ABSTRACT
Numb is a conserved protein plays important roles in the development of cancer. Two Numb isoforms have been found produced by alternative splicing and play contrast roles in regulating cellular functions. It is reported that the expression of Numb long isoform (Numb-L) was increased in various kinds of cancers, but in endometrial cancer, the condition is still unknown. The level of two Numb transcripts and protein isoforms were detected by semiquantitative polymerase chain reaction and immunoblotting in 47 paired endometrial tumor and adjacent non-tumor control tissues. The level of three alternative splicing related proteins: RBM5, RBM6, and RBM10 was determined by immunoblotting. MiRNAs targeting RBM10 were predicted by bioinformatics tools and their interaction with RBM10 was confirmed by luciferase assay and immunoblotting. The function of miR-335 in endometrial cancer was examined in xenograft mouse model. Numb-L level was increased in tumors and negatively correlated with RBM10 protein level. RBM10 mRNA level was not significantly altered in endometrial tumors suggesting its expression may regulated by post transcriptional regulators such as miRNAs. We identified miR-133a, miR-133b, and miR-335 directly target RBM10, but only miR-335 level increased in tumors and negatively correlated with RBM10 protein level. miR-335 overexpression promoted tumor growth by downregulating RBM10 and upregulating Numb-L level in xenograft mouse model. miR-335 overexpression promoted Numb-L expression via targeting RBM10 in endometrial cancer, which may provide new biomarkers for EC diagnosis.
Subject(s)
Alternative Splicing/physiology , Endometrial Neoplasms/metabolism , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , Adult , Alternative Splicing/genetics , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation/genetics , Cell Proliferation/physiology , Endometrial Neoplasms/genetics , Female , Humans , Mice , MicroRNAs/genetics , Middle Aged , RNA-Binding Proteins/geneticsABSTRACT
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.
