Examining MYBPH and LILRB5 as mediators of metastasis in Renal Medullary Carcinoma Restricted; Files Only

Abstract

Renal medullary carcinoma (RMC) is a subtype of non-clear cell renal cell carcinoma that accounts for approximately 0.02-0.06% of all pediatric renal cancers. This cancer mainly occurs in male adolescents and young adults of African ancestry, especially those who are carriers of the sickle cell trait or those affected by sickle cell disease. Most patients of RMC present with metastasis at diagnosis. To identify potential mechanisms of metastasis and the epithelial-to-mesenchymal transition (EMT) in RMC, we performed differential gene expression analysis comparing PEDS005M_SUS cells that grow in suspension to PEDS005M_ADH cells that grow as an adherent monolayer. From this analysis, we identified that MYBPH, SLC13A5, and LILRB5 were significantly upregulated in PEDS005M_SUS cells. Furthermore, GSEA analysis revealed downregulation of the EMT pathway, indicating mesenchymal-to-epithelial transition (MET) in the PEDS005M_SUS cells. We then overexpressed MYBPH and LILRB5 in our PEDS005M_ADH cells. We hypothesized that MYBPH and LILRB5 overexpression in PEDS005M_ADH cells would lead to an increase in proliferative and migratory ability compared to the parental PEDS005M_ADH cells. Our findings suggest that MYBPH plays no role in cell proliferation or wound healing properties of our PEDS005M_ADH cells but may increase clonogenicity and cell migratory properties towards a chemoattractant. Furthermore, we observed that LILRB5 overexpression has no significant effect on wound healing or cell migration but promoted clonogenicity in our cells. These findings suggest that MYBPH and LILRB5 may play a role in metastasis in RMC. 

Table of Contents

Introduction 1

Methods 4

Table 1. List of cell lines used 4

Table 2. List of primers used for qRT-PCR 7

Results 11

Figure 1. Quantitative analysis of parental PEDS005M_ADH and PEDS005M_SUS cells 13

Table 3. Log2FoldChange and adjusted p-value values from RNA-Seq of parental PEDS005M_SUS as compared to PEDS005M_ADH cells. 14

Figure 2. qRT-PCR analysis of parental PEDS005M cell lines 14

Figure 3. Analysis of PEDS005M_ADH cells post lentiviral transduction with genes MYBPH, SLC13A5, and LILRB5. 16

Figure 4. Cell proliferation assays of PEDS005M_ADH parental, PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 17

Figure 5. Cell proliferation assay in poly-HEMA coated plates with PEDS005M_ADH parental, PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 18 Figure 6. In vitro wound healing scratch assay with PEDS005M_ADH parental, PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 20

Figure 7. Images of cell migration assays of PEDS005M_ADH parental cells and PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 21

Figure 8. Transwell in vitro cell migration assays of PEDS005M_ADH parental, PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 22

Figure 9. Images of colony formation wells of PEDS005M_ADH parental cells and PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 23

Figure 10. Colony formation assays of PEDS005M_ADH parental, PEDS005M_ADH LUC, PEDS005M_ADH MYBPH, and PEDS005M_ADH LILRB5 cells 24

Figure 11. Verification of transduced PEDS005M_ADH biological replicates 25 Figure 12. Bulk RNA-Seq of transduced PEDS005M_ADH cells 27

Figure 13. GSEA of PEDS005M_ADH MYBPH cells compared to PEDS005M_ADH LUC cells 30

Figure 14. GSEA of PEDS005M_ADH LILRB5 cells compared to PEDS005M_ADH LUC cells 31

Figure 15. Summary of cell proliferation, in vitro scratch, transwell migration, and colony formation assay results in PEDS005M_ADH MYBPH cells compared to PEDS005M_ADH LUC cells. 32

Figure 16. Summary of cell proliferation, in vitro scratch, transwell migration, and colony formation assay results in PEDS005M_ADH LILRB5 cells compared to PEDS005M_ADH LUC cells. 32

Discussion 33

References 44 

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Oncology Biology, Cell
Parola chiave	cancer biology renal medullary carcinoma
Committee Chair / Thesis Advisor	Andrew L. Hong, Emory University
Committee Members	Jennifer M. Spangle, Emory University William G. Kelly, Emory University

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