CELF1 promotes matrix metalloproteinases gene expression at transcriptional level in lens epithelial cells

Abstract

Background: RNA binding proteins (RBPs)-mediated regulation plays important roles in many eye diseases, including the canonical RBP CELF1 in cataract. While the definite molecular regulatory mechanisms of CELF1 on cataract still remain elusive.

Methods: In this study, we overexpressed CELF1 in human cultured lens epithelial SRA01/04 cells and applied whole transcriptome sequencing (RNA-seq) method to analyze the global differences mediated by CELF1. We then analyzed public RNA-seq and CELF1-RNA interactome data to decipher the underlying mechanisms.

Results: The results showed that transcriptome profile was globally changed by CELF1 overexpression (CELF1-OE). Functional analysis revealed CELF1 specifically increased the expression of genes in extracellular matrix disassembly, extracellular matrix organization, and proteolysis, which could be classified into matrix metalloproteinases (MMPs) family. This finding was also validated by RT-qPCR and public mouse early embryonic lens data. Integrating analysis with public CELF1-RNA interactome data revealed that no obvious CELF1-binding peak was found on the transcripts of these genes, indicating an indirectly regulatory role of CELF1 in lens epithelial cells.

Conclusions: Our study demonstrated that CELF1-OE promotes transcriptional level of MMP genes; and this regulation may be completed by other ways except for binding to RNA targets. These results suggest that CELF1-OE is implicated in the development of lens, which is associated with cataract and expands our understanding of CELF1 regulatory roles as an RNA binding protein.

Keywords: CELF1; Cataract; MMPs; RNA-seq; Transcriptional regulation.

Fig. 1

Assessment of CELF1-overxpression in SRA01/04 cells. A Validation of CELF1-overexpression by Western Blot. B Validation of CELF1-overexpression by qPCR. C The FPKM values of CELF1 were calculated in RNA-seq. D Principal component analysis of all expressed genes between the control and CELF1 overexpression samples. Data are represented as the mean ± standard deviation. Student’s test was performed to compare CELF1-overexpression SRA01/04 cells and control with significance set at a p value of less than 0.05. *P < 0.05, ***P < 0.001

Fig. 2

RNA-seq analysis of CELF1 regulated transcriptome in SRA01/04 cells. A Identification of CELF1 regulated genes. Red dots indicated up-regulated genes, whereas blue dots indicated down-regulated genes. B Heatmap of all DEGs in control and CELF1 overexpression samples. The most representative GO biological processes of up-regulated genes (C) and down-regulated genes (D)

Fig. 3

Elevated CELF1 extensively regulates expression of genes involved in proteolysis. A Relative expression level of CELF1 determined by RNA-seq between E10.5 and E14.5 in mouse lens. B The top 15 representative GO biological processes of up-DEGs between E10.5 and E14.5 in mouse lens. C Hierarchical clustering heatmap of up-DEGs from proteolysis term in (B). D PCA result showed the expression pattern of proteolysis-related genes in CELF1-OE and control samples from this study. E Hierarchical clustering heatmap of proteolysis-related genes in CELF1-OE and control samples from this study. F Hierarchical clustering heatmap showed the expression pattern of overlapped DEGs in E10.5 and E14.5 in mouse lens (left panel) and CELF1-OE and control samples from this study (right panel)

Fig. 4

Validation of CELF1-regulated genes. Validation of DEGs in SRA01/04 cells measured by RNA-seq (A) and qPCR (B). C Relative expression levels of MMP9 in mouse lens measured by RNA-seq. For qPCR, GAPDH was used as the reference gene

Fig. 5

Function analysis of CELF1-bound genes. A CELF1-regulated DEGs overlapped with the CELF1-bound genes from SRR503. B The most representative GO biological processes of overlapped genes between DEGs and CELF1-bound genes from SRR503. C The top representative KEGG pathways of overlapped genes between DEGs and CELF1-bound genes from SRR503. D CELF1-regulated DEGs overlapped with the CELF1-bound genes from PRJEB12208. E The top representative KEGG pathways of overlapped genes between DEGs and CELF1-bound genes from PRJEB12208