Abstract
MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR-106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome-wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed-binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of five proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of several members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology.
- List of abbreviations
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- DAPI
- 4’,6-diamidine-2’-phenylindole dihydrochloride
- CLASH
- Crosslinking, ligation, and sequencing of hybrids
- DR5
- Death receptor-5
- DMEM
- Dulbecco’s Modified Eagle’s Medium
- FDR
- False discovery rate
- FBS
- Fetal bovine serum
- KLF
- Kruppel-like factor
- LNA
- Locked-nucleic acid
- qRT-PCR
- Quantitative reverse-transcription PCR
- SDS-PAGE
- Sodium dodecylsulfate-polyacrylamide gel electrophoresis
- UTR
- Untranslated region
