Extensive research on circRNAs has raised many new insights into cancer development. Researchers now use advanced sequencing and micro-array technology to better investigate circRNAs and verify their function and molecular mechanism[14–19]. CircRNA are considered potential biomarkers and therapeutic targets for cancer because of their special stable structure[20, 21]. However, the mechanisms by which circRNAs affect OSCC progression are still unclear. Our study was based on a previous study in which 407 differentially expressed circRNAs were detected between OSCC and AHT using high-throughput sequencing analysis, with 134 highly expressed and 273 lowly expressed in OSCC. Circ_0005232 was an implicated highly expressed circRNA. Following preliminary qRT-PCR experiments, we chose circ_0005232 as the focus of this study. The relationship between circ_0005232 and OSCC was confirmed through bioinformatic analysis and functional experiments. Circ_0005232 is a newly discovered circRNA which has not yet been reported in any other cancers. QRT-PCR results from 53 pairs of OSCC tissue confirmed that circ_0005232 has significantly higher expression in OSCC. Correlation analysis of clinical pathological characteristics indicated that high circ_0005232 is related to lymphatic metastasis. To better define the relationship between circ_0005232 and OSCC, we knocked down circ_0005232 in OSCC cell lines by transfecting with si-circ_0005232 then performed colony formation, transwell migration and invasion, and wound healing assays. We found that circ_0005232 silencing inhibited proliferation, migration, and invasion in OSCC cells, suggesting that circ_0005232 plays an important role in malignant OSCC development. To determine functional effects of circ_0005232 in OSCC cells, we overexpressed circ_0005232 then repeated the functional experiments, finding that overexpression of circ_0005232 significantly improved proliferation, migration, and invasion ability. These results suggest that circ_0005232 may serve as a cancer-promoting gene that affects biological functions of CAL27 and SCC9 in OSCC, consistent with the results of clinical and pathological data analysis.
Epithelial-to-mesenchymal transformation (EMT) refers to the process by which cells transform from normal epithelial cells to abnormal mesenchymal cells[23, 24]. EMT is a typical event in the development of malignant tumors including OSCC. MMP2 and MMP9 mainly play roles in cell invasion and migration progression. Western blot and qRT-PCR for EMT markers, MMP2, and MMP9 after circ_0005232 knockdown indicated that si-circ_0005232 inhibits EMT progression and MMP family expression, thus inhibiting OSCC progression.
We also examined the TCGA database, but circ_0005232 was not included in the database, so our results could not be confirmed by a larger data set. We used 53 clinical samples to test the expression of circ_0005232 in OSCC vs AHT. While this is a sizeable sampling, it is insufficient to generalize the expression of circ_0005232 in all of OSCC. Therefore, we hope that future studies can expand the sample size for a more detailed overview of the effects of circ_0005232 on OSCC.
A large amount of evidence has shown that circRNAs can adsorb miRNAs by acting as sponges to affect miRNA activity and regulate expression of their target genes, ultimately promoting or suppressing cancer. This process of regulation is a competitive mechanism[27, 28]. Potential miRNAs targets of circ_0005232 were predicted using Circ-interactom and Circ-bank software. We choose to overlay the results of the two databases then combine it with experimental results. Through dual luciferase assays, we confirmed that circ_0005232 negatively regulates miR-1299. Presently reported studies implicate miR-1299 in non-small cell lung cancer (NSCLC), prostate cancer, ovarian cancer, liver cancer, breast cancer, osteosarcoma, esophageal squamous cell cancer (ESCC), colon cancer, bile duct cancer, gastric cancer, and renal cell cancer. For example, circ_0006528 could sponge miR-1299 in breast cancer to regulate CDK8 expression. In NSCLC and ESCC, miR-1299 acted as a sponge of EGFR (epidermal growth factor receptor) and negatively regulated the expression of EGFR[29, 35].
Our study is the first to verify that circ_0005232 acts as a sponge of miR-1299 in OSCC. To further verify the relationship between circ_0005232 and miR-1299 in OSCC, we conducted rescue experiments which showed that inh-miR-1299 can restore the influence of si-circ_0005232 on cell biological functions. In addition, protein and RNA expression results were consistent with functional results, verifying that circ_0005232 exerts its biological function in OSCC cells by sponging miR-1299.
We predicted target proteins of miR-1299 by TargetScan and selected CDK6 for further study. CDK6 is encoded by the CDK6 gene, which is a cell division protein kinase regulated by cyclins. Reports in breast cancer, liver cancer and osteosarcoma show that miR-1299 acts on CDK6 through sponging to play a role in tumor progression. RIP verified that miR-1299 binds to CDK6 in squamous cell carcinoma cell lines, so we speculated that circ_0005232 might regulate the miR-1299/CDK6 axis in OSCC. Mechanistic experiment results verified that miR-1299 could restore the effects of circ_0005232 on CDK6 RNA and protein expression and that CDK6 could restore the effects of circ_0005232 on proliferation in SCC9 cells. Taken together, our results suggest that circ_0005232 regulates expression of CDK6 and affects the malignant function of OSCC cells through sponging miR-1299.