Novel circular RNA circNF1 acts as a molecular sponge, promoting gastric cancer by absorbing miR-16

in Endocrine-Related Cancer
Correspondence should be addressed to S J Meltzer: smeltzer@jhmi.edu
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Circular RNAs (circRNAs) are a new class of RNA involved in multiple human malignancies. However, limited information exists regarding the involvement of circRNAs in gastric carcinoma (GC). Therefore, we sought to identify novel circRNAs, their functions and mechanisms in gastric carcinogenesis. We analyzed next-generation RNA sequencing data from GC tissues and cell lines, identifying 75,201 candidate circRNAs. Among these, we focused on one novel circRNA, circNF1, which was upregulated in GC tissues and cell lines. Loss- and gain-of-function studies demonstrated that circNF1 significantly promotes cell proliferation. Furthermore, luciferase reporter assays showed that circNF1 binds to miR-16, thereby derepressing its downstream target mRNAs, MAP7 and AKT3. Targeted silencing or overexpression of circNF1 had no effect on levels of its linear RNA counterpart, NF1. Taken together, these results suggest that circNF1 acts as a novel oncogenic circRNA in GC by functioning as a miR-16 sponge.

Downloadable materials

  • Supplementary Table S1: Primers for Real-time PCR and RT-PCR.
  • Supplementary Table S2: Samples studied by next-generation RNA sequencing.
  • Supplementary Table S3: Top 20 circRNA candidates from screening results.
  • Supplementary Table S4: Descriptions of patients studied for differential expression of circNF1.
  • Supplementary Figure 1. Preliminary screening of 10 circRNA candidates. A and B, Reverse transcription of RNA from NCI-N87 cells amplifying 10 circRNA candidates using divergent or convergent primers. Red: verified correct; black: not verified.
  • Supplementary Figure 2. Correlation between circNF1 (x) and linear NF1 (y) levels in 6 GC cell lines. Consistent with results in GC tissues, expression levels of circNF1 and linear NF1 RNA were also poorly correlated in cell lines.

 

      Society for Endocrinology

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    Flowchart depicts work steps used to identify circRNA candidates of interest from RNAseq data.

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    Verification strategy of circRNA selection. (A) RT-PCR amplification products of circMYH9 and circNF1 using divergent (‘Div’) or convergent (‘Con’) primers. PCR products with divergent primers contain backsplice junction sites. Convergent primers’ amplicons reflecting full-length circMYH9 and circNF1 are approximately 500 and 800 bp, respectively. (B) Head-to-tail splicing of circMYH9 and circNF1 was confirmed by Sanger sequencing of divergent PT-PCR products. The red arrows refer to backsplice junction sites. (C) Expression levels of circMYH9 in gastric cancer cells vs the immortalized normal gastric epithelial cell line, HFE145. (D) Expression levels of circNF1 in gastric cancer cells vs HFE145. (E) RNAs extracted from NCI-N87 and MKN28 gastric cancer cell lines were treated without or with (−/+) RNase R prior to reverse transcription, then amplified with circNF1 or linear NF1 primers. Only circNF1 resists RNase R treatment. (F) RT-PCR with forward primer on exon 1 and reverse primer on exon 9 (to amplify exons 1–9 of linear NF1) and exon-skipped transcripts (exons 1/9, not detected) in NCI-N87 and MKN28 cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.00001. A full-colour version of this figure is available at https://doi.org/10.1530/ERC-18-0478.

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    Silencing of circNF1 inhibits GC cell proliferation and migration but promotes apoptosis. (A) Schematic representation of backsplice junction and sequence of siRNA targeted to junction site of circNF1. (B) Results of qRT-PCR for circNF1 in MKN28 and NCI-N87 cells treated with either si-NC or si-circNF1. (C) and (D) Results of WST-1 assay for proliferation after transfection with si-NC or si-circNF1. Inhibition of proliferation by knockdown of circNF1 in MKN28 and NCI-N87 cells at day 5 (P = 0.0079 and P = 0.0212, respectively). (E) Representative images and quantification results of colony formation of knock-down circNF1. (F) Representative images of scratch assays in MKN28 and NCI-N87 cells transfected with control or circNF1 siRNAs. (G) Line chart depicting scratch healing rates of MKN28 and NCI-N87 cells after silencing of circNF1. At hour 48t, wound closure in si-circNF1 group is significantly lower than in si-NC group in both MKN28 and NCI-N87 cells (P = 0.0196 and 0.0188, respectively). (H) Representative images and quantification results of flow cytometry to evaluate apoptosis induction by circNF1 in MKN28 and NCI-N87 cells transfected with si-NC or si-circNF1. *P < 0.05; **P < 0.01; ***P < 0.001. A full-colour version of this figure is available at https://doi.org/10.1530/ERC-18-0478.

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    Over-expression of circNF1 promotes cell proliferation. (A) Results of qRT-PCR for circNF1 in MKN28 and HFE145 cells without or with exogenous overexpression of circNF1. (B and C) WST-1 proliferation assay results in MKN28 and HFE145 cells transfected with either circNF1 (circNF1) or empty vector (EV) at day 0, 1, 3 and 5. Proliferation increase at day 5 in cells treated by exogenous circNF1 overexpression. (P = 0.0128 and P = 0.0181, respectively). (D) Representative images for colony formation after transfecting circNF1 or EV. *P < 0.05; ***P < 0.001. A full-colour version of this figure is available at https://doi.org/10.1530/ERC-18-0478.

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    circNF1 functions as a sponge of miR-16 in GC cells. (A) Schematic flowchart shows pipelines for screening miRNAs binding to circNF1. (B) Luciferase activity of luciferase vector containing each candidate miRNA binding site to detect endogenous miRNAs that were able to competitively bind to corresponding miRNA binding sequences in MKN28 cells. (C) Activity of luciferase plasmid (‘Luc’) containing or lacking a miR-16 binding site in MKN28 cells with vs without circNF1 overexpression. (D) Results of qRT-PCR for miR-16 in MKN28 gastric cancer cells with either exogenous overexpression of circNF1 or si-circNF1. (E) Representative images and quantitative analysis from Western blot of MAP7 protein with either over-expressed or under-expressed circNF1 in MKN28 cells. (F) Representative images and quantitative analysis from Western blot of P-AKT and AKT3 in MKN28 cells treated with overexpression or silencing of circNF1, respectively. β-Actin antibody served as a control in these experiments. (G) WST-1 proliferation assay in cells transfected with circNF1 or miR-16 as indicated. Proliferation decrease at day 5 in cells treated by combination of circNF1 vector and miR-16 mimic relative to circNF1 overexpression (P = 0.022). (H) Representative images for colony formation after either transfecting circNF1 or miR-16 independently or co-transfecting circNF1 and miR-16. *P < 0.05, **P < 0.01. A full-colour version of this figure is available at https://doi.org/10.1530/ERC-18-0478.

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    Expression of circNF1 and linear NF1 in GC tissues and cell lines. (A and B) Results of qRT-PCR of circNF1 and linear NF1 in 23 GC tissues. β-Actin served as an internal control. (C) Pearson correlation coefficient analysis of correlation between expression levels of circNF1 (x) and linear NF1 (y) (r = −0.1348, P = 0.5397). (D) Results of qRT-PCR for linear NF1 transcript levels in MKN28 and NCI-N87 cells transfected with either si-NC or si-circNF1. (E) Results of qRT-PCR for linear NF1 transcript levels in MKN28 and HFE145 cells treated with either empty vector (EV) or circular NF1 vector (circNF1). A full-colour version of this figure is available at https://doi.org/10.1530/ERC-18-0478.

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