RNA binding protein RBM3 modulates novel lncRNAs to increase colon cancer tumor progression

RNA binding proteins play a significant role in regulating gene expression, and dysregulation in their expression may skew cells to various pathophysiological conditions. In previous studies, the Anant laboratory has shown that RNA binding protein RBM3 may function as a protooncogene, with its expression increasing in colon cancers. To get a better understanding of how RBM3 works, we took an unbiased approach and performed total RNA-sequencing (RNAseq) and RNA-immunoprecipitation-coupled sequencing (RIPseq), to identify transcripts targeted by the protein. We identified several genes involved in cell movement and angiogenesis, including VEGF, ZEB1, TWIST1 and Slug mRNAs, which we validated by Real time RT-PCR analyses. Coupled with this, we observed that RBM3 overexpression increased stemness, migration and invasion in vitro. A second discovery from the RNA seq and RNA-IP studies was that RBM3 overexpression increased the levels of multiple long noncoding RNAs (lncRNAs), a class of regulatory RNAs that play important roles in tumorigenesis. Of these, we chose four lncRNAs, two known (HOTAIR, TUG1) and two novel ones (lnc-Flii-1 and lnc-LSAMP-3) based on their ability to interact with VEGF, ZEB1, TWIST1 and Slug mRNAs using the RNAfold program. Knocking down the lncRNAs using a combination of specific siRNAs and locked nucleic acid (LNA) oligonucleotides showed reduced levels of the four mRNAs, and their corresponding protein expression. The consequence of this was reduced cell migration and invasion in scratch plate and Boyden chamber assays. To determine the role of RBM3 in vivo, we performed three sets of experiments in various mouse models, including RBM3 overexpression, RBM3 knockout and a tumor xenograft model. For RBM3 overexpression, we developed a mouse where RBM3 is encoded in the ROSA locus and driven by the cytomegalovirus immediate early gene promoter (RBM3tg). LncRNA (HOTAIR, TUG1, lnc-Flii-1 and lnc-LSAMP-3) levels were higher in the colons of the RBM3tg animals, when compared to matched controls. We also generated a mouse line where RBM3 is deleted specifically in the intestine (intRBM3ko mouse). Here, the four lncRNA levels were lower than wild type controls. To determine the effect of RBM3 loss on tumorigenesis, we treated the wild type and intRBM3ko mice with azoxymethane (AOM)-dextran sodium sulfate (DSS) to induce colitis-associated colorectal cancer. There was significantly lower number of tumors in colons of intRBM3ko mice, when compared to wild type controls. Moreover, there were lower levels of the four lncRNAs and mRNAs for VEGF, ZEB1, TWIST1 and Slug in the tumors from the intRBM3ko mice. Finally, we performed studies in a tumor xenograft model, where HCT116 and DLD1 cells overexpressing RBM3 (HR and DR, respectively) and compared them to vector controls (HG and DG, respectively). Overexpression of RBM3 in the two cell lines resulted in a significant increase tumor xenograft growth as compared to vector control cells. RBM3 overexpressing HR and DR xenograft tumors also showed higher levels of expression of lncRNAs as well as VEGF, ZEB1, TWIST1 and Slug mRNAs and proteins. Furthermore, intra-tumoral knockdown of the lncRNAs decreased the size and volume of HG and DG tumor xenografts along with a decrease in Slug and VEGF protein expression. We used the IntaRNA program to analyze lncRNA-mRNA interaction in silico. The lncRNAs formed a bridge between stem-loop structures in the 5’ untranslated (5’UTR) and 3’ untranslated (3’UTR) regions of the four mRNAs (VEGF, ZEB1, TWIST1 and Slug) resulting in formation of a closed circle. We confirmed the interaction of RBM3 with the lncRNAs using RIP-RT-PCR. The association results in the formation of the kissing loop interaction between lncRNA and mRNA increasing translation. Based on these results, we conclude that RNA binding protein RBM3 enhances tumor progression by modulating novel lncRNAs