RNA Binding Protein RBM3 Modulates Novel LncRNAs to Increase Tumor Progression in Colon Cancer Cells
Authors: Sayed Afreen , Choudhury Sonali , Subramaniam Dharmalingam , Gunewardena Sumedha , Ponnurangam Sivapriya , Dandawate Prasad , Umar Shahid , Jensen Roy , Thomas Sufi , Anant Shrikant 
Published: April 2020

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, we demonstrated that RNA binding protein RBM3 acts as a protooncogene, with its expression. Increasing in a stage dependent manner 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. We validated these results 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 lncRNAs 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 protein expression. The consequence of this was reduced cell migration and invasion in vitro, in scratch plate and Boyden chamber assays. We also confirmed these studies in vivo. We generated transgenic mice overexpressing RBM3. LncRNA levels were higher in the colons of the transgenic animals. We also generated intestine specific RBM3 knockout mice. Here, lncRNA levels were lower. We further treated the wild type and RBM3−/− mice with azoxymethane (AOM)‐dextran sodium sulfate (DSS) to induce colitis‐associated colorectal tumorigenesis. There was significantly lower number of tumors in colons of RBM3−/− mice, coupled with lower levels of the four lncRNAs and mRNAs. In addition, RBM3 expression significantly increased tumor xenograft growth as compared to vector control cells. RBM3 overexpressing xenograft tumors also showed increased expression of lncRNAs along with EMT and angiogenic markers. Intratumoral knockdown of the lncRNAs decreased the tumor size and volume of tumor xenograft along with a decrease in Slug and VEGF protein expression. We used IntaRNA to analyze lncRNA‐mRNA interaction in silico. The lncRNAs formed a bridge between stem‐loop structures in the 5’ untranslated and 3’ untranslated regions of the four mRNAs (VEGF, ZEB1, TWIST1 and Slug) resulting in a closed circle formation. The interaction results in the formation of kissing loop interaction between lncRNA‐mRNA that contributes to the increased translation of the mRNA. Based on these results, we conclude that RNA binding protein RBM3 enhances tumor progression by modulating novel lncRNAs.