miR-335 modulates Numb alternative splicing via targeting RBM10 in endometrial cancer.

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.