RBM24 regulates apoptosis rates by modulating global transcriptome profile in CAL27 cells

Abstract

RNA binding proteins (RBPs) are key factors regulating post-transcriptional events. Categorized as RBPs, RBM24 expression levels have been shown to be a prognosis-related pivotal gene in oral squamous cell carcinoma. We analyzed the binding targets and regulated post-transcriptional events of RBM24 in RBM24-overexpressing cell lines and controls using iRIP-seq and RNA-seq. RBM24-overexpressing cells showed significant changes in gene expression, which are involved in biological pathways related to apoptosis and immune inflammation. RBM24-regulated genes that undergo alternative splicing are primarily engaged in biological processes related to DNA damage repair and RNA metabolism. More notably, we found that RBM24 binds to lncRNAs in addition to pre-mRNAs. These results indicated that RBM24 could play a key role in cancer progression by finding clinical therapeutic targets for Oral squamous cell carcinoma.

Keywords: Alternative splicing; Apoptosis; Cancer therapy; Oral squamous cell carcinoma; RBM24.

Fig. 1

RBM24 inhibits apoptosis in CAL27 cell. (A, B) RT-PCR (A) and Western blotting. (B) analysis of LTA4H expression. (C, D) Flow-type apoptosis results of CAL27 cells after RBM24 overexpression. (E) Cell proliferation was determined by CCK-8 assay after RBM24 overexpression. Error bars represent mean ± SEM. **** P-value < 0.0001, *** P-value < 0.001.

Fig. 2

Analysis of RBM24-regulated differential genes by RNA-seq. (A) The expression pattern of DEGs for RBM24. (B) PCA base on FPKM value of all detected genes RBM24 overexpression. (C) All DEGs between RBM-OE and NC samples. (D) Hierarchical clustering of DEGs in RBM24-OE and NC samples. (E)The top 10 representative GO Biological Process results of the up-regulated and down-regulated DEGs. (F) RNA-seq and RT-qPCR showed that RBM24 regulates several differentially expressed genes. Error bars represent mean ± SEM.*** P-value < 0.001.

Fig. 3

RBM24 regulates multiple RASE. (A) Alternative splicing event regulated by RBM24. (B) PCA based on PSI value of all different expression levels of RBM24 regulated RASE. (C) Hierarchical clustering of RASE in RBM-OE and NC samples. (D) The top 10 representative pathways of the alternative splicing genes regulated by RBM24 in GO database. (E) Validation of gene expression of RASE using RNA-seq and RT-qPCR. Error bars represent mean ± SEM.*** P-value < 0.001,** P-value < 0.01.

Fig. 4

Analysis of the targets bound by RBM24 in iRIP-seq. (A) Western blot experiment to validate IP efficiency. (B) Heat map showing two immunoprecipitated samples clustered (C) Reads distribution across reference genome. (D) Motif enrichment of RBM24-bound peaks by HOMER (E) RBM24 binding peak genes of NPM1. (F) RBM24 binding peak genes of NEAT1. Error bars represent mean ± SEM.*** P-value < 0.001.

Fig. 5

Comprehensive analysis of downstream targets and alternative splicing genes regulated by RBM24. (A) The overlapped genes between RASGs and peak genes from the two biological replicates. (B) RBM24 regulates alternative splicing of ENOSF1. Error bars represent mean ± SEM. * * * P-value < 0.001, * P-value < 0.05. (C) RBM24 binding peak genes of ENOSF1. Error bars represent mean ± SEM. * * * P-value < 0.001.