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Analysis of post-transcriptional regulation during cancer progression using a donor-derived isogenic model of

Laura S Bisogno¹, Jack D Keene¹

¹Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC 27710, United States.

Methods (San Diego, Calif.)

|May 23, 2017

Abstract:

Post-transcriptional regulation of gene expression by RNA binding proteins (RBPs) and non-coding RNAs plays an important role in global gene expression. Many post-transcriptional regulators are misexpressed and misregulated in cancers, resulting in altered programs of protein biosynthesis that can drive tumor progression. While comparative studies of several RBPs and microRNAs expressed in various cancer types have been reported, a model system that can be used to quantify RBP regulation and functional outcomes during the initiation and early stages of tumorigenesis is lacking. It was previously demonstrated that oncogenic transformation of normal human cells can be induced by expressing hTERT, p53^DD, cyclin D1, CDK4^R24C, C-MYC^T58A and H-RAS^G12^V. Here we describe a user-friendly method for generating this genetically defined model of step-wise tumorigenesis beginning with normal donor-derived human cells. This method immortalizes a donor's normal cells in about a week, reducing the chances of senescence. The entire stable system can be established in less than 12weeks. We then demonstrate the utility of such a system in elucidating the expression of multiple RBPs at an early step of tumor formation. We identify significant changes in the expression levels of transcripts encoding RBPs prior to transformation, suggesting that our described donor-derived isogenic system can provide insight about post-transcriptional regulation during the earliest stages of tumorigenesis in the context of diverse genetic backgrounds.

Insights

A new model system allows researchers to study RNA binding proteins (RBPs) during early cancer development. This system tracks RBP changes in human cells before tumor formation, offering insights into gene regulation in tumorigenesis.

Area of Science:

Molecular Biology
Cancer Research
Gene Regulation

Background:

Post-transcriptional regulation by RNA binding proteins (RBPs) and non-coding RNAs is crucial for gene expression.
Dysregulation of these factors in cancer drives tumor progression through altered protein biosynthesis.
Existing models lack the ability to quantify RBP regulation during early tumorigenesis.

Purpose of the Study:

To develop a user-friendly, genetically defined model for studying step-wise tumorigenesis.
To investigate the role of RBPs in the initiation and early stages of cancer development.
To analyze RBP expression changes prior to oncogenic transformation.

Main Methods:

Genetically engineered a model system using normal human cells.
Induced oncogenic transformation by expressing specific genes (hTERT, p53DD, cyclin D1, CDK4R24C, C-MYC T58A, H-RASG12V).
Established a stable, donor-derived isogenic system for step-wise tumorigenesis within 12 weeks.

Main Results:

Successfully generated a stable, genetically defined model of tumorigenesis from normal human cells.
Demonstrated the system's utility in analyzing RBP expression during early tumor formation.
Identified significant alterations in RBP transcript levels preceding cellular transformation.

Conclusions:

The developed donor-derived isogenic system provides a robust platform for studying early tumorigenesis.
This model offers valuable insights into post-transcriptional regulation by RBPs at the earliest stages of cancer.
The findings highlight the dynamic changes in RBP expression during the transition to a transformed state.

Read full analysis

Keywords:

Isogenic models Post-transcriptional regulation RNA binding proteins RNA regulons Tumorigenesis

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