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

Abstract

Background: Long non-coding RNAs (lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics. The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, results in reproductive defects in many species including pigs. The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells.

Results: One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells. The identified lncRNAs were found to be shorter than mRNAs. In addition, the number of exons was lower in lncRNAs than in mRNAs and their exons were longer. TCDD affected the expression of 22 lncRNAs (differentially expressed lncRNAs [DELs]; log₂ fold change ≥ 1, P-adjusted < 0.05) in the examined cells. Potential functions of DELs were indirectly predicted via searching their target cis- and trans-regulated protein-coding genes. The co-expression analysis revealed that DELs may influence the expression of numerous genes, including those involved in cellular response to xenobiotics, dioxin metabolism, endoplasmic reticulum stress and cell proliferation. Aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1 (CYP1A1) were found among the trans-regulated genes.

Conclusions: These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells. To our knowledge, this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile. These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.

Keywords: AVG-16 cell line; Granulosa cells; Pig; RNA-Seq; TCDD; lncRNAs.

Fig. 1

Screening of candidate long non-coding RNAs (lncRNAs) in the porcine granulosa cell transcriptome and classification of novel lncRNAs. a Schematic diagram of the pipeline used for the identification of lncRNAs in porcine granulosa cells. b Venn diagram presenting the results of the coding potential analysis of the obtained 4,160 long transcripts. Please note that five different tools (CPC, CNCI, PLEK, Pfam, FEELnc) were employed to analyze the coding potential. In consequence, 1,689 potentially non-coding long transcripts were identified and designated as candidate lncRNAs. c Classification of the obtained novel lncRNAs according to their genomic positions. *Customized algorithms of the authors were applied for the lncRNA identification in this step

Fig. 2

The percentage distribution of the identified lncRNAs and mRNAs according to their (a) transcript length, (b) exon length, (c) exon number and (d) expression level (the latter expressed as fragments per kilobase of exon per million fragments mapped [FPKM] values). The assumed nucleotide (nt) ranges as well as ranges of FPKM values are depicted in the X axis

Fig. 3

The comparison of genomic features of the identified lncRNAs and mRNAs. The lncRNAs and mRNAs were compared in respect to average (a) transcript length, (b) exon length, (c) exon number and (d) expression level. Data are expressed as mean ± SE. Statistical analysis was performed using Welch’s t-test in R package. Asterisks designate statistical differences (P < 0.05)

Fig. 4

Heatmap illustrating the expression profile of differentially expressed lncRNAs (presented as Z-score values) in porcine granulosa cells treated with TCDD for 3, 12 or 24 h. Red blocks represent up- and green blocks represent down-regulated lncRNAs. The color scale of the heatmap shows the expression level where the brightest green stands for − 2.0 Z-score and the brightest red stands for + 2.0 Z-score. Z-score was calculated using logFPKM and the following equation: [x - mean(x)] / sd(x) where ‘sd’ means standard deviation. C 3–24: untreated porcine granulosa cells (control) cultured for 3, 12 or 24 h. TCDD 3–24: porcine granulosa cells treated with TCDD (100 nmol/L) for 3, 12 or 24 h. *transcript expression was not detected in control (untreated) cells; ○ transcripts with expression level altered in two or three examined incubation times

Fig. 5

Presentation of differential expression of exons (E) and the occurrence of splice junction (J) in the exemplary selected lncRNA XLOC (XLOC_020835) identified in porcine granulosa cells treated with TCDD. The upper panel shows the expression level estimates for the mean normalized read counts for each exon or splice junction of XLOC_020835 identified in TCDD-treated (blue) and control (red) cells. The lower panel shows the exonic regions (boxes, labelled E001-E003) and known splice junctions (solid line, labelled J004). Statistically significant differences (P-adjusted < 0.05) of usage exons and junctions are drawn in violet. C3: untreated porcine granulosa cells (control) cultured for 3 h. TCDD3: porcine granulosa cells treated with TCDD (100 nmol/L) for 3 h

Fig. 6

Real-time PCR validation of three selected differentially expressed lncRNAs (TCONS_00016901, TCONS_00031035, TCONS_00034713) which were identified in TCDD-treated porcine granulosa cells (treated vs. untreated cells) by RNA-Seq. Data are expressed as mean ± SEM (n = 3–4). Statistical analysis was performed using Student’s t-test. Asterisks designate statistical differences (P < 0.05). AU: arbitrary units; C: control; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin