Abstract
BackgroundAlthough both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. CCAT2 (Colon cancer-associated transcript-2), a long noncoding RNA (lncRNA), has been reported to be over-expressed in CRC and is found to promote tumor growth. miRNAs, a class of naturally occurring short RNAs negatively control the expression of target genes by cleaving mRNA or through translation repression. Recently, we reported that miR-145 and miR-21 cooperate to regulate colon cancer stem cell (CSC) proliferation and differentiation. Considering that CCAT2 is mainly located in the nucleus and miRNA maturation process begins in the nucleus, we hypothesize that CCAT2 selectively blocks miR-145 maturation process, resulting in decreased mature miR-145 affecting colon CSC proliferation and differentiation.MethodsThe levels of CCAT2 were manipulated by transfection of CCAT2 expression plasmid or knockdown by siRNA or by CRISPR/Cas9. Quantitative RT-PCR was performed to examine the expression of CCAT2 and pri-, pre- and mature miR-145/21. Fluorescence in situ hybridization (FISH) was used to visualize CCAT2 in the cells. In vitro processing of pri-miRNA-145 was performed using T7 RNA polymerase and recombinant human Dicer.ResultsWe have observed that modulated expression of CCAT2 regulates the expression of miR-145 in colon cancer HCT-116 and HT-29 cells. Knockout of CCAT2 increases miR-145 and negatively regulates miR-21 in HCT-116 cells, impairs proliferation and differentiation. In contrast, stable up-regulation of CCAT2 decreases mature miR-145 and increases the expression of several CSC markers in colon cancer cells. We have also observed that CCAT2 is enriched in the nucleus and correlates with the expression of pre-miR-145 but not pre-miR-21 in HCT-116 cells. These results indicate CCAT2 selectively blocks miR-145 maturation by inhibiting pre-miR-145 export to cytoplasm. Further, we revealed that CCAT2 blocks cleavage of pre-miR-145 by Dicer in vitro.ConclusionsOur results identify CCAT2 as a negative regulator of miRNA-145 biogenesis, and expose a novel mechanism of lncRNA-miRNA crosstalk.
Highlights
Both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood
Mature miRNA is generated through two-step cleavage process, first nuclear and subsequent cytoplasmic cleavage events [6]. miRNAs are initially transcribed from the genome by RNA polymerase II (RNAP II) into primary transcripts and processed in the nucleus to hairpin structures of about ~70 nucleotide precursors miRNA by the ribonuclease (RNAase) III family enzyme Drosha
These pre-miRNAs subsequently are exported to the cytoplasm by exportin-5 [7] or exportin-1 [8] where the loop sequence is removed from the hairpin by Dicer to produce an RNA duplex analogous [9]
Summary
Both long and micro RNAs are emerging as important functional components in colorectal cancer (CRC) progression and metastasis, the mechanism of their interaction remains poorly understood. MiRNAs are initially transcribed from the genome by RNA polymerase II (RNAP II) into primary transcripts (pri-miRNA) and processed in the nucleus to hairpin structures of about ~70 nucleotide precursors miRNA (pre-miRNAs) by the ribonuclease (RNAase) III family enzyme Drosha. These pre-miRNAs subsequently are exported to the cytoplasm by exportin-5 [7] or exportin-1 [8] where the loop sequence is removed from the hairpin by Dicer to produce an RNA duplex analogous [9]. Tumor-specific miRNA expression profiles are functionally relevant because many miRNAs act as tumor suppressors or as oncogenes
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