Alternative splicing of VEGFA is regulated by RBM10 in endometrial cancer

Abstract

Vascular endothelial growth factor A (VEGFA) gene has three alternative exons which results in multiple isoforms. VEGFA has been found overexpressed in patients with endometrial cancer, but the VEGFA expression pattern and how it is regulated are still unknown. The level of VEGFA transcripts and protein isoforms were detected by semi-quantitative Polymerase chain reaction (PCR) and immunoblotting in 29 paired endometrial tumor and adjacent nontumor control tissues. The level of three alternative splicing related proteins: RBM5, RBM6, and RBM10 was determined by immunoblotting. The H3K27Ac level in RBM10 promoter region was detected by ChIP-PCR. The RBM10 promoter region methylation level were quantified by methylation-sensitive high resolution melting. VEGFA165a was overexpressed and VEGFA165b level was reduced in tumors. RBM10 level was reduced in tumors. RBM10 level was negatively correlated with VEGFA165a level and positively correlated with VEGFA165b level in tumors. Using HEC-1-A and RL95-2 cells, we confirmed that VEGFA165a/b expressed pattern was controlled by RBM10. MALAT1 level was increased in tumors but not involved in VEGFA alternative splicing. Reduced H3K27Ac level and increased DNA methylation in the promoter region controlled RBM10 expression in tumors. VEGFA alternative splicing in endometrial cancer was regulated by RBM10, the expression of which was controlled by histone acetylation and DNA methylation.

Keywords: MALAT1; RBM10; VEGFA; alternative splicing; endometrial cancer.

Figure 1

VEGFA and RBMs expression pattern in endometrial tumor samples. The VEGFA expression pattern was detected by PCR followed by electrophoresis. The protein level of VEGFA165a/b, RBM5, RBM6, and RBM10 was determined by immunoblotting with the signal of β‐actin as loading control. The band density was quantified by Quantity One software and results were analyzed by paired t test with P < .05 was considered as significant. *P < .05, **P < .01. VEGFA, vascular endothelial growth factor A

Figure 2

The correlations between RBM10 and VEGFA165a/b. Pearson correlation analysis was performed to investigate the linear relationship between protein levels. The findings were considered significant at a P‐value <.05. VEGFA, vascular endothelial growth factor A

Figure 3

VEGFA165a/b expression pattern is regulated by RBM10. RBM10 was knocked down by siRNAs or overexpressed in HEC‐1‐A and RL95‐2 cells. The level of VEGFA165a, VEGFA165b, and RBM10 was determined by immunoblotting. The relative level of VEGFA165a, VEGFA165b, and RBM10 were quantifed by Quantity One (BioRad)

Figure 4

MALAT1 is not involved in VEGFA alternative splicing. MALAT1 level was determined by qRT‐PCR. Pearson correlation analysis was performed to investigate the linear relationship between MALAT1 and protein level. Pearson correlation analysis was performed to investigate the linear relationship between protein levels. VEGFA, vascular endothelial growth factor A

Figure 5

RBM10 was regulated by H3K27Ac and DNA methylation. The level of RBM10 mRNA was quantified by qRT‐PCR. The H3K27Ac level was determined by ChIP‐PCR and the DNA methylation percentage was determined by MS‐HRM. Results were analyzed by paired t test with P < .05 as statistically significant. *P = .05, *P = .1. MS‐HRM, methylation‐sensitive high resolution melting