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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
P53 regulation of osteoblast differentiation is mediated through specific microRNAs
Shivang Shah1, Elisha Pendleton1, Oliver Couture1
1Department of Biochemistry, College of Graduate Studies, Midwestern University, 555, 31st, Street, Downers Grove, IL60515, USA.
Abstract:
In order to understand the role of the p53 tumor suppressor gene in microRNA expression during osteoblast differentiation, we used a screen to identify microRNAs that were altered in a p53-dependent manner. MicroRNAs from MC3T3-E1 preosteoblasts were isolated from day 0 (undifferentiated) and day 4 (differentiating) and compared to a p53 deficient MC3T3-E1 line treated similarly. Overall, one fourth of all the microRNAs tested showed a reduction of 0.6 fold, and a similar number of them were increased 1.7 fold with differentiation. P53 deficiency caused 40% reduction in expression of microRNAs in differentiating cells, while a small percent (0.03%) showed an increase. Changes in microRNAs were validated using real-time PCR and two microRNAs were selected for further analysis (miR-34b and miR-140). These two microRNAs were increased significantly during differentiation but showed a dramatic reduction in expression in a p53 deficient state. Stable expression of miR-34b and miR-140 in MC3T3-E1 cells resulted in decreases in cell proliferation rates when compared to control cells. There was a 4-fold increase in p53 levels with miR-34b expression and a less dramatic increase with miR-140. Putative target binding sites for bone specific transcription factors, Runx2 and Osterix, were found for miR-34b, while Runx2, beta catenin and type 1 collagen were found to be miR-140 targets. Western blot analyses and functional assays for the transcription factors Runx2, Osterix and Beta-catenin confirmed microRNA specific interactions. These studies provide evidence that p53 mediated regulation of osteoblast differentiation can also occur through specific microRNAs such as miR-34b and miR-140 that also directly target important bone specific genes.
Insights
The p53 tumor suppressor influences microRNA expression during bone cell differentiation. p53 deficiency significantly reduces microRNAs, including miR-34b and miR-140, which are crucial for osteoblast differentiation and target bone-specific genes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The p53 tumor suppressor gene plays a critical role in cellular processes, including differentiation.
- MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are implicated in various biological functions.
- Osteoblast differentiation is a complex process involving specific gene expression patterns.
Purpose of the Study:
- To investigate the role of the p53 tumor suppressor gene in regulating microRNA expression during osteoblast differentiation.
- To identify specific microRNAs whose expression is dependent on p53 during the differentiation of bone cells.
Main Methods:
- MC3T3-E1 preosteoblasts were used, comparing wild-type and p53-deficient cells at different differentiation stages (day 0 and day 4).
- MicroRNA expression profiling was performed, followed by validation using real-time PCR.
- Stable expression of selected microRNAs (miR-34b, miR-140) was introduced, and their effects on cell proliferation were assessed.
- Western blot analyses and functional assays were conducted to confirm interactions with target genes.
Main Results:
- Differentiation led to significant changes in microRNA expression, with many miRNAs decreasing and some increasing.
- p53 deficiency resulted in a substantial reduction (40%) in microRNA expression during osteoblast differentiation.
- miR-34b and miR-140 levels increased during differentiation but were significantly decreased in p53-deficient cells.
- Stable expression of miR-34b and miR-140 reduced cell proliferation and modulated p53 levels.
- Functional assays confirmed that miR-34b and miR-140 directly target key bone-related transcription factors (Runx2, Osterix, beta-catenin) and genes (collagen type I).
Conclusions:
- p53 regulates osteoblast differentiation partly through modulating specific microRNA expression, including miR-34b and miR-140.
- These p53-regulated microRNAs directly influence the expression of critical genes involved in bone formation.
- The findings highlight a novel mechanism of p53-mediated control over bone cell differentiation via microRNA pathways.
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