Insulin-like growth factor 2 mRNA-binding protein 2-regulated alternative splicing of nuclear factor 1 C-type causes excessive granulosa cell proliferation in polycystic ovary syndrome

Abstract

Objectives: Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder. Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) serves as an HMGA2 target gene to promote the proliferation of granulosa cells (GCs). However, it is still unclear whether IGF2BP2 participates in the pathogenesis of PCOS as RNA binding protein (RBP). In this study, we aimed to elucidate IGF2BP2-interacting transcripts, global transcriptome together with alternative splicing in GCs to eventually uncover potential mechanisms of PCOS pathogenesis.

Materials and methods: The expression of IGF2BP2 in GCs from PCOS patients was detected using quantitative reverse transcription PCR (RT-qPCR) and western blot. We captured IGF2BP2-interacting transcripts, global transcriptome together with alternative splicing by RNA immunoprecipitation sequencing (RIP-seq) and RNA sequencing (RNA-seq). KGN cells transfected with IGF2BP2 overexpressing plasmids and nuclear factor 1 C-type (NFIC) siRNAs, were applied to CCK-8, EdU and TUNEL assays.

Results: IGF2BP2 was highly expressed in GCs from PCOS patients. As an RBP, it preferentially bound to the 3'and 5'UTRs of mRNAs with GGAC motif and a newly found GAAG motif. The overexpression of IGF2BP2 changed the transcriptome profile of KGN cells. IGF2BP2 functioned to regulate alternative splicing events and promote cell proliferation through inhibiting exon skipping events of NFIC.

Conclusion: In conclusion, we demonstrated that IGF2BP2 promotes GC proliferation via regulating alternative splicing of NFIC in PCOS. The findings help to better understand the roles of IGF2BP2 in the pathogenesis of PCOS.

FIGURE 1

Upregulated insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) in granulosa cells (GCs) from polycystic ovary syndrome (PCOS) patients increases cell viability and proliferation. (A) IGF2BP2 mRNA detection by RT‐qPCR in GCs from Control and PCOS groups. Data were normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Control: n = 20; PCOS: n = 20. *P < 0.05. (B) IGF2BP2 protein detection by western blot in GCs from PCOS and Control women. (C) RT‐qPCR showing expression of IGF2BP2 mRNA in IGF2BP2 overexpression (OE) KGN cells. ***P < 0.001. (D) IGF2BP2 protein detection by western blot in IGF2BP2 OE KGN cells. Experiments were performed in triplicate. (E) KGN cells were transfected with vector control or IGF2BP2 OE plasmids for 24, 48, or 72 h, and cell viability was determined by the CCK‐8 assay. ***P < 0.001. (F) EdU staining of IGF2BP2 OE cells. Nuclei were stained by using DAPI. EdU positive cells, red; cell nuclei, blue; the data shown were representative of three independent experiments with similar results. Scale bar, 100 μm. (G) Statistics of EdU positive cells quantified by counting the cells with fluorescent signal using the software Image J. Experiments were performed in triplicate. *P < 0.05. (H) Apoptosis was reflected by TUNEL staining of the cells from the indicated groups. Representative images of the apoptotic cells were shown. Scale bar, 100 μm. (I) Statistics of TUNEL positive cells quantified by counting the cells with fluorescent signal using the software Image J. *P < 0.05. Ctrl, control. OE, IGF2BP2 overexpression. Error bars represent the mean ± SEM

FIGURE 2

RNA‐seq analysis of insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) regulated transcriptome profile in KGN cells. (A) IGF2BP2 expression quantified by RNA‐seq data. Fragments per kilobase of transcript per million fragments mapped (FPKM) values were calculated following the described in Section 2. Error bars represent the mean ± SEM. *P < 0.05. (B) Heat map showing a hierarchically clustered Pearson's correlation matrix resulting from the comparison between transcript expression levels for control and IGF2BP2‐overexpressed samples. (C) Identification of IGF2BP2‐regulated genes. Volcano plot shows up‐ and down‐regulated genes labelled in red and blue, respectively. (D) Hierarchical clustering of differentially expressed genes (DEGs) in control and IGF2BP2‐overexpressed samples. FPKM values were log2‐transformed and then median‐centred by each gene. (E) The top 10 GO biological processes of IGF2BP2 up‐ and down‐regulated genes. (F) The top 10 KEGG pathways of IGF2BP2 up‐ and down‐regulated genes. (G) The relative expression of DEGs and qPCR validation. Error bars represent the mean ± SEM. ***P < 0.001, *P < 0.05

FIGURE 3

RNA immunoprecipitation sequencing analysis of insulin‐like growth factor 2 mRNA‐binding protein 2's (IGF2BP2) binding profile and binding motifs. (A) IGF2BP2 protein detection by western blot in KGN cells. (B) Read distribution across the reference genome. Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The peak reads density for 5′UTR, CDS and 3′UTR for input and IGF2BP2 IP samples. (D) Motif analysis using the software HOMER showing the top 10 preferred binding motifs of IGF2BP2. (E) The binding sites of the GGAC and GAAG binding motifs

FIGURE 4

Differentially expressed genes (DEGs) regulated by insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) overexpression showed no direct bindings of IGF2BP2. (A) Venn diagram showing the overlap between peaks in two RNA immunoprecipitation sequencing (RIP‐seq) sample replicates. (B) Bubble plot showing the top 10 biological processes associated with IGF2BP2‐bound genes using a GO enrichment analysis. (C) Bubble plot showing the top 10 biological processes associated with IGF2BP2‐bound genes using a KEGG enrichment analysis. (D) Bubble plot showing the top 10 pathways associated with IGF2BP2‐bound genes using a reactome enrichment analysis. (E) Venn diagram showing genes overlapping between RIP‐seq and DEGs

FIGURE 5

Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2)‐regulated alternative splicing events in KGN cells. (A) Classification of IGF2BP2‐regulated alternatively spliced events. (B) The overlap between IGF2BP2‐regulated differentially expressed genes and regulated alternative splicing genes (RASGs). (C) The top 10 biological processes associated with IGF2BP2‐regulated alternatively spliced genes using a GO enrichment analysis. (D) The top 10 pathways associated with IGF2BP2‐regulated alternatively spliced genes using a KEGG analysis. (E) Overlap between binding genes and RASGs. (F) Bubble plot showing the biological processes of the overlapped genes between binding genes and RASGs using a GO enrichment analysis. (G) Bubble plot showing the top KEGG pathways of the overlapped genes between binding genes and RASGs

FIGURE 6

Insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) overexpression (OE) regulates alternative splicing of nuclear factor 1 C‐type (NFIC). (A) IGF2BP2 binds to the mRNAs of NFIC. Genome visualization showing IGF2BP2‐regulated ASEs and relative binding on the left and right panels, respectively. The peak ranges were highlighted with purple or green colours. (B) Quantification of NFIC binding using RNA immunoprecipitation sequencing data. ***P < 0.001 (C) Schematic diagram of the NFIC AS (ENST00000341919.7). Boxes represent exons, lines between exons represent introns. (D) The altered ratios of ASEs in RNA‐seq were plotted. **P < 0.01. (E) RT‐qPCR validation of AS of NFIC in IGF2BP2 OE KGN cells. *P < 0.05. (F) The relative expression of CTF5 was detected using RT‐qPCR following IGF2BP2 OE in KGN cells. **P < 0.01. ASEs, alternative splicing events; ES, exon skipping. Error bars represent the mean ± SEM

FIGURE 7

Knockdown of nuclear factor 1 C‐type (NFIC) promotes cell proliferation in KGN cells. (A) The relative expression of CTF5 mRNA is detected following NFIC knockdown in KGN cells. **P < 0.01. (B) Western blot analysis of CTF5 following NFIC knockdown. (C) KGN cells were transfected with siRNA‐NC or siRNA‐NFIC#1/ #2/ #3 for 24, 48, or 72 h, and cell viability was determined by the CCK‐8 assay. *P < 0.05, **P < 0.01, ***P < 0.001. (D) EdU staining of NFIC knockdown cells. Nuclei were stained by using DAPI. EdU positive cells, red; cell nuclei, blue; the data shown were representative of three independent experiments with similar results. Scale bar, 100 μm. (E) Statistics of EdU positive cells quantified by counting the cells with fluorescent signal using the software Image J. Experiments were performed in triplicate. *P < 0.05, **P < 0.01