Table 2.) PCR produced was electrophoresed
on a 2% agarose gel and PCR fragments were visualized by SYBR green
(Applied bio systems).
Fig. 4. Egr1 gene expression in B16 cells. Total RNA was
isolated from the cells and the reverse transcription reaction
was performed using the High Capacity cDNA Reverse
Transcriptase Kits Protocol (Applied bio systems). The cDNA
was then amplified by PCR using the Egr1 primer (Table 2).
PCR produced was electrophoresed on a 2% agarose gel
and PCR fragments were visualized by SYBR green.