For my STEP experience, I conducted undergraduate research on the study of Hepatocellular Carcinoma. I am a part of Dr. Jacob and Dr. Ghoshal’s 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 our 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, Epithelial Mesenchymal Transition, and are tumor suppressors.
Data obtained from real-time PCR (qPCR) showed significant upregulation of SNAI2 and LIMK1 in miR-122 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 data, microarray analysis, and qPCR 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:
- Tail Extraction
- Gel Electrophoresis
- Polymerase Chain Reaction (PCR)
- Real-Time Polymerase Chain Reaction (qPCR)
- Primer Design
- Cell Culture
- Vector Cloning & Luciferase Reporter Assay
Because of STEP, I was able to utilize the $2000.00 funding to cover living expenses May through August. 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.
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:
- 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. I will then conduct a final Luciferase Assay with a mutant miR-122 TIMP2 binding site.