Faculty Mentor
Name: Michael J. Bouchard, Ph.D.
Faculty Mentor Email: mjb93@drexel.edu
Department Administrative Contact: Joshua Stevenson
Department Administrative Email: jds336@drexel.edu
Department Account Code: 130555-5876
Research Statement: A short description of the project the student will undertake, including the research or create question the student will explore, the experience the student has with this topic/project, the background or significance of this work, and the methods used to explore this topic:
Hepatocellular carcinoma (HCC) incidence continues to rise and poses an evolving global issue [1-6]. The molecular mechanisms that underlie HCC development are poorly understood, and approved therapies are rarely curative [1-6]. Using publicly available RNAseq data to assess the levels of long noncoding (lnc) RNAs in normal livers and HCCs, the Bouchard research team discovered that the lncRNA – FAM99A – is highly expressed in normal liver but absent in HCCs [7,8,9]. This indicates potential tumor-suppressor capabilities; however, the function of FAM99A in hepatocytes is currently own. Therefore, our research interest lies in defining FAM99A activities in hepatocytes and determining if FAM99A is a true tumor suppressor. We hypothesize that FAM99A helps maintain the differentiated status of hepatocytes by acting as a tumor suppressor. Our objectives are to define functions of FAM99A, including a role in hepatocyte differentiation; identify interacting partners of FAM99A; and assess the effect of FAM99A on the transformed phenotype of a panel of HCC cell lines. During the Spring Term, the student will determine if FAM99A expression is required to maintain the differentiated status of human hepatocytes and assess FAM99A tumor-suppressor activity in HCC cell lines. Studies will be conducted in cultured primary human hepatocytes to assess the effect of FAM99A overexpression or knockdown on hepatocyte differentiation. Studies, such as analyzing colony formation in soft agar, will be conducted in a panel of HCC cell lines to assess the effect of over-expressed FAM99A on the transformed phenotype of these cells. The student has limited experience with mammalian tissue culturing and knowledge of HCC, which permits for a perfect learning opportunity. Furthermore, the student has taken lower and upper biology and chemistry courses with an extensive background in wet-bench experience, that gives the fundamental skills needed for this project. Techniques and skills from molecular biology and microbiology will be greatly built upon throughout the project.
References:
1. Singal, A.G., P. Lampertico, and P. Nahon, Epidemiology and surveillance for hepatocellular carcinoma: New trends. J Hepatol, 2020. 72(2): p. 250-261.
2. Hou, J., et al., The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications. J Hepatol, 2020. 72(1): p. 167-182.
3. Zhu, C., et al., The fusion landscape of hepatocellular carcinoma. Mol Oncol, 2019. 13(5): p. 1214-1225.
4. Yang, J.D., et al., A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol, 2019. 16(10): p. 589-604.
5. Keenan, B.P., L. Fong, and R.K. Kelley, Immunotherapy in hepatocellular carcinoma: the complex interface between inflammation, fibrosis, and the immune response. J Immunother Cancer, 2019. 7(1): p. 267.
6. Hlady, R.A., et al., Integrating the Epigenome to Identify Drivers of Hepatocellular Carcinoma. Hepatology, 2019. 69(2): p. 639-652.
7. The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science, 2020. 369(6509): p. 1318-1330.
8. Zhao, B., et al., HIF-1alpha and HDAC1 mediated regulation of FAM99A-miR92a signaling contributes to hypoxia induced HCC metastasis. Signal Transduct Target Ther, 2020. 5(1): p. 118.
9. Mo, M., et al., A liver-specific lncRNA, FAM99B, suppresses hepatocellular carcinoma progression through inhibition of cell proliferation, migration, and invasion. J Cancer Res Clin Oncol, 2019. 145(8): p. 2027-2038.
Timeline of Work: A short description of how the project will progress over the Spring Term, including when and how the student will prepare to participate in the Week of Undergraduate Excellence:
Preliminary studies showed that while FAM99A expression is absent in HCCs, it is highly expressed in normal primary human hepatocytes (PHHs), but its expression is lost by 24 hours after plating PHHs, which also correlates with de-differentiation of the cultured hepatocytes. This suggests that FAM99A might be required to maintain the differentiated status of hepatocytes and thus acts as a tumor suppressor.
Therefore, the student will:
1) Knockdown and overexpress FAM99A in plated PHHs and assess effects on PHH differentiation.
2) Overexpress FAM99A in a panel of HCC cell lines and assess effects on colony formation in soft agar (an indicator of a cell’s ability to form a tumor) and cell proliferation and death.
De-identified PHHs will be purchased from commercial sources and cultured as in our previous studies [1,2]. PHHs will be infected with a recombinant Adenovirus (recAd) expressing green fluorescent protein (GFP), or AdGFP-FAM99A in suspension prior to plating by incubating the cells with the recAds. Cells will then be analyzed at 24, 48, and 72 hours post infection. Albumin and urea levels in the cell-culture supernatant will be analyzed as indicators of hepatocyte function [3-5]. We will also use western blot analysis to assess levels of transferrin, hepatocyte nuclear factor 4a, and transthyretin, markers of differentiated hepatocytes [3-8, 9, 10]. HCC cell lines will also be assessed for effects on colony formation in soft agar. When an effect on colony formation is observed, we will assess cell proliferation and death using well established protocols [11-12]. The student will be conducting full-time research in the Bouchard lab for the Spring Term, thus they will see many facets of this project at whatever capacity 10 weeks may grant. The first ~2 weeks are expected to consist of literature review and laboratory familiarization. Weeks ~2-4 will roughly be focused on lab technique monitoring and development, as well as preliminary project design, development, and implementation. Weeks 4-8 will then consist of semi-independent research and laboratory group work. The last few weeks of the term will be spent on poster development for the Week of Undergraduate Excellence as well as final data analysis.
References
1. Lamontagne, J., J.C. Mell, and M.J. Bouchard, Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression. PLoS Pathog, 2016. 12(2): p. e1005438.
2. Arrigoni, A., et al., Analysis RNA-seq and Noncoding RNA. Methods Mol Biol, 2016. 1480: p. 125-35.
3. Kang, Y.B., Rawat, S., Duchemin, N., Bouchard, MJ., Noh, M., Human liver sinusoid on a chip for hepatitis B viral replication study. Micromachines, 2017. In press.
4. Kang, Y.B., et al., Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms. Biotechnol Bioeng, 2015. 112(12): p. 2571-82.
5. Kang, Y.B., et al., Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid. Biofabrication, 2013. 5(4): p. 045008.
6. Kang, Y.B., et al., Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid. Biofabrication, 2013. 5(4): p. 045008.
7. Sodunke, T.R., M.J. Bouchard, and H.M. Noh, Microfluidic platform for hepatitis B viral replication study. Biomed Microdevices, 2008. 10(3): p. 393-402.
8. Clippinger, A.J. and M.J. Bouchard, The Hepatitis B Virus Hbx Protein Localizes to Mitochondria in Primary Rat Hepatocytes and Modulates Mitochondrial Membrane Potential. J Virol, 2008. 82: p. 6798-6811.
9. Clippinger, A.J., T.L. Gearhart, and M.J. Bouchard, Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappaB and the mitochondrial permeability transition pore. J Virol, 2009. 83(10): p. 4718-31.
10. Casciano, J.C., et al., Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes. PLoS One, 2017. 12(2): p. e0168328.
11. Crowley, L.C., et al., Measuring Cell Death by Trypan Blue Uptake and Light Microscopy. Cold Spring Harb Protoc, 2016. 2016(7).
12. Crowley, L.C., et al., Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry. Cold Spring Harb Protoc, 2016. 2016(7).
Personal statement of interest: A short description of the student’s interest in this work, including how this project may fit into the student’s academic plan/goals, what drew them to this work, and/or why they are passionate about this work:
I have been involved in many research endeavors throughout my time in high school and undergrad; each has propelled me toward biochemical and molecular biological research. In high school, I worked on individual projects and competed internationally three times, including environmental projects which won national awards. Since high school, I have had two experiences with the United States Department of Agriculture – Agricultural Research Services. The first was an internship, focused on enzyme identification and bioinformatic database mining, and the second was with the Molecular Characterization of Foodborne Pathogens unit, developing a rapid identification method using next-generation sequencing. I also participated in the Student Tackling Advanced Research program and worked with a professor to investigate a new polymerization technique. I am currently working in a neuroengineering lab through Vertically Integrated Projects, where I have been the lead in establishing a research records database that is Institutional Review Board and HIPAA (Health Insurance Portability and Accountability) compliant. Upon looking at my research history, most would say that these topics are varied in field and show no direction or culmination. However, I argue that each experience has allowed me to build an arsenal of skills that could not be developed from one area alone. There is value in these skills, especially as I find myself aiming for a career as a principal investigator with the National Institutes of Health. As I reflect on my time in the laboratory and my contributions towards the advancement of knowledge, I find myself yearning for research that directly reflects the future I want. Furthermore, research that can one day be applied to alleviate health disparities or address disproportions in minority communities is a driving force for why I want to work at the NIH. Although the studies outlined in this proposal are not designed to directly address a specific health disparity, the incidence of HCC is higher in Asian, African Americans, Hispanics, and American Indians/Alaskan Natives populations (REFS). Therefore, any identification of novel biomarkers or treatment methods would be directly applicable to both general health and the higher incidence of HCC in these specific minority groups. With the Bouchard Lab at the Drexel University College of Medicine, I would gain experience with mammalian cell tissue culturing, molecular biological techniques, and biochemical techniques. I also find the research fascinating – knowing that I’m directly contributing to knowledge that can help my community and many others that face similar issues. I believe that this research will enhance the skills I’ve developed and can show me how research can surpass the fundamental sciences and begin to translate into something that directly benefits humankind. I have aggressively pursued research as a student, but topics similar to this project are what I foresee myself doing for the rest of my career. Proposal title: Investigating the Tumor-suppressing Potential of Long noncoding RNA FAM99A in Primary Human Hepatocytes
Faculty Mentor Email: mjb93@drexel.edu
Department Administrative Contact: Joshua Stevenson
Department Administrative Email: jds336@drexel.edu
Department Account Code: 130555-5876
Research Statement: A short description of the project the student will undertake, including the research or create question the student will explore, the experience the student has with this topic/project, the background or significance of this work, and the methods used to explore this topic:
Hepatocellular carcinoma (HCC) incidence continues to rise and poses an evolving global issue [1-6]. The molecular mechanisms that underlie HCC development are poorly understood, and approved therapies are rarely curative [1-6]. Using publicly available RNAseq data to assess the levels of long noncoding (lnc) RNAs in normal livers and HCCs, the Bouchard research team discovered that the lncRNA – FAM99A – is highly expressed in normal liver but absent in HCCs [7,8,9]. This indicates potential tumor-suppressor capabilities; however, the function of FAM99A in hepatocytes is currently own. Therefore, our research interest lies in defining FAM99A activities in hepatocytes and determining if FAM99A is a true tumor suppressor. We hypothesize that FAM99A helps maintain the differentiated status of hepatocytes by acting as a tumor suppressor. Our objectives are to define functions of FAM99A, including a role in hepatocyte differentiation; identify interacting partners of FAM99A; and assess the effect of FAM99A on the transformed phenotype of a panel of HCC cell lines. During the Spring Term, the student will determine if FAM99A expression is required to maintain the differentiated status of human hepatocytes and assess FAM99A tumor-suppressor activity in HCC cell lines. Studies will be conducted in cultured primary human hepatocytes to assess the effect of FAM99A overexpression or knockdown on hepatocyte differentiation. Studies, such as analyzing colony formation in soft agar, will be conducted in a panel of HCC cell lines to assess the effect of over-expressed FAM99A on the transformed phenotype of these cells. The student has limited experience with mammalian tissue culturing and knowledge of HCC, which permits for a perfect learning opportunity. Furthermore, the student has taken lower and upper biology and chemistry courses with an extensive background in wet-bench experience, that gives the fundamental skills needed for this project. Techniques and skills from molecular biology and microbiology will be greatly built upon throughout the project.
References:
1. Singal, A.G., P. Lampertico, and P. Nahon, Epidemiology and surveillance for hepatocellular carcinoma: New trends. J Hepatol, 2020. 72(2): p. 250-261.
2. Hou, J., et al., The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications. J Hepatol, 2020. 72(1): p. 167-182.
3. Zhu, C., et al., The fusion landscape of hepatocellular carcinoma. Mol Oncol, 2019. 13(5): p. 1214-1225.
4. Yang, J.D., et al., A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol, 2019. 16(10): p. 589-604.
5. Keenan, B.P., L. Fong, and R.K. Kelley, Immunotherapy in hepatocellular carcinoma: the complex interface between inflammation, fibrosis, and the immune response. J Immunother Cancer, 2019. 7(1): p. 267.
6. Hlady, R.A., et al., Integrating the Epigenome to Identify Drivers of Hepatocellular Carcinoma. Hepatology, 2019. 69(2): p. 639-652.
7. The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science, 2020. 369(6509): p. 1318-1330.
8. Zhao, B., et al., HIF-1alpha and HDAC1 mediated regulation of FAM99A-miR92a signaling contributes to hypoxia induced HCC metastasis. Signal Transduct Target Ther, 2020. 5(1): p. 118.
9. Mo, M., et al., A liver-specific lncRNA, FAM99B, suppresses hepatocellular carcinoma progression through inhibition of cell proliferation, migration, and invasion. J Cancer Res Clin Oncol, 2019. 145(8): p. 2027-2038.
Timeline of Work: A short description of how the project will progress over the Spring Term, including when and how the student will prepare to participate in the Week of Undergraduate Excellence:
Preliminary studies showed that while FAM99A expression is absent in HCCs, it is highly expressed in normal primary human hepatocytes (PHHs), but its expression is lost by 24 hours after plating PHHs, which also correlates with de-differentiation of the cultured hepatocytes. This suggests that FAM99A might be required to maintain the differentiated status of hepatocytes and thus acts as a tumor suppressor.
Therefore, the student will:
1) Knockdown and overexpress FAM99A in plated PHHs and assess effects on PHH differentiation.
2) Overexpress FAM99A in a panel of HCC cell lines and assess effects on colony formation in soft agar (an indicator of a cell’s ability to form a tumor) and cell proliferation and death.
De-identified PHHs will be purchased from commercial sources and cultured as in our previous studies [1,2]. PHHs will be infected with a recombinant Adenovirus (recAd) expressing green fluorescent protein (GFP), or AdGFP-FAM99A in suspension prior to plating by incubating the cells with the recAds. Cells will then be analyzed at 24, 48, and 72 hours post infection. Albumin and urea levels in the cell-culture supernatant will be analyzed as indicators of hepatocyte function [3-5]. We will also use western blot analysis to assess levels of transferrin, hepatocyte nuclear factor 4a, and transthyretin, markers of differentiated hepatocytes [3-8, 9, 10]. HCC cell lines will also be assessed for effects on colony formation in soft agar. When an effect on colony formation is observed, we will assess cell proliferation and death using well established protocols [11-12]. The student will be conducting full-time research in the Bouchard lab for the Spring Term, thus they will see many facets of this project at whatever capacity 10 weeks may grant. The first ~2 weeks are expected to consist of literature review and laboratory familiarization. Weeks ~2-4 will roughly be focused on lab technique monitoring and development, as well as preliminary project design, development, and implementation. Weeks 4-8 will then consist of semi-independent research and laboratory group work. The last few weeks of the term will be spent on poster development for the Week of Undergraduate Excellence as well as final data analysis.
References
1. Lamontagne, J., J.C. Mell, and M.J. Bouchard, Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression. PLoS Pathog, 2016. 12(2): p. e1005438.
2. Arrigoni, A., et al., Analysis RNA-seq and Noncoding RNA. Methods Mol Biol, 2016. 1480: p. 125-35.
3. Kang, Y.B., Rawat, S., Duchemin, N., Bouchard, MJ., Noh, M., Human liver sinusoid on a chip for hepatitis B viral replication study. Micromachines, 2017. In press.
4. Kang, Y.B., et al., Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms. Biotechnol Bioeng, 2015. 112(12): p. 2571-82.
5. Kang, Y.B., et al., Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid. Biofabrication, 2013. 5(4): p. 045008.
6. Kang, Y.B., et al., Layered long-term co-culture of hepatocytes and endothelial cells on a transwell membrane: toward engineering the liver sinusoid. Biofabrication, 2013. 5(4): p. 045008.
7. Sodunke, T.R., M.J. Bouchard, and H.M. Noh, Microfluidic platform for hepatitis B viral replication study. Biomed Microdevices, 2008. 10(3): p. 393-402.
8. Clippinger, A.J. and M.J. Bouchard, The Hepatitis B Virus Hbx Protein Localizes to Mitochondria in Primary Rat Hepatocytes and Modulates Mitochondrial Membrane Potential. J Virol, 2008. 82: p. 6798-6811.
9. Clippinger, A.J., T.L. Gearhart, and M.J. Bouchard, Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappaB and the mitochondrial permeability transition pore. J Virol, 2009. 83(10): p. 4718-31.
10. Casciano, J.C., et al., Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes. PLoS One, 2017. 12(2): p. e0168328.
11. Crowley, L.C., et al., Measuring Cell Death by Trypan Blue Uptake and Light Microscopy. Cold Spring Harb Protoc, 2016. 2016(7).
12. Crowley, L.C., et al., Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry. Cold Spring Harb Protoc, 2016. 2016(7).
Personal statement of interest: A short description of the student’s interest in this work, including how this project may fit into the student’s academic plan/goals, what drew them to this work, and/or why they are passionate about this work:
I have been involved in many research endeavors throughout my time in high school and undergrad; each has propelled me toward biochemical and molecular biological research. In high school, I worked on individual projects and competed internationally three times, including environmental projects which won national awards. Since high school, I have had two experiences with the United States Department of Agriculture – Agricultural Research Services. The first was an internship, focused on enzyme identification and bioinformatic database mining, and the second was with the Molecular Characterization of Foodborne Pathogens unit, developing a rapid identification method using next-generation sequencing. I also participated in the Student Tackling Advanced Research program and worked with a professor to investigate a new polymerization technique. I am currently working in a neuroengineering lab through Vertically Integrated Projects, where I have been the lead in establishing a research records database that is Institutional Review Board and HIPAA (Health Insurance Portability and Accountability) compliant. Upon looking at my research history, most would say that these topics are varied in field and show no direction or culmination. However, I argue that each experience has allowed me to build an arsenal of skills that could not be developed from one area alone. There is value in these skills, especially as I find myself aiming for a career as a principal investigator with the National Institutes of Health. As I reflect on my time in the laboratory and my contributions towards the advancement of knowledge, I find myself yearning for research that directly reflects the future I want. Furthermore, research that can one day be applied to alleviate health disparities or address disproportions in minority communities is a driving force for why I want to work at the NIH. Although the studies outlined in this proposal are not designed to directly address a specific health disparity, the incidence of HCC is higher in Asian, African Americans, Hispanics, and American Indians/Alaskan Natives populations (REFS). Therefore, any identification of novel biomarkers or treatment methods would be directly applicable to both general health and the higher incidence of HCC in these specific minority groups. With the Bouchard Lab at the Drexel University College of Medicine, I would gain experience with mammalian cell tissue culturing, molecular biological techniques, and biochemical techniques. I also find the research fascinating – knowing that I’m directly contributing to knowledge that can help my community and many others that face similar issues. I believe that this research will enhance the skills I’ve developed and can show me how research can surpass the fundamental sciences and begin to translate into something that directly benefits humankind. I have aggressively pursued research as a student, but topics similar to this project are what I foresee myself doing for the rest of my career. Proposal title: Investigating the Tumor-suppressing Potential of Long noncoding RNA FAM99A in Primary Human Hepatocytes