CircAgtpbp1 Acts as a Molecular Sponge of miR-543-5p to Regulate the Secretion of GH in Rat Pituitary Cells

Abstract

CircRNAs have been identified to be expressed differently and stably in numerous species and tissues, but their functions in growth hormone (GH) secretion are still largely unknown. In summary, we have revealed a circRNA-miRNA-mRNA network that may play a biological role in the rat pituitary gland. First, we verified the chromosome location information of circAgtpbp1 according to sequencing analysis. The circAgtpbp1 characteristics were authenticated through PCR, qRT-PCR, treating with RNase and fluorescent in situ hybridization (FISH). Second, we detected the expression pattern of circAgtpbp1 in the rat anterior pituitary by qRT-PCR. We also designed circAgtpbp1 siRNA and constructed overexpression plasmid to evaluate the effect of circAgtpbp1 function on GH secretion by qRT-PCR, ELISA and Western blot. CircAgtpbp1 is a stable, truly circular molecule. We found that circAgtpbp1 interacted with miR-543-5p and can regulate GH secretion in pituitary cells through a circAgtpbp1-miR-543-5p-GH axis. Overall, the evidence generated by our study suggests that circAgtpbp1 can act as a sponge of miR-543-5p to reduce the inhibitory effect of miR-543-5p on Gh1 and further promote GH secretion. These findings expand our existing knowledge on the mechanisms of hormone regulation in the pituitary gland.

Keywords: GH; animal growth; circRNA; miRNA sponge; pituitary.

Figure 1

Identification and validation of circular RNAs (circRNA). (A) Schematic illustration demonstrates the binding sites of miR-543-5p in rno_circ_0001059. CircAgtpbp1 is produced by the Agtpbp1 gene of exons in chromosome 17. (B) The presence of rno_circ_0001059 was validated by PCR followed by agarose gel electrophoresis and Sanger sequencing. (C) Arrows represent divergent primers binding to the genomic region of circAgtpbp1. (D) RT–PCR assay with divergent or convergent primers indicated the existence of circAgtpbp1 in rat pituitary cells. GAPDH was used as a negative control. (E) RT–qPCR analysis of circAgtpbp1 and Agtpbp1 mRNA the relative expression level after treatment with RNase R. (F) The relative circAgtpbp1 and Agtpbp1 mRNA levels were analyzed by RT–qPCR after the reverse transcription with Random hexamer or oligo (dT)18 primers. (G) RNA fluorescent in situ hybridization (FISH) for circAgtpbp1. CircAgtpbp1 probes were labeled with Cy3. Nuclei were stained with DAPI. At least three replicates of each experiment were performed. Mean values and standard deviations (SD) of data are shown. One-way ANOVA was applied to determine statistical significance. p < 0.05 was considered significant. *, p < 0.05.

Figure 2

CircAgtpbp1 expression pattern in rat anterior pituitary. (A) RT–qPCR analysis of circAgtpbp1 the relative expression in different stages. (B) The relative expression level of circAgtpbp1 in mature rat different tissues. (C) CircAgtpbp1 expression in rat pituitary primary cell and GH3, MMQ cell lines. At least three replicates of each experiment were performed. Mean values and standard deviations (SD) of data are shown. One-way ANOVA and independent-samples t-test were applied to determine statistical significance. p < 0.05 was considered significant. Different letters (a, b and c) indicate significant differences between groups (p < 0.05). *, p < 0.05.

Figure 3

Effects of circAgtpbp1 knockdown on Gh1 transcription. (A) Schematic illustration showing three siRNAs that the back-splicing junction site of circAgtpbp1.Schematic illustration showing siRNAs and circAgtpbp1 expression vectors. (B) The expressions of circAgtpbp1 were determined with RT–qPCR in GH3 cells transfected with siRNAs. (C) The expressions of circAgtpbp1 were determined with RT–qPCR in pituitary cells transfected with siRNAs. (D) The expressions of Gh1 were determined with RT–qPCR in GH3 cells transfected with siRNA-2. (E) The expressions of Gh1 were determined with RT–qPCR in pituitary cells transfected with siRNA-2. (F) The expressions of Gh1 were determined with WB in GH3 cells transfected with siRNA-2. (G) The expressions of circAgtpbp1 were determined with WB in pituitary cells transfected with siRNA-2. (H) ELISA experiment detected GH secretion in GH3 cells after transfected with circAgtpbp1 siRNA-2. (I) ELISA experiment detected GH secretion in pituitary cells after transfected with circAgtpbp1 siRNA-2. At least three replicates of each experiment were performed. Mean values and standard deviations (SD) of data are shown. One-way ANOVA was applied to determine statistical significance. p < 0.05 was considered significant. Different letters (a,b) indicate significant differences between groups (p < 0.05). *, p < 0.05.

Figure 4

Effects of overexpressing circAgtpbp1 on Gh1 transcription. (A) The expressions of circAgtpbp1 were determined with RT–qPCR in GH3 cells transfected with circAgtpbp1 overexpression vector. (B) The expressions of circAgtpbp1 were determined with RT–qPCR in pituitary cells transfected with circAgtpbp1 overexpression vector. (C) The expressions of Gh1 were determined with RT–qPCR in GH3 cells transfected with circAgtpbp1 overexpression vector. (D) The expressions of Gh1 were determined with RT–qPCR in pituitary cells transfected with circAgtpbp1 overexpression vector. (E) The expressions of Gh1 were determined with WB in GH3 cells transfected with circAgtpbp1 overexpression vector. (F) The expressions of Gh1 were determined with WB in pituitary cells transfected with circAgtpbp1 overexpression vector. (G) ELISA experiment detected GH secretion in GH3 cells after transfected with circAgtpbp1 overexpression vector. (H) ELISA experiment detected GH secretion in pituitary cells after transfected with circAgtpbp1 overexpression vector. At least three replicates of each experiment were performed. Mean values and standard deviations (SD) of data are shown. One-way ANOVA was applied to determine statistical significance. p < 0.05 was considered significant. Different letters (a,b) indicate significant differences between groups (p < 0.05).

Figure 5

CircAgtpbp1 acts as a sponge for miR-543-5p in rat pituitary primary cells. (A) Luciferase assays were performed to detect the luciferase activities of 293 T cells to confirm the interaction between miR-543-5p and circAgtpbp1. (B) The Ago2 RIP assay showed that Ago2 significantly enriched miR-543-5p and circAgtpbp1 in rat pituitary cells. (C) MiR-543-5p expression increased upon circAgtpbp1 knockdown by siRNA-2 in GH3 cells. (D) MiR-543-5p expression increased upon circAgtpbp1 knockdown by siRNA-2 in rat pituitary cells. (E) MiR-543-5p expression decreased after circAgtpbp1 overexpression in GH3 cells (F) MiR-543-5p expression decreased after circAgtpbp1 overexpression in rat pituitary cells. At least three replicates of each experiment were performed. Mean values and standard deviations (SD) of data are shown. One-way ANOVA and independent-samples t-test were applied to determine statistical significance. p < 0.05 was considered significant. Different letters (a,b) indicate significant differences between groups (p < 0.05).

Figure 6

CircAgtpbp1 regulates Gh1 expression and GH secretion via targeting miR-543-5p. (A,C) The expressions of Gh1 were detected by qRT–PCR after regulating circAgtpbp1 or miR-543-5p in GH3 cells. (B,D) The expressions of Gh1 were detected by qRT–PCR after regulating circAgtpbp1 or miR-543-5p in pituitary cells. At least three replicates of each experiment were performed. Mean values and standard deviations (SD) of data are shown. One-way ANOVA and independent-samples t-test were applied to determine statistical significance. p < 0.05 was considered significant. *, p < 0.05.

Figure 7

The schematic diagram shows the mechanism underlying circAgtpbp1 as a ceRNA for miR-543-5p.