CircRNAs, a new class of noncoding RNAs (ncRNAs), has gradually gained attention. It has been reported that exonic-originated circRNAs located mainly in the cytoplasm usually function as miRNA ‘sponges’6. CircRNAs also participate in transcriptional or post-transcriptional regulation28,29. Moreover, several circRNAs containing internal ribosome entry sites could be translated into peptides30. However, the acknowledged circRNAs and their regulatory mechanism in GC have not been thoroughly elaborated. CircRNA_100290, one of circRNAs discovered recently, is located on chromosome 1, and its parental gene is SLC30A7. A recent study reported that circRNA_100290 was abnormally highly expressed in colorectal cancer and promoted the proliferation of colorectal cells15. In the present study, circRNA_100290 was significantly upregulated in GC, and was closely related to invasion depth, lymph node metastasis, and TNM stage. Functionally, silencing circRNA_100290 in AGS and HGC-27 cells significantly inhibited cell viability, colony formation, migration, and invasion ability, and induced the G0/G1 phase arrest in vitro. These results suggested that circRNA_100290 might serve as an oncogene in GC.
CircRNAs might function as miRNA sponges via binding miRNAs and regulate downstream target genes, also known as competing endogenous RNA regulatory mechanism. In our study, the expression of miR-29b-3p was found to be decreased in GC and this trend was validated by analyzing data from the EBI database. MiR-29b-3p is a member of miR-29 family, decreased expression of miR-29 family members has been reported in various tumors, including lung cancer, esophageal cancer, hepatocellular carcinoma, and so on31–33. The bioinformatic prediction and correlation analysis indicated miR-29b-3p might share the complementary binding sites with circRNA_100290, which was confirmed by the dual-luciferase reporter assay, suggesting that circRNA_100290 could function as a sponge for miR-29b-3p. Taken together, our results suggested that miR-29b-3p acted as a tumor suppressor and interacted with circRNA_100290 by sponging in GC.
ITGA11, a candidate target of miR-29b-3p, was further studied. ITGA11 is a member of the integrin family. It is unregulated in various tumors, such as lung cancer, breast cancer, meningeal glioma and so on34–36. However, the role of ITGA11 in GC has not been reported so far. The present study reported an elevated expression of ITGA11 in GC, which was reversed to the expression of miR-29b-3p. Pathological factors analysis and survival analysis indicated that high expression of ITGA11 predicted a worse prognosis of GC patients. ITGA11 might serve as an oncogene in GC. Furthermore, silencing circRNA_100290 in AGS and HGC-27 cells led to the increased miR-29b-3p and diminished ITGA11, suggesting circRNA_100290 could sponge miR-29b-3p to increase the expression of ITGA11, thereby promoting GC cell proliferation, migration and invasion.
Growing evidence demonstrates that circRNAs usually regulate tumor progression and metastasis by affecting EMT37,38. In our study, knocking down of circRNA_100290 induced altered expression of several EMT markers, accompanied by the release of miR-29b-3p and blocking of ITGA11. Previous studies have reported the involvement of miR-29b family members in EMT39. In addition, there is report reveals that miR-29b could inhibit EMT and metastasis by targeting a network of pro-metastatic motivators involved in angiogenesis, collagen remodeling, and proteolysis40. Shin et al. reported that exogenous miR-29b mediated an anticancer effect by impeding the activation of ITGA1136. In our study, by using PPI and GO analyses, ITGA11 was found to have a close connection with EMT-related proteins and could be involved in EMT. These results reflected that circRNA_100290 promoted EMT mainly via the miR-29b-3p/ITGA11 axis.
CircRNAs are formatted via back-splicing regulated by RNA splicing factors or RBPs41,42. In this study, EIF4A3 was found to have the most predicted binding sites among other RBPs with both flanking regions and circRNA_100290 itself, and RIP assay confirmed the direct interaction between flanking regions of circRNA_100290 and EIF4A3. As a member of the DEAD-box protein family, EIF4A3 is located mainly in the nucleus, and is a part of the exon junction complex necessary for nonsense-mediated mRNA decay43. In addition, EIF4A3 is also involved in various biological processes, including mRNA translation initiation and RNA splicing44,45. It was inferred that EIF4A3 was probably involved in the transcriptional regulation of circRNA_100290. Wang et al. reported that EIF4A3 could induce circMMP9 cyclization and increased expression of circMMP9 in GBM by binding to the MMP9 mRNA transcript46. Inconsistent with that, we found silenced EIF4A3 led to the elevated circRNA_100290, indicated that EIF4A3 could inhibit the formation of circRNA_100290. The regulatory mechanisms of circRNAs was complicated. Inverted Alu repeats could promote circRNA formation by facilitating back-splicing of pre-mRNAs, while DHX9, an RBP, bound specifically to inverted-repeat Alu elements, thereby inhibiting the formation of circRNAs. The loss of DHX9 led to an increase in the number of circular RNAs47. In addition, Ivanov et al. reported that ADAR1 could decrease the expression of circRNA by competitively binding with reverse complementary sequences highly enriched in introns bracketing circRNAs48. EIF4A3 might inhibit the formation of circRNA_100290 by binding the flanking regions of circRNA_100290.These studies suggested that the role of EIF4A3 in different circRNAs’ cyclization is distinct, the underlying mechanism remains further explore.
Additionally, we found the expression of EIF4A3 was down regulated in GC. Moreover, low expression of EIF4A3 was found to predict a worse prognosis and was closely related to Lauren’s type, invasion depth, and TNM staging by analyzing pathological factors in GSE62254, a dataset from the GEO database, which contained 300 GC cases. By comprehensive sequencing, 300 GC cases were classified into four subtypes, including MSI, MSS/EMT, MSS/TP53+ and MSS/TP53−, according to the ACRG molecular type49. Interestingly, EIF4A3 high-expression group had the lowest percentage of MSS/EMT subtype of ACRG genotyping. Previous study reported that GC patients with MSS/EMT subtype had the worst OS and the highest rate of recurrence, especially involving peritoneal dissemination50. The results indicated that EIF4A3 functioned as a tumor suppressor and negative regulator of circRNA_100290 in GC.