title: Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells.
abstract: BACKGROUND
  An abundant class of intronic microRNAs (miRNAs) undergoes atypical Drosha-independent biogenesis in which the spliceosome governs the excision of hairpin miRNA precursors, called mirtrons. Although nearly 500 splicing-dependent miRNA candidates have been recently predicted via bioinformatic analysis of human RNA-Seq datasets, only a few of them have been experimentally validated. The detailed mechanism of miRNA processing by the splicing machinery and the roles of mirtronic miRNAs in cancer are yet to be uncovered.
  
  
  METHODS
  We experimentally examined whether biogenesis of certain miRNAs is under a splicing control by analyzing their expression levels in response to alterations in the 5'- and 3'-splice sites of a series of intron-containing minigenes carrying appropriate miRNAs. The expression levels of the miRNAs processed from mirtrons were determined by quantitative real-time PCR in five digestive tract (pancreas PANC-1, SU.86.86, T3M4, stomach KATOIII, colon HCT116) and two excretory system (kidney CaKi-1, 786-O) carcinoma cell lines as well as in pancreatic, stomach, and colorectal tumors. Transiently expressed SRSF1 and SRSF2 splicing factors were quantified by western blotting in the nuclear fractions of HCT116 cells.
  
  
  RESULTS
  We found that biogenesis of the human hsa-miR-1227-3p, hsa-miR-1229-3p, and hsa-miR-1236-3p is splicing-dependent; therefore, these miRNAs can be assigned to the class of miRNAs processed by a non-canonical mirtron pathway. The expression analysis revealed a differential regulation of human mirtronic miRNAs in various cancer cell lines and tumors. In particular, hsa-miR-1229-3p is selectively upregulated in the pancreatic and stomach cancer cell lines derived from metastatic sites. Compared with the healthy controls, the expression of hsa-miR-1226-3p was significantly higher in stomach tumors but extensively downregulated in colorectal tumors. Furthermore, we provided evidence that overexpression of SRSF1 or SRSF2 can upregulate the processing of individual mirtronic miRNAs in HCT116 cells.
  
  
  CONCLUSIONS
  An interplay of different splicing factors, such as SRSF1 or SRSF2, may alter the levels of miRNAs of mirtron origin in a cell. Our findings underline the specific expression profiles of mirtronic miRNAs in colorectal, stomach, and pancreatic cancer.
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Stasė
authors.initials: S
authors.lastname: Butkytė
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Laurynas
authors.initials: L
authors.lastname: Čiupas
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Eglė
authors.initials: E
authors.lastname: Jakubauskienė
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Laurynas
authors.initials: L
authors.lastname: Vilys
authors.affiliation: Institute for Digestive Research, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Paulius
authors.initials: P
authors.lastname: Mocevicius
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Arvydas
authors.initials: A
authors.lastname: Kanopka
authors.affiliation: Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania.
authors.forename: Giedrius
authors.initials: G
authors.lastname: Vilkaitis
mesh_terms: D045744:Cell Line, Tumor; D004067:Digestive System Neoplasms / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D012326:RNA Splicing; D000072260:RNA Splicing Factors / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors / Q000378:metabolism
chemical_list: C571208:MIRN1236 microRNA, human; D035683:MicroRNAs; D000072260:RNA Splicing Factors; C000595779:SRSF1 protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors
keywords: Human cancer; Mirtron; Post-transcriptional RNA processing; Splicing factor; miRNA; microRNA