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. 2019 Jun;16(6):770-784.
doi: 10.1080/15476286.2019.1585738. Epub 2019 Mar 23.

The Origin of Exosomal miR-1246 in Human Cancer Cells

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Free PMC article

The Origin of Exosomal miR-1246 in Human Cancer Cells

Yi-Fan Xu et al. RNA Biol. .
Free PMC article

Abstract

miR-1246 is considered an oncomiR in various cancer types. However, the origin and biogenesis of miR-1246 remain controversial which often leads to misinterpretation of its detection and biological function, and inevitably masking its mechanisms of action. Using next generation small RNA sequencing, CRISPR-Cas9 knockout, siRNA knockdown and the poly-A tailing SYBR qRT-PCR, we examined the biogenesis of exosomal miR-1246 in human cancer cell model systems. We found that miR-1246 is highly enriched in exosomes derived from human cancer cells and that it originates from RNU2-1, a small nuclear RNA and essential component of the U2 complex of the spliceosome. Knockdown of Drosha and Dicer did not reduce exosomal miR-1246 levels, indicating that exosomal miR-1246 is generated in a Drosha- and Dicer-independent manner. Direct digestion of cellular lysate by RNase A and knockdown of the RNU2-1 binding protein SmB/B' demonstrated that exosomal miR-1246 is a RNU2-1 degradation product. Furthermore, the GCAG motif present in the RUN2-1 transcript was shown to mediate miR-1246 enrichment in cancer exosomes. We conclude that exosome miR-1246 is derived from RNU2-1 degradation through a non-canonical microRNA biogenesis process. These findings reveal the origin of an oncomiR in human cancer cells, providing guidance in understanding miR-1246 detection and biological function. Abbreviations: CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; miRNA, microRNA; PDAC, pancreatic ductal adenocarcinoma; RNU2-1, U2 small nuclear RNA; RT-PCR, Reverse transcription polymerase chain reaction; sgRNA, single-guide RNA.

Keywords: Exosome; RNU2-1; biogenesis; cancer; microRNA.

Figures

Figure 1.
Figure 1.
The sequence overlap of the precursor miR-1246, RNU2-1 and exosomal miR-1246, and comparison of two qRT-PCR detection strategies. (a). Exosomal miR-1246, precursor miR-1246, and RNU2-1 sequences. Mature miR-1246 as registered in miRBase (miRBase.org) is red colored. Schematic illustration of miR-1246 and RNU2-1 detection using the stem-loop qRT-PCR method (b and c). This method cannot qualitatively differentiate the detection of miR-1246 and RNU2-1. Schematic illustration of miR-1246 and RNU2-1 detection using the poly A tailing qRT-PCR method (d and e). This method can differentiate the detection of miR-1246 and RNU2-1.
Figure 2.
Figure 2.
miR-1246-like sequences are highly enriched in PANC-1 exosomes. (a and b), qRT-PCR analysis using the poly A tailing method on miR-1246 expression in hTERT-HPNE, PANC-1 and MIA-PaCa-2 cells and their exosomes (means ± SEM, n = 3). The melting curves showed detection of different fragments in cells versus exosomes with the same set of primers. (c), Representative sequencing results from three individual clones of the detected exosomal miR-1246 and cellular RUN2-1. (d), miR-1246 target gene reporter assay. Mimics of mature miR-1246 and the exosomal miR-1246 variant suppress reporter gene activity as compared to scramble mimics (means ± SEM, n = 3). * p < 0.05, one-way ANOVA, followed by Dunnett analysis. (e), Northern blot analysis using a labeled miR-1246 probe against RNAs from several human cancer cell lines. There was no precursor miR-1246 and mature miR-1246 being detected and the primary band migrating at a size of RNU2-1 in all cell lines examined.
Figure 3.
Figure 3.
Effects of the MIR1246 gene knockout on exosomal miR-1246 expression. (a), The strategy of CRISPR-Cas9 HDR knockout of the MIR1246 gene. Ensembl gene ID and the precise sequence location are indicated. (b), GFP detection of the homozygous MIR1246 knockout PANC-1 cells by fluorescence microscopy. (c), Agarose gel electrophoresis of the PCR products of DNA from wild type PANC-1 and the MIR1246 knockout PANC-1 cells, using primers covering the DNA regions upstream and downstream of the MIR1246 gene. (d), qRT-PCR analysis using the poly A tailing method detecting miR-1246 in exosomes derived from wild type and MIR1246 knockout PANC-1 cells (means ± SEM, n = 3).
Figure 4.
Figure 4.
Effects of RNU2-1 knockdown on exosomal miR-1246 expression. (a), qRT-PCR analysis of RNU2-1 expression in scramble siRNA or RNU2-1 siRNA transfected PANC-1 cells. (b), qRT-PCR analysis of miR-1246 expression in exosomes derived from scramble siRNA or RNU2-1 siRNA transfected PANC-1 cells (means ± SEM, n = 3). ** p < 0.01, Student t-test, performed using ∆CT values.
Figure 5.
Figure 5.
Effects of forced expression of the precursor miR-1246 or RNU2-1 on exosomal miR-1246 expression. (a), Agarose gel electrophoresis of the RT-PCR products detecting the precursor miR-1246 in PANC-1 exosomes and cells transfected with the precursor miR-1246 expression plasmid. (b), qRT-PCR detection of RNU2-1 in wild type and the precursor miR-1246 expression plasmid transfected PANC-1 cells. (c), qRT-PCR detection of miR-1246 in exosomes derived from wild type and the precursor miR-1246 expression plasmid transfected PANC-1 cells. (d), qRT-PCR detection of RNU2-1 in wild type and the RNU2-1 expression plasmid transfected PANC-1 cells. e, qRT-PCR detection of miR-1246 in exosomes derived from wild type and RNU2-1 expression plasmid transfected PANC-1 cells (n = 3, means ± SEM, for b. c, d, and e). * p < 0.05, Student t-test, performed using ∆CT values.
Figure 6.
Figure 6.
Effects of knockdown of Drosha and Dicer in PANC-1 cells on exosomal miR-1246 expression. (a), Western blot analysis confirming Dicer (left) and Drosha (right) knockdown in PANC-1 cells. (b), qRT-PCR analysis of RNU2-1 expression in Dicer and Drosha knockdown PANC-1 cells. (c), qRT-PCR analysis of miR-1246 expression in exosomes derived from Dicer and Drosha knockdown PANC-1 cells (means ± SEM, n = 3).
Figure 7.
Figure 7.
Exosomal miR-1246 is generated through RNU2-1 transcript degradation. (a and b), PANC-1 cell lysate was digested with RNase A at various concentrations for 3 min and the RNA isolated from the lysate was analyzed using Agilent 2100 Bioanalyzer. (c), The melting curves of qRT-PCR analysis showing the detection of RNU2-1 in control and 12.5 μg/ml RNase A-treated lysate, detection of miR-1246 in 250 μg/ml RNase A-treated lysate, and detection of both RNU2-1 and miR-1246 in 50 μg/ml RNase A-treated lysate. (d), Western blot analysis confirming SmB/B’ knockdown in PANC-1 cells. (e), qRT-PCR analysis of miR-1246 expression in exosomes derived from control and SmB/B’ knockdown PANC-1 cells (means ± SEM, n = 3). * p < 0.05, one-way ANOVA, followed by Dunnett analysis.
Figure 8.
Figure 8.
The GCAG motif in the RNU2-1 transcript is responsible for miR-1246 enrichment in PANC-1 exosomes. (a), Sequences of RNU2-1 and RNU2-2 with homology (green), miR-1246 fragment (red), and differences (black) highlighted. (b), small RNA sequencing reads match to RNU2-1 and RNU2-2 in PANC-1 cells and exosomes. (c), RNA motif mutants constructed using RNU2-1 expression plasmid. (d), qRT-PCR analysis of miR-1246 expression in exosomes derived from wild type and RUN2-1 mutants transfected PANC-1 cells (means ± SEM, n = 3). ** p < 0.05, one-way ANOVA, followed by Dunnett analysis.

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