The role of MIR-199A in BAO-induced transgenerational bone deformities in Japanese Medaka

Date

2023-05

Authors

Jayarajan, Rijith

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Abstract

Benzo[a]pyrene (BaP), a ubiquitous environmental pollutant and known carcinogen, has been evidenced to cause bone disorders in a transgenerational manner by altering epigenetic mechanisms like microRNAs (miRNA). Transcriptome analysis of bone tissue in the ancestrally BaP-exposed F3 generation indicated a prominent role of miR-199a in the regulation of significantly enriched molecular pathways for bone formation and oxidative stress. To elucidate the function of miR-199a during bone development wildtype (Orange Red) and transgenic (twist:dsred/col10a1:gfp and col10a1:gfp/osx:mCherry) freshwater medaka (Oryzias latipes) were assessed for calcification and cell-subpopulation level changes in response to miR-199a agonist and antagonist microinjections at the onset of vertebra development. To further characterize the role of miR-199a on bone development, mineralization and osteoblast differentiation, the effects of miR199a-3p agonist and antagonist injections (700 pg) at the beginning of bone formation in medaka embryos 3 days post fertilization (dpf) were studied. The area and intensity of calcification were assessed in wild-type medaka. Medaka transgenic lines - twist:dsred/col10a1:gfp and col10a1:gfp/osx:mCherry were used to assess changes during osteoblast differentiation. No impact on the calcified area and bone thickness upon miR-199a-3p agonist or antagonist injection was identified. The spatial distribution and the cellular density of twist+ mesenchymal stem cells (MSCs) were not affected. A significant reduction upon agonist injection was noted in the cellular density of col10+ osteochondro progenitors (OPCs). No change in spatial distribution and density of osx+ premature osteoblast cells (POCs) was seen upon miR-199a-3p treatment. Assessment of bone gene expression levels in 25 dpf old medaka larvae revealed a potential inhibitory effect of miR-199a-3p on bone development. The tissue, cellular and molecular level experiments confirmed the role of miR-199a-3p in bone maturation and osteoblast differentiation. This study provides a novel understanding of miRNA regulation during two critical windows of bone development. The provided data can be used to delineate the effect of BaP exposure on regulating specific miRNAs. Moreover, developmental levels of miR-199a may be useful as a potential biomarker for later life-stage bone impairment, such as osteoporosis development.

Description

A Thesis Submitted In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in Marine Biology from Texas A&M University-Corpus Christi.

Keywords

BaP, epigenetics, medaka fish, MiRNA, transgenerational

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