Abstract:

Objective

To explore the effects of circular RNA CCDC66 （Circ-CCDC66）on colorectal cancer progression through miR-618/phosphatase and tensin homolog deleted on chromosome ten （PTEN）.

Methods

Ten pairs of colorectal cancer tissues and adjacent normal tissues were collected.Bioinformatics methods were used to predict the targets of Circ-CCDC66， and miR-618 and PTEN were selected for subsequent experiments.Real- time fluorescence quantitative PCR （RT-qPCR） was used to detect the expression of Circ- CCDC66 in NCM620， SW480， SW620，and HCT116 cells.The expression of CCDC66 and Circ-CCDC66 in HCT116 as well as SW620 cells were detected after RNase R treatment.The expression of miR-618 in SW620 and HCT116 cells were detected after transfection with miR- 618 mimic or lentivirus vector transfection of Circ- CCDC66.Western blot was used to detect the expression levels of PTEN， AKT， and BCL- 2 proteins in cells.Dual-luciferase reporter gene assay was used to detect the targeted relationship between Circ- CCDC66 and miR-618， miR-618 and PTEN.Cell Counting Kit-8 （CCK- 8） assay was used to detect the changes in cell proliferation after different treatments， scratch assay and Transwell invasion assay were used to detect the invasion and migration of colorectal cancer cells after differenttreatments.Independent sample t test was used to compare the measurement data between the two groups， one-way analysis of variance was used for comparison among multiple groups， and LSD-t test was used for pairwise comparison between groups.

Results

Compared with the adjacent tissues of colorectal cancer， the expression of Circ-CCDC66 （0.69 ± 0.20 vs 1.00 ± 0.21） and PTEN mRNA （0.26 ± 0.13 vs 1.00 ± 0.48） and PTEN protein （0.67 ± 0.23 vs 1.00 ± 0.13） in colorectal cancer tissues were decreased （all P ＜ 0.05）.Compared with the normal colon epithelial cell line NCM460， the expression of Circ-CCDC66 （0.50 ± 0.03， 0.18 ± 0.02， 0.62 ± 0.05 vs 1.00 ± 0.10）was decreased in colorectal cancer cell lines HCT116， SW620， and SW480 （all P ＜ 0.01）， and the expression of Circ-CCDC66 in HCT116 and SW620 cells was lower than that in SW480 cells （all P ＜0.05）.Bioinformatics analysis and lentivirus transfection experiments indicated that miR- 618 was a downstream target of Circ-CCDC66.Dual-luciferase reporter assays showed that miR-618 targeted the PTEN gene in colorectal cancer cell lines.After overexpression of miR-618， the expression of PTEN proteins in colorectal cancer SW620 and HCT116 cells （0.49 ± 0.01 vs 1.00 ± 0.01， 0.52 ±0.00 vs 1.01 ± 0.00） were decreased （all P ＜ 0.001）.CCK-8， cell scratch， and Transwell assays demonstrated that overexpression of Circ-CCDC66 could inhibit the proliferation （0.48 ± 0.09 vs 0.83 ± 0.05， 0.41 ± 0.05 vs 0.68 ± 0.05）， migration ［（25.33 ± 2.08）% vs （41.00 ± 3.61）%， （28.00 ±2.00）% vs （40.00 ± 5.00）%］， and invasion ［（42.33 ± 4.93） vs （61.33 ± 4.16）cells， （25.00 ± 5.00）vs （41.00 ± 3.61） cells］ abilities of colorectal cancer SW620 and HCT116 cells at 48 h （all P ＜ 0.05）.However， miR-618 could reverse the inhibitory effects of Circ-CCDC66 overexpression ［proliferation（0.78 ± 0.05 vs 0.48 ± 0.09， 0.58 ± 0.05 vs 0.41 ± 0.05）； migration ［（35.00 ± 2.00）% vs （25.33 ±2.08）%， （40.00 ± 1.73）% vs （28.00 ± 2.00）%］； invasion ［（68.67 ± 5.03） vs （42.33 ± 4.93）cells， （39.33 ± 5.13） vs （25.00 ± 5.00） cells］ （all P ＜ 0.05）.

Conclusion

Circ-CCDC66 could influence the progression of colorectal cancer cells by binding to miR-618 and targeting PTEN

Key words: miR-618, PTEN, Colorectal cancer, Target, CircRNA