RNA-binding proteins potentially regulate the alternative splicing of apoptotic genes during knee osteoarthritis progression

Abstract

Background: Alternative splicing (AS) is a principal mode of genetic regulation and one of the most widely used mechanisms to generate structurally and functionally distinct mRNA and protein variants. Dysregulation of AS may result in aberrant transcription and protein products, leading to the emergence of human diseases. Although considered important for regulating gene expression, genome-wide AS dysregulation, underlying mechanisms, and clinical relevance in knee osteoarthritis (OA) remain unelucidated. Therefore, in this study, we elucidated and validated AS events and their regulatory mechanisms during OA progression.

Results: In this study, we identified differentially expressed genes between human OA and healthy meniscus samples. Among them, the OA-associated genes were primarily enriched in biological pathways such as extracellular matrix organization and ossification. The predominant OA-associated regulated AS (RAS) events were found to be involved in apoptosis during OA development. The expression of the apoptosis-related gene BCL2L13, XAF1, and NF2 were significantly different between OA and healthy meniscus samples. The construction of a covariation network of RNA-binding proteins (RBPs) and RAS genes revealed that differentially expressed RBP genes LAMA2 and CUL4B may regulate the apoptotic genes XAF1 and BCL2L13 to undergo AS events during OA progression. Finally, RT-qPCR revealed that CUL4B expression was significantly higher in OA meniscus samples than in normal controls and that the AS ratio of XAF1 was significantly different between control and OA samples; these findings were consistent with their expected expression and regulatory relationships.

Conclusions: Differentially expressed RBPs may regulate the AS of apoptotic genes during knee OA progression. XAF1 and its regulator, CUL4B, may serve as novel biomarkers and potential therapeutic targets for this disease.

Keywords: Alternative splicing; Apoptosis; Covariation; Differentially expressed genes; Knee osteoarthritis; RNA binding protein; Transcriptome.

Fig. 1

RNA sequencing of human meniscus cells in osteoarthritis (OA) and healthy samples. (A) Volcano plots presenting all differential expressed genes (DEGs) between OA and healthy samples. false discovery rate (FDR) ≤ 0.01 and fold change ≥ 2 or ≤ 0.5. (B) Principal component analysis (PCA) based on fragments per kilobase of exon per million mapped fragments (FPKM) value of all DEGs. The ellipse for each group is the confidence ellipse. (C) Hierarchical clustering heatmap showing the expression of all DEGs. (D) Bar plot showing the most enriched GO pathways of the upregulated genes. (E) Bar plot showing the most enriched GO pathways of the downregulated genes. (F) Boxplot showing FPKM of 5 DEGs. ^*: p < 0.05, ^**: p < 0.01, ^***: p < 0.001

Fig. 2

Identification of predominant regulatory alternative splicing (RAS) events between OA and healthy samples. (A) Bar plot showing the number of different RAS types identified by SUVA in OA samples. (B) Bar plot showing RAS with different pSAR (proportion of each SUVA AS event reads). RAS with pSAR ≥ 50% were labeled. (C) Principal component analysis (PCA) based on RAS with pSAR ≥ 50%. The ellipse for each group is the confidence ellipse. (D) The heatmap showing the splicing ratio of RAS of all samples (pSAR ≥ 50%). (E) Bar plot exhibiting the most enriched GO pathways of the RAS with pSAR ≥ 50%.

Fig. 3

Alternative splicing (AS) of apoptotic genes in osteoarthritis. (A) The heatmap showing splicing ratio of RAS events of all samples in apoptotic pathway. (B-C) The read distribution and splicing ratio of apoptotic genes XAF1, BCL2L13. The red boxes in the left panel indicate the splicing area of the difference; the boxplots in the right panel show the splicing ratio. ^*p < 0.05, ^**p < 0.01

Fig. 4

Covariation analysis of differentially expressed RNA binding protein (DERBP) and regulatory alternative splicing (RAS) in apoptotic pathway of OA. (A) Venn diagram showing the overlap of RBP and DEG. (B) Heatmap showing the expression profile of DERBP in apoptotic pathway of osteoarthritis. (C) Covariation network of DERBPs and apoptosis-associated RAS (pSAR ≥ 50%). Cutoffs of p ≤ 0.01 and Pearson coefficient ≥ 0.6 ≤ -0.6 were applied to identify the covariation pairs. (D) The read distribution of the apoptotic RAS gene MTFP1 is showing in the left panel. The red box indicates the splicing area of the difference. The boxplots in the right panel show the FPKM of the coexpressed RBP gene AFF2 and the splicing ratio of MTFP1. ^*: p < 0.05, ^**: p < 0.01, ^***: p < 0.001. (E) The reads distribution of the apoptotic RAS gene CARD8 is showing in the left panel. The red boxes indicate the splicing area of the difference. The boxplots in the right panel show the FPKM of the coexpressed RBP genes LAMA2 and CUL4B and the splicing ratio of CARD8. ^*: p < 0.05, ^**: p < 0.01, ^***: p < 0.001

Fig. 5

Expression of RBP genes LAMA2 and CUL4B in normal control (black bars) and OA meniscus samples (gray bars); NC: normal control; OA: osteoarthritis; ^*: p < 0.05, ^**: p < 0.01, ^***: p < 0.001, ^****: p < 0.0001

Fig. 6

AS ratio of RAS genes XAF1 and BCL2L13 in normal control (black bars) and OA meniscus samples (gray bars); NC: normal control; OA: osteoarthritis; ^*: p < 0.05, ^**: p < 0.01, ^***: p < 0.001, ^****: p < 0.0001