miRNA Expression and Cancer Development

miRNA Expression and Cancer Development



The papers for this module were selected because:
miRNAs are an important form of eukaryotic gene regulation that students sometimes struggle to understand in a classic lecture format. Discussion of miRNAs nicely tie in the idea of how DNA formerly believed to be “junk” because it did not code for a traditional gene that would become a protein has now been found the be very important for regulatory functions.
miRNAs are found to be very important in both the cause of and the treatment of various diseases. This module will focus mainly on cancer, but the involvement of miRNAs in other human diseases will also be discussed.
The discovery of miRNAs is a unique one that illustrates the unexpected nature of science and is a nice discussion to have with students.
This progression illustrates how many genetic regulatory mechanisms can function both positively and negatively.
Cancer is a major topic discussed in Cell Biology, and a discussion of the steps involved in cancer formation from the papers will serve as a good reinforcement of the topics discussed in class.
Cancer is always one of the topics students find most interesting in Cell Biology, so it is hoped they will find the articles related to the miRNA causes and miRNA potential treatment of cancer interesting.
These papers review some of the major signaling pathways whose dysfunctional regulation (or deregulation) can result in progression or inhibition of the cell cycle.
Important aspects of the cell cycle are discussed, forcing students to draw on knowledge learned in both Cell Biology and Introductory Biology.
Techniques in the paper range from genetic, molecular, and biochemical to effects on cell culture growth and physiological properties all the way to in silico analysis.


This module contains four papers from various laboratories. Two of these papers focus on the ability of one specific microRNA (miRNA) to inhibit the development of cancerous properties in two different cell types, and the other two papers describe two different miRNA whose overexpression results in the promotion of tumor and metastatic properties in the same cancerous cell type. This module is intended for an upper level Cell Biology course, which will contain a partial implementation of C.R.E.A.T.E. in the second half of the semester during the laboratory portion.

Applicable for Courses:

Cell Biology

Educational Level:


Roadmap Objectives:

    • Article: miR-449a causes Rb-dependent cell cycle arrest and senescence in prostate cancer cells. Noonan, E. J., R. F. Place, S. Basak, D. Pookot, and L-C. Li. (2010) Oncotarget. 1(5):349-358.
    • Content area/major concepts: This paper demonstrates that miR-449a is a component of the Rb pathway and functions as a tumor suppressor dependent on the functional status of Rb in prostate cancer cells.

      Cell growth requirements, cell cycle and how it is affected by the Rb signaling pathway, tumor suppressors, molecular biology and reporter genes, restriction enzymes, antibody binding, miRNA and gene expression and over-expression from a plasmid or retroviral construct, wild-type vs. mutant, protein biochemistry, 3’UTR, and complementary nucleotide sequence hybridization.
    • Methods or technology used to obtain data: Growth in cell culture, miRNA and siRNA transfection, cell staining with propidium iodide, flow cytometry, β-galactosidase staining and assay, phase contrast microscopy, SDS-PAGE, membrane transfer, immunoblotting (Western blot), use of secondary HRP-linked antibodies and chemiluminescence, in silico analysis cloning into a reporter vector, and luciferase reporter assay.
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Keeping different cell lines and sublines straight (abbreviated jargon), distinguishing between different forms of miRNA transfected, and a complicated immunoblotting figure (Fig. 6)
    • Article: MicroRNA-449a Is Downregulated in Non-Small Cell Lung Cancer and Inhibits Migration and Invasion by Targeting c-Met. Luo, W., B. Huang, Z. Li, H. Li, L. Sun, Q. Zhang, X. Qui, and E. Wang. (2013) PLOS One. 8(5):e64759.
    • Content area/major concepts: This paper demonstrates that miR-449a regulated cell migration and invasion in Non-small cell lung cancer cells as a tumor suppressor by targeting c-Met.

      Informed consent, PCR, chemoattractants, melting curve analysis, paired-samples T-test, Chi-square test, Mann-Whitney U test, Spearman-determined correlation, Kaplan-Meier method for survival curves, Cox regression, independent samples T-test, and one-way ANOVA.
    • Methods or technology used to obtain data: Patient-obtained samples, use of formalin-fixed paraffin-embedded tissues (FFPETs), diagnoses and histological grades, RNA extraction, real-time quantitative polymerase chain reaction, miRNA assays, cell migration and invasion assays, RT-qPCR, fluorescent reporter assay, and use of fluorescence spectrophotometer.
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Understanding the plethora of statistical analyses as needed to interpret the data, very busy and somewhat needlessly complicated figures and tables (particularly related to the 84 samples taken from patients with non-small cell lung cancer, interpreting the correlation between miR-449a expression levels and pathological cancer stage, keeping cell line abbreviations straight, interpreting effects of miR-449a expression on cell migration and invasion, and understanding the many different controls.
    • Article: MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1. Li, Y., D. Zhang, C. Chen, Z. Ruan, Y. Li, and Y. Huang. (2012) Mol. Biol. Cell. 23:1423-1434.
    • Content area/major concepts: This paper demonstrates that miR-212 is involved in tumorigenesis through an oncogenic mode of action that involves the suppression of PTCH1.

      miRNA Illumina ChIP, GFP fluorescence, hedgehog signaling pathway, human transcriptome, 96-well plate, and microplate reader, normalization to a control, and use of a reverse microscope.
    • Methods or technology used to obtain data: Microarray analysis, formaldehyde-agarose gel electrophoresis, fluorescence microscopy, BLAST, colony-formation assay, and cell proliferation assay.
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Interpretating flow cytometry data visualized in Figures 2A-C and 4A, interpreting combined real-time PCR and Western blotting figure, and many figures/experiments.
    • Article: MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN. Liu, Z., H. Wang, J. Liu, and Z-X. Wang. (2013) Mol. Cell Biochem. 372:35-45.
    • Content area/major concepts: This paper demonstrates that miR-21 promotes non-small cell lung cancer development through the PTEN signaling pathway.

      PTEN signaling pathway, chemotherapeutic agents, and comparison of viable vs. non-viable cell physiology.
    • Methods or technology used to obtain data: Use of tissue samples (as compared to cell culture), tumor sectioning, immunohistochemistry, snap-freezing in liquid nitrogen and storage at -80°C, site-directed mutagenesis, semi-quantitative RT-PCR assay, cell viability MTT assay, analysis of apoptosis, and clonogenic survival assay
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Many figures, remembering cell type abbreviations, interpreting busy figures with statistical data awkwardly placed, and understanding what is happening in Figure 9.

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