Identification and characterization of long non-coding RNAs in porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Ruszkowska, Monika; Nynca, Anna; Paukszto, Łukasz; Sadowska, Agnieszka; Świgońska, Sylwia; Orlowska, Karina; Molcan, Tomasz; Jastrzebski, J; Ciereszko, Renata E.

doi:10.1186/s40104-018-0288-3

Cited by 22 publications

(24 citation statements)

References 72 publications

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“…The AVG-16 cell line was derived from granulosa cells of medium porcine follicles (Horisberger, 2006), and the cells were reported to exhibit AhR expression, both at mRNA and protein level (Sadowska et al, 2015). Previously, we employed the intact (untransfected) AVG-16 cells to study the impact of TCDD on the gene (Sadowska et al, 2017;Nynca et al, 2019) and lncRNA expression profiles (Ruszkowska et al, 2018) as well as on the cell proteome (Orlowska et al, 2018). The AhR gene and protein abundance in the cultured AhR knock-down AVG-16 cells was significantly reduced in comparison to control untransfected cells.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study, we were focused on identifying all possible pathways (molecules) involved in the mechanism of TCDD action in porcine granulosa cells and, therefore, the selected dose of TCDD (100 nM) moderately exceeded its environmentally relevant concentrations. The concentration of 100 nM of TCDD was previously reported to affect porcine granulosa cells steroidogenesis (Gregoraszczuk et al, 2000;Gregoraszczuk, 2002;Jablonska et al, 2014), gene expression (Piasecka-Srader et al, 2016;Sadowska et al, 2017;Nynca et al, 2019), lncRNA expression (Ruszkowska et al, 2018) and proteome (Orlowska et al, 2018) without affecting cell viability and morphology. Moreover, 100 nM was one of the two most frequently used TCDD concentrations in in vitro experiments performed on porcine ovarian cells.…”

Section: Tcdd Treatment Of Granulosa Cellsmentioning

confidence: 98%

“…Since TCDD influences the production of estrogens and progesterone by porcine granulosa cells (Gregoraszczuk, 2002;Jablonska et al, 2011;Jablonska et al, 2014), it is of importance to identify its molecular targets in follicular cells. The results of our previous studies, performed on porcine granulosa cells, demonstrated, among others, that TCDD affected the expression of transcripts involved in the follicular atresia as well as cell proliferation and cell cycle regulation (Sadowska et al, 2017;Ruszkowska et al, 2018;Nynca et al, 2019). The granulosa cells of pigs were found to express the Ah receptor (Sadowska et al, 2015).…”

Section: Introductionmentioning

confidence: 93%

“…Total RNA isolation and evaluation of RNA integrity were performed as described previously (Sadowska et al, 2017;Ruszkowska et al, 2018). The RNA samples with integrity number greater than 8.0 were designated for NGS.…”

Section: Total Rna Isolation and Evaluation Of Rna Integritymentioning

confidence: 99%

“…In addition, real-time PCR was used to validate the results of NGS by analysing the expression of three selected DEGs identified in AhR knock-down granulosa cells treated with TCDD for 3 h. The validation was performed on the same RNA samples which were used for NGS (n= two biological replicates per one time point). The reverse transcription reaction and real-time PCR were performed as previously reported (Sadowska et al, 2017;Ruszkowska et al, 2018). Primers and probes (Thermofisher Scientific, Waltham, MA, USA) for particular genes are presented in Table S2.…”

Section: Real-time Pcrmentioning

confidence: 99%

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The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells

Ruszkowska

Sadowska

Nynca

et al. 2020

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Background 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a toxic man-made chemical, adversely affecting reproductive processes. The well-characterized canonical mechanism of TCDD action involves the activation of aryl hydrocarbon receptor (AhR) pathway, but AhR-independent mechanisms were also suggested. By applying RNA interference technology and Next Generation Sequencing (NGS) we aimed to identify genes involved in the mechanism of TCDD action in AhR knock-down porcine granulosa cells. Methods Porcine granulosa cells were transfected with small interfering RNAs targeting mRNA of AhR. After transfection, medium was exchanged and the AhR knock-down cells were treated with TCDD (100 nM) for 3, 12 or 24 h, total cellular RNA was isolated and designated for NGS. Following sequencing, differentially expressed genes (DEGs) were identified. To analyze functions and establish possible interactions of DEGs, the Gene Ontology (GO) database and the Search Tool for the Retrieval of Interacting Genes (STRING) database were used, respectively. Results The AhR gene expression level and protein abundance were significantly decreased after AhR-targeted siRNAs transfection of the cells. In TCDD-treated AhR knock-down cells we identified 360 differentially expressed genes (DEGs; P-adjusted < 0.05 and log2 fold change [log2FC] ≥ 1.0). The functional enrichment analysis of DEGs revealed that TCDD influenced the expression of genes involved, among other, in the metabolism of vitamin A, follicular development and oocyte maturation, proliferation and differentiation as well as inflammation, stress response, apoptosis and oncogenesis. The three-time point study demonstrated that TCDD-induced changes in the transcriptome of AhR knock-down porcine granulosa cells were especially pronounced during the early stages of the treatment (3 h). Conclusions TCDD affected the transcriptome of AhR knock-down porcine granulosa cells. The molecules involved in the AhR-independent action of TCDD were indicated in the study. The obtained data contribute to better understanding of molecular processes induced by xenobiotics in the ovary.

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