Circ_0026344 overexpression inhibits the migration, invasion, and enhances apoptosis of colorectal cancer cells by sponging miR-31

Background: Circ_0026344 was reported to be associated with the metastasis of colorectal cancer (CRC). This study aimed to investigate the expression of circ_0026344 in CRC and the effect mechanisms of circ_0026344 on CRC. Methods: The expressions of circ_0026344 and miR-31 in clinical CRC tissues or CRC cell lines were analyzed by qPCR. The target of circ_0026344 was predicted and veried by CircInteractome and dual-luciferase reporter assays. The correlation between circ_0026344 and miR-31 expression was analyzed using Pearson analysis. After the CRC cells were overexpressed circ_0026344 or miR-31 or silenced circ_0026344, the viability, apoptosis, migration, and invasion of CRC cells were evaluated by CCK-8, ow cytometry, wound healing, and transwell. Also, the expressions of miR-31, Bcl-2, Bax, E-cadherin, and N-cadherin in the cells were detected by qPCR or Western blot. Results: Circ_0026344 was low-expressed in CRC tissues and cell lines. Circ_0026344 sponged miR-31 which was high-expressed in CRC tissues. The expression of circ_0026344 was negatively correlated to the expression of miR-31. The miR-31 expression could be down-regulated by circ_0026344 overexpression. Circ_0026344 overexpression inhibited the cell viability, migration, and invasion; and enhanced the apoptosis of CRC cells. Circ_0026344 overexpression decreased the expressions of Bcl-2 and N-cadherin and increased the expressions of Bax and E-cadherin in CRC cells. Circ_0026344 silencing and miR-31 overexpression had an opposite effect on CRC cells as circ_0026344 overexpression. Furthermore, miR-31 overexpression counteracted the effect of circ_0026344 overexpression.

by regulating APC2 (8). Circ_0026344, a newly discovered circRNA, was previously proved to be lowexpressed in gastric cancer and it can be an independent predictor for CRC patients' overall survival (9).
What's more, circ_0026344 was suggested that its abnormal expression was associated with the metastasis of CRC (10), although the expression level and effect of circ_0026344 on CRC needed more exploration.
Scholars have proved that circRNA is able to function as a sponge of miRNA and thereby to regulate the progression of different kinds of diseases and cancers, including CRC (11)(12)(13). In particular, circ-ITGA7 inhibits the proliferation of CRC cells by sponging miR-3187-3p (14), circ_0009361 acts as a sponge of miR-582 to inhibit the processes of CRC (8), circ_0000523 modulates the proliferation and apoptosis of CRC cells through acting as a sponge of miR-31 (13). MiR-31, highly expressed in CRC, is widely proved that it is correlated to the metastasis of CRC and has a high value in CRC diagnosis (15,16). Whether circ_0026344 had an effect on CRC by sponging miR-31 needs further exploration and veri cation.
Then, the 2.5 × 10 5 SW480 and HCT116 cells in 300 µL medium were placed into per well of a 48-well plate, respectively. After the cell con uence arrived at about 80%, the cells were transfected with miR-31 mimic and circ_0026344-WT or circ_0026344-MUT together. After transfection nished, the cells were collected and proceed with the dual-luciferase-reporter-kit (ab228530, Abcam, Cambridge, UK). Finally, the proceeded cells were placed into a SpectraMax reader (Molecular Devices, Shanghai, China) to measure the activity of the luciferase.

CCK-8 assay
After transfection, 1.0 × 10 4 SW480 and HCT116 in 100 µL medium were placed into a 96-well plate respectively and further cultured for 24 h, 48 h, or 72 h. Then, 10 µL of CCK-8 buffer (A56097, OKA, Beijing, China) was added into each well of the 96-well plate to further culture the cells for 4 h. Finally, the absorbance of per well of the 96-well plate was measured by a microplate reader (Imark; Bio-Rad, Hercules, California, USA) under 570 nm.

Flow cytometry
Cell apoptosis of SW480 and HCT116 cells after transfection was detected using Annexin V-FITC/Propidium Iodide (PI) Kit (BB-4101-2, BestBio, Nanjing, China) by ow cytometry. In brief, after being transfected, SW480 and HCT116 cells were harvested and washed with PBS (PB180327, Procell, Wuhan, China) for three times. Then the cells were suspended in 500 µL Working Buffer and stained with 5 µL of Annexin V-FITC solution and 5 µL of PI solution for 25 min in the dark. Finally, the cell apoptosis was analyzed by a FACSCaliburTM ow cytometer (BD Biosciences, Franklin Lake, New Jersey, USA).

Wound healing assay
After transfection, 2.0 × 10 6 SW480 and HCT116 in 2 mL medium were placed into a 6-well plate respectively and cultured until the cell con uence arrived over 95%. Then a wound was created in the each well, and each wound had equal width. Meantime, the previous medium was refreshed with 2 mL

Statistical analysis
One-way ANOVA was used for comparison among multiple groups, Two-way ANOVA was used for comparison of cell viability among multiple groups, and Dunnet's test was used as post-hoc tests. Correlation between miR-31 expression with circ_0026344 expression in clinical samples was analyzed using Pearson analysis. All statistical analyses were implemented using Graphpad 8.0 software, measurement data were described by Mean ± SD, and P < 0.05 was considered statistically signi cant.

Results
Circ_0026344 was low-expressed in CRC tissues and cell lines CRC tissues and its corresponding adjacent normal tissues were rstly collected for the analysis of circ_0026344 expression level. As the data displayed in Fig. 1A, the expression of circ_0026344 in CRC tissues was signi cantly lower than that in normal tissues (P < 0.001). Similarly, the expression of circ_0026344 in CRC cell lines (HCT-15, SW480, SW620, HCT116) were also lower than that in human normal colorectal mucosal cell line FHC (Fig. 1B, P < 0.01). Among these CRC cell lines, the expression of circ_0026344 in the SW480 was the highest and in the HCT116 was the lowest, hence, we chose the two cell lines for later use.
Circ_0026344 regulated the expressions of factors related to apoptosis and epithelial-mesenchymal transformation (EMT) in SW480 and HCT116 cells To con rm the present discovery, we further detected the expressions of factors related to apoptosis and EMT (Fig. 4). After overexpression circ_0026344, the expressions of Bcl-2 and N-cadherin both in SW480 (Fig. 4A-B) and HCT116 (Fig. 4C-D) cells were down-regulated (P < 0.001), and the expressions of the Bax and E-cadherin were up-regulated (P < 0.001). On the contrary, the expressions of Bcl-2 and N-cadherin both in SW480 and HCT116 cells were up-regulated by si-circ_0026344 (P < 0.001) while the expressions of the Bax and E-cadherin were up-regulated by si-circ_0026344 (P < 0.001). Circ_0026344 acted as sponge of miR-31 After the binding sites between circ_0026344 with miR-31 were predicted by CircInteractome (Fig. 5A), we further performed dual-luciferase reporter assays to verify that circ_0026344 might a sponge of miR-31. As shown in Fig. 5B-C, the luciferase activities both in SW480 and HCT116 cells were decreased after transfected with circ_0026344-WT and miR-31 mimic together (P < 0.001), while, no difference were found in the luciferase activities of the two cell lines after transfected with circ_0026344-MUT and miR-31 mimic together, which indicated that circ_0026344 might act as a sponge of miR-31 in CRC.
MiR-31 was high-expressed in CRC tissues and its expression was negative correlated to circ_0026344 expression both in CRC tissues and normal tissues Then we evaluated the expression of miR-31 in CRC tissues (Fig. 6A) and found that miR-31 expression was higher than that in the normal tissues (P < 0.001). Furthermore, the expression of miR-31 in normal tissues (Fig. 6B) and in CRC tissues (Fig. 6C) were both negative correlated to the expression of circ_0026344. These results made us wonder that the effect of circ_0026344 on CRC cells was mediated by sponging miR-31.
MiR-31 overexpression counteracted the effect of circ_0026344 on the cell viability, migration, invasion, and the expression of miR-31 in SW480 and HCT116 cells To further make the deep mechanism underlying the effect of circ_0026344 on CRC, the miR-31 mimic was used to overexpress the expression of miR-31 both in SW480 (Fig. 7A) and HCT116 (Fig. 7B) cells. Then the expressions of miR-31 in the two cells were evaluated (Fig. 7C-D), as the results revealed that the expression of miR-31 was down-regulated by circ_0026344 overexpression (P < 0.001) and upregulated by miR-31 mimic (P < 0.001) when compared with the Vector + MC group; while, after cooverexpressed with circ_0026344 and miR-31 (the circ_0026344 + M group), the decreased miR-31 which down-regulated by circ_0026344 overexpression was further decreased by miR-31 mimic as compared to the circ_0026344 + MC group (P < 0.001). Cell viability was illustrated in Fig. 7E-F, the cell viabilities were decreased by circ_0026344 overexpression (P < 0.05) and increased by miR-31 overexpression (P < 0.01) as compared to the Vector + MC groups, while after co-overexpressed circ_0026344 and miR-31, the effect of circ_0026344 overexpression on cell viabilities was offset by miR-31 overexpression (P < 0.01). Similarly, the cell migration (Fig. 8A-D) and invasion (Fig. 8E-H) were also analyzed. As depicted in Fig. 8, the abilities of cells to migrate and invade were decreased by circ_0026344 overexpression (P < 0.05) and increased by miR-31 overexpression (P < 0.001) as compared to the Vector + MC group, while after cooverexpressed circ_0026344 and miR-31, the effects of circ_0026344 overexpression on cell migration and invasion were offset by miR-31 overexpression (P < 0.001). All these discoveries suggested that the effects of circ_0026344 on cell viability, migration, and invasion of CRC cells might be mediated by sponging miR-31.
MiR-31 overexpression counteracted the effect of circ_0026344 on the expressions of factors related to apoptosis and EMT in SW480 and HCT116 cells Lastly, we evaluated the expressions of factors related to cell apoptosis and EMT to further con rm our above discoveries (Fig. 9). The effects of circ_0026344 overexpression on these factors were the same as the results showed in Fig. 4. Meantime, miR-31 mimic up-regulated the expressions of Bcl-2 and Ncadherin (P < 0.005), and down-regulated the expressions of Bax and E-cadherin (P < 0.05) both in SW480 (Fig. 9A-B) and HCT116 (Fig. 9C-D) cells. Furthermore, after co-overexpressed circ_0026344 and miR-31, the effects of circ_0026344 overexpression on these factors were offset by miR-31 overexpression.

Discussion
The growing number of pieces of evidence have proved that circRNA with abnormal expression regulates the development of different types of diseases and cancers, including CRC (4,17). In CRC, circ_00038646, circ-ZNF609, circ_0004585, and circ_102958 are high-expressed and further promote the development of CRC (5, 6, 18, 19); circ_0000523, circDDX7, circ_0014717, and circ-ITGA7 are low-expressed and the silencing of these circRNAs can enhance the malignant abilities of CRC cells (7,13,14,17). In the present study, we also found that circ_0026344 was down-regulated both in CRC tissues and cell lines, which suggested that the process of CRC might be regulated by circ_0026344 although the effects and mechanisms were needed more exploration.
The extremely high recurrence rate and mortality of CRC can be attributed to the strong migration and invasion abilities of cancer cells and less apoptosis in cancer cells (20). Therefore, the key to treatment CRC is to inhibit the migration and invasion and induce the apoptosis of cancer cells. The biological activities of CRC cells including migration, invasion, proliferation, and apoptosis also can be regulated by circRNAs such as circ-100876, circ_00038646, circ_0000523, circ-ZNF609, and so on (5,6,13,21). In the present study, we discovered that circ_0026344 overexpression suppressed the migration and invasion, and induced the apoptosis of CRC cells, while, circ_0026344 silencing did the opposite. The cell apoptosis, migration, as well as invasion, are occurred after the expression changes of the factors which are related to these biological activities (22,23). Apoptosis can be successfully carried out only after Bcl-2 (one of the anti-apoptotic factors) binding with Bax (one of the pro-apoptotic factors) (22). During the intrinsic apoptosis of cells, the expression of Bcl-2 is decreased while Bax is up-regulated (24,25). This study further proved that circ_0026344 overexpression down-regulated the expression of Bcl-2 and upregulated the expression of Bax in CRC cells, while, circ_0026344 silencing did the opposite. These phenomena further con rmed that circ_0026344 regulated the apoptosis of CRC cells. Equally important, EMT plays an important role in the process of cancer cells to migrate and invade (26). During this process, the cancer cells gradually lose the characteristics of epithelial cells with the expression decreasing of epithelial marker (E-cadherin), while, the cells gradually acquired the characteristics of mesenchymal cells with the expression increasing of mesenchymal markers (N-cadherin) (27). Thereby, the cancer cells can migrate and invade into the surrounding tissues and organs (28). Our study further discovered that circ_0026344 overexpression up-regulated the expression of E-cadherin and up-regulated the expression of N-cadherin in CRC cells, while, circ_0026344 silencing did the opposite. These phenomena also con rmed that circ_0026344 did regulate the migration and invasion of CRC cells.
Since the role of "miRNA sponges" was put forward, circRNA was widely researched and proved that it can sponge some miRNAs to play its role in various biological activities (12). Theoretically, circ-ITGA7, circ_0009361, and circ_0000523 respectively sponge miR-3187-3p, miR-582, and miR-31 in CRC (8, 13,14). In the current study, we discovered that binding sites have existed between circ_0026344 with miR-31, also, circ_0026344 down-regulated the expression of miR-31 in CRC cells, which indicated that the effect of circ_0026344 on CRC might be mediated by sponging miR-31. MiR-31 is previously found that it's highly expressed in CRC (15). Consistent with the report above, our results in this study also discovered that miR-31 was high-expressed in CRC tissues. What's more, miR-31 is proved to be correlated to the metastasis of CRC and can regulate autophagy, proliferation, and chemoresistance of CRC cells (16,29,30). Similarly, in this study, the results con rmed that miR-31 overexpression not only promoted the migration, invasion, and suppressed the apoptosis of CRC cells but also counteracted the effect of circ_0026344. All the evidence revealed that circ_0026344 acted as a sponge of miR-31 to regulate the migration, invasion, and apoptosis of CRC cells.

Conclusion
To sum up, our study proved that circ_0026344 overexpression inhibited the migration and invasion, and induced apoptosis of CRC cells through sponging miR-31. Our ndings in this study provided a novel