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.
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.