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Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor
Milena Rizzo1, Monica Evangelista, Marcella Simili
1Laboratory of Gene and Molecular Therapy, Institute of Clinical Physiology, CNR, Pisa, Italy.
Abstract:
The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary MEF senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity.
Insights
Cellular senescence, or the Hayflick limit, is variable. Inactivating cell proliferation genes and miRNAs can delay senescence and promote immortalization, suggesting tumor suppressor roles for these miRNAs.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- The Hayflick limit, or cellular senescence, defines the finite number of times a primary cell population can divide.
- Variability in the escape from the Hayflick limit in primary mouse embryo fibroblasts (MEF) is not well understood.
Purpose of the Study:
- To investigate if the escape from the Hayflick limit is variable.
- To identify genetic and miRNA changes associated with delayed immortalization of MEF.
Main Methods:
- MEF were cultured with delayed expansion (6T15) versus standard expansion (6T3) to assess lifespan and immortalization.
- Expression levels of senescence-related genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) were analyzed.
- Karyotyping was performed to detect chromosomal changes (ploidy).
Main Results:
- Delayed expansion (6T15) extended MEF lifespan and delayed immortalization.
- A switch in p21 gene expression from up-regulation to down-regulation was observed.
- Down-regulation of specific miRNAs and a shift towards hyperdiploidy coincided with renewed cell proliferation.
- Transfection of down-regulated miRNAs into immortalized MEF inhibited proliferation, indicating tumor suppressor activity.
Conclusions:
- Inactivation of critical cell proliferation genes and miRNAs appears to be a determinant factor in escaping the Hayflick limit.
- Specific down-regulated miRNAs exhibit tumor suppressor-like activity, influencing cell proliferation and potentially preventing immortalization.
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