Establishment of a non.coding RNAomics screening platform for the regulation of KRAS in pancreatic cancer by RNA sequencing

Abstract

KRAS oncogene point mutations occur in >95% of patients with pancreatic cancer. The KRAS protein can activate various downstream effector molecules that affect proliferation and differentiation. MS2 binding sites (MS2bs) are RNAs of 19 bp in length that can bind MS2 coat proteins with their specific stem-loop structure. The MS2 binding site sequence of the 19-nucleotide stem-loop is ACATGAGGATCACCCATGT. We constructed an expression vector that expresses the KRAS non.coding region coupled with 12 copies of MS2bs in series and established a high-throughput library for collecting microRNA (miRNA or miR)- and long non.coding RNA (lncRNA)-omics that regulate KRAS. To the best of our knowledge, this is the first study to combine RNA-protein interactions with RNA sequencing to obtain KRAS-associated non.coding RNAs. As a result, we identified several miRNA precursors that belong to the let-7 and miR-34, -30 and -143 families, as well as relevant lncRNAs and their families (MALAT1, MEG3-2 and TUG1-1-4). Our databank of non.coding RNAs (mainly miRNAs and lncRNAs) that regulate KRAS is expected to greatly enhance our understanding of KRAS regulation-associated tumorigenesis and may aid in the development of gene therapies for pancreatic cancer.