Dysregulated microRNA expression contributes to emergence of cancer stem cell subpopulations in human colorectal cancers
Date
2021
Authors
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Publisher
University of Delaware
Abstract
MicroRNAs (miRNAs) have a role in the regulation of different biological pathways in the body. Dysregulation of these noncoding fragments can lead to disease, developmental defects, and various cancer types. In this study we investigate the miRNAs that are implicated to target genes that encode different stem cell (SC) genes during tumorigenesis of colorectal cancer (CRC). CRC is one of the leading causes of cancer-related death in the United States. One possible reason behind the lack of efficacy in cancer drug therapeutics might be the result of the tumor heterogeneity, as proposed by the Big Bang Model of cancer heterogeneity. Previous research has been done to track the colonic SCs present at the bottom of colonic crypts during tumorigenesis. The Boman research team demonstrated that aldehyde dehydrogenase1 (ALDH1) is a specific biomarker for tracking colonic SCs (SCs), and that SC overpopulation drives CRC development and growth. They also found that CRC contains multiple cancer SC (CSC) subpopulations. Other essential biomarkers include LRIG1, LGR5, and CD166 and it was postulated they mark different CSC subpopulations in the emergence of tumor heterogeneity in CRC. The gap in our knowledge is that we do not know what causes tumor heterogeneity. The research objective is to investigate whether the different miRNAs target the CSC genes in each of the CSC subpopulations. Hypothesis: Specific miRNAs target the different CSC subpopulations (LRIG1, ALDH, LGR5, and CD166) which leads to multiple CSC subpopulations that establishes intra-tumoral heterogeneity in CRCs. Specific Aims: Aim 1: To analyze the different cancer stem cell (CSC) subpopulations using flow cytometry. Aim 2: To determine the expression of miRNAs in each CSC subpopulation and identify the mRNAs predicted to be targeted by the upregulated miRNAs. Aim 3: To determine the plasticity of ALDH and LGR5 CSC subpopulations from HT29 cells by growing them in 3D cultures to see if the subpopulations interconvert and if the miRNA signatures remain unchanged. Accordingly, different CSC sub-populations were isolated using fluorescence activated cell sorting (FACS), differential expression of miRNAs between CSC subpopulations was measured using NanoString profiling, and mRNAs targeted by the miRNAs as well as their enriched functional-related mRNA groups was identified using bioinformatics analysis. My results depict 4 different subpopulations when running the CSC combination sorts between ALDH, LGR5, LRIG1, and CD166. I quantified different percentages of the CSC genes in their respective combination sorts via flow cytometry. Moreover, I determined the expression of miRNAs and predicted, using bioinformatics analyses, their respective mRNA targets for the CSC subpopulations. The LGR5/ALDH sort resulted in 326 significant miRNAs of interest. Thus, I focused on LGR5 and ALDH CSCs to assess cell plasticity studies because LGR5 and ALDH have major roles in CRC progression. In conclusion, the presence of multiple subpopulations and different mRNA targets provides a mechanism that explains how intra-tumor heterogeneity arises in CRC. It also illustrates that CRC is highly complex in its different miRNA and mRNA targets. This study is significant as we aim to understand tumor heterogeneity through miRNA dysregulation and how to therapeutically target CSC populations.
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Keywords
Cancer, Cancer stem cells, Colorectal cancer, MicroRNAs, Stem cells