Download STEP Experience Reflection – Chelsey Williams

STEP Reflection
Name: Chelsey Williams
For your reflection of your STEP Experience, please do the following:
1. Fill in the Reflection Form below and post it on your respective STEP
experience page at u.osu.edu.
2. Upload 1 – 2 pictures from your experience to your u.osu.edu post if
you have some.
3. If you created a separate blog, video, digital story, etc. about your
experience, please attach a link to your creation to your u.osu.edu
post as well.
STEP Experience: Undergraduate Research
What? – A detailed description of what you did during your STEP experience.
For my STEP experience, I conducted undergraduate research on the study of Hepatocellular Carcinoma.
I am a part of Jacob Laboratory in the Comprehensive Cancer Research Center at The Ohio State
University under the instruction of Dr. Samson Jacob and Dr. Kalpana Ghoshal.
The purpose of Jacob Laboratory is to identify microRNAs highly expressed in the liver, and to determine
the cancerous consequences if those microRNAs are deregulated. Primary hepatocytes, cell culture,
diseased liver tissues, and mouse models are utilized to better understand the biological roles of
microRNAs, and to aid in development of therapies for hepatocellular carcinoma (HCC).
MicroRNA 122 (miR-122) is the most abundant liver- specific microRNA. After undergoing cellular
processing, it is expressed during late stages of embryonic development. In the laboratory of Dr. Jacob
and Dr. Ghoshal, miR-122 was found to be reduced in both rodent and human HCCs. Furthermore, loss
of miR-122 in the liver causes HCC to spontaneously develop in liver-specific knockout (LKO) mice.
Overexpression of miR-122 in HCC cell lines reduces tumerogenesis, which suggests that it functions as
a tumor suppressor. MiR-122 also targets small molecules in signaling pathways associated with
apoptosis and metastasis.
My contribution to these goals included researching four miR-122 targets. Those gene targets are
TIMP2, Prakaca, Snai2, and LIMK1. The reason that these particular four genes are important is because
of their involvement with apoptotic pathways (cell death), Epithelial Mesenchymal Transition
(movement of cancer cells throughout the body), and are tumor suppressors.
Data obtained from real-time PCR (qPCR) showed significant upregulation of SNAI2 and LIMK1 in miR122 KO mice. Since gene expression of SNAI2 and LIMK1 is downregulated in normal, healthy mice, this
upregulation shown in miR-122 KO mice is suggestive that miR-122 functions to suppress these genes.
Based on HITSCLIP, microarray analysis, and real-time data, SNAI2 and LIMK1 are both potential miR-122
targets. As targets, this indicates that these two genes are involved in the HCC pathway, and contribute
to the development of liver cancer in cells.
To complete my own experiments and to aid in the experiments of others in our laboratory, I learned
and mastered the following techniques:
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Tail Extraction
Used to purify cDNA from mouse tail samples
cDNA later used in PCR
Gel Electrophoresis
5-10 microliters of final PCR product is loaded into wells of 1.5% agarose gel and run through the
gel as an electrical field is applied
Depending on the PCR products size in nucleotides, distinct bands will separate, allowing
determination of mouse genotype
Polymerase Chain Reaction (PCR)
Initialization, Denaturation, Annealing, Elongation
Used for DNA replication and quantification for mouse genotyping purposes in conjunction with
gel electrophoresis
Real-Time Polymerase Chain Reaction (qPCR)
DNA quantification detected in real time
Three-step thermal cycle consisting of denaturation, annealing, and elongation stages
Primer Design
MacVector program used to design specific primers for certain genes to be used in qPCR
Cell Culture
Hepatocellular Carcinoma (HCC) cell lines grown and maintained for use in experiments
Cell lines utilized for vector cloning and drug testing experiment
Vector Cloning & Luciferase Reporter Assay
Foreign DNA integrated into psi-Check 2 vector with restriction enzymes and DNA ligase for
cloning purposes
Luciferase activity acts as a reporter gene to ensure experimental validity
Transformation: DH5α bacterial cells used as factories to make multiple copies
Transfection: DNA introduced into normal cells and
So What? – A personal response to your STEP experience, including feelings, thoughts,
judgments, and what you have learned about yourself and your assumptions from what you did
and how you reacted.
Because of STEP, I was able to utilize the $2000.00 funding to cover living expenses May through August
while living in the Ohio Staters apartment complex. By living in Columbus, I was able to effectively
commute to the Tzagournis building on Ohio State’s campus. I was also able to successfully complete
courses Physics 1201, History 3642, and Classics 2210 during this time period. If not for the STEP
program, I would not have had an enriched academic experience during the summer, nor would I have
been able to develop new skills through research.
One of the most incredible parts of the STEP program included being able to interact one-on-one with
faculty. I felt that my interactions with not only my cohort faculty member, but also the faculty members
of my entire house really enhanced my experience.
I learned that my personal strengths include verbal and written communication. I am proud of the
poster I was able to present at the STEP expo, and that I was able to convey the meaning and relevance
of my project. Being awarded the Dean’s Best In Show Award was an incredible honor, and was
personally inspiring to me to continue forward in my research with even greater enthusiasm than
before.
Now What? – Discuss how the things you experienced and learned during your STEP
experience will affect your academic, personal, and life goals moving forward.
Moving forward, I am currently continuing my research in Ohio State’s Department of Pathology. I will
continue to research and identify other gene targets of mir122. My future directions include:
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Determine if TIMP2 and Prakaca are also potential targets using qPCR
Complete TIMP2 Luciferase Reporter Assay to confirm as target
Conduct Luciferase Reporter Assays for Prakaca, SNAI2, LIMK1
Continue to contribute to overall laboratory purpose through continued genotyping of mice
Following the STEP Expo, I have since completed the TIMP2 Luciferase Reporter Assay, and have
confirmed with statistically significant evidence that TIMP2 is a miR-122 target. We are continuing to
investigate the TIMP2 target gene region since it appears that the region has several properties unique
to itself, including a “bulge” binding region.