Knockdown of lncRNA CCAT1 Inhibits the Progression of Colorectal Cancer via hsa-miR-4679 Mediating the Downregulation of GNG10

Abstract

Great concerns have raised crucial roles of long noncoding RNAs (lncRNAs) on colorectal cancer progression due to the increasing number of studies in cancer development. Previous studies reveal that lncRNA CCAT1 plays an important role in the progression of a variety of cancers. However, the role of lncRNA CCAT1 in colorectal cancer is still unclear. In this study, we found that in both colorectal tissues and cell lines the level of lncRNA CCAT1 was increased. Downregulation of lncRNA CCAT1 inhibited the proliferation, migration, and invasion of colorectal cell lines and promoted apoptosis. We then found that hsa-miR-4679 could bind to lncRNA CCAT1 directly, and with further functional analyses, we confirmed that lncRNA CCAT1 sponged hsa-miR-4679 to promote the progression of colorectal cancer. Next, we found that hsa-miR-4679 was directly bound to 3'UTR of GNG10 (guanine nucleotide-binding protein, gamma 10). GNG10 overexpression promoted the progression of colorectal cancer, and this phenotype could be reversed by miR-4679 mimics. At last, we knocked down CCAT1 in vivo and found that sh-CCAT1 reduced the tumor size and the number of proliferating cells. In summary, our findings revealed that lncRNA CCAT1 facilitated colorectal cancer progression via the hsa-miR-4679/GNG10 axis and provided new potential therapeutic targets for colorectal cancer.

Figure 1

Knockdown of lncRNA CCAT1 inhibited the progression of colorectal cancer progression. (a) Relative expression of CCAT1 in CRC and normal tissues. (b) Relative expression of CCAT1 in FHC, a human colon immortalized cell line, and HT-29 and HCT-116, two colorectal cancer cell lines. (c) Relative expression of CCAT1 in HT-29 and HCT-116 after knockdown of CCAT1. (d) EdU staining was performed to detect the proliferation ability after the knockdown of CCAT1. (e) The statistical result of (d). (f) WB was performed to detect the levels of proliferation markers. (g) Transwell migration and invasion assays were performed to access the migration and invasion abilities after the knockdown of CCAT1. (h) The statistical results of (g). (i) FACS was performed to analyze the apoptosis rate after the knockdown of CCAT1. (j) The statistical result of (i). ^∗∗p < 0.01 and ^∗∗∗p < 0.001.

Figure 2

hsa-miR-4679 directly bound to lncRNA CCAT1. (a) The relative expression level of hsa-miR-7-5p, hsa-miR-1246, hsa-miR-8063, hsa-miR-6879-5p, and hsa-miR-4679 after knockdown of CCAT1. (b) The relative expression level of hsa-miR-4679 in CRC and normal tissues. (c) The strategy for the construction of luciferase reporter vectors to detect the binding between CCAT1 and hsa-miR-4679. (d) Relative luciferase activity in cotransfection of CCAT1 and hsa-miR-4679 group was significantly reduced. (e) RIP assay with anti-AGO2 antibody was performed to confirm the binding between CCAT1 and hsa-miR-4679. ^∗∗p < 0.01 and ^∗∗∗p < 0.001.

Figure 3

lncRNA CCAT1 functioned as an hsa-miR-4679 sponge. (a) EdU staining was performed to detect the proliferation ability after upregulation of hsa-miR-4679. (b) The statistical result of (a). (c) WB was performed to detect the levels of proliferation markers. (d) Transwell migration and invasion assays were performed to access the migration and invasion abilities after upregulation of hsa-miR-4679. (e) The statistical results of (d). (f) FACS was performed to analyze the apoptosis rate after upregulation of hsa-miR-4679. (g) The statistical results of (f). (h) WB was performed to detect the levels of proliferation markers. (i) EdU staining was performed to detect the proliferation ability after the treatment with sh-CCAT1, hsa-miR-4679 inhibitors, and sh-CCAT1+hsa-miR-4679 inhibitors. (j) Transwell migration and invasion assays were performed to access the migration and invasion abilities after the treatment with sh-CCAT1, hsa-miR-4679 inhibitors, and sh-CCAT1+hsa-miR-4679 inhibitors. (k) FACS was performed to analyze the apoptosis rate after the treatment with sh-CCAT1, hsa-miR-4679 inhibitors, and sh-CCAT1+hsa-miR-4679 inhibitors. (l) The statistical result of (i). (m) The statistical result of (j). (n) The statistical result of (k). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 4

hsa-miR-4679 regulated the expression of GNG10. (a) Relative expression of AKR1B10, CDA, KLK7, and GNG10 in CRC and normal tissues was quantified using qPCR. (b) WB was performed to detect the level of GNG10 in CRC tissues. (c) The expression level of GNG10 was negatively correlated with the overall survival rate. (d) The size of tumor tissues was significantly increased with GNG10 overexpression. (e) The strategy for the construction of luciferase reporter vectors to detect the binding between hsa-miR-4679 and GNG10. (f) Relative luciferase activity in cotransfection of hsa-miR-4679 and GNG10 group was significantly reduced. (g) Relative expression of GNG10 was regulated by hsa-miR-4679 mimics and hsa-miR-4679 inhibitors. (h) WB was performed to detect the level of GNG10 after the treatment of hsa-miR-4679 mimics and hsa-miR-4679 inhibitors. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

hsa-miR-4679 reversed the phenotype of GNG10 on promoting CRC. (a) WB was performed to detect the level of GNG10 after GNG10 overexpression. (b) EdU staining was performed to detect the proliferation ability after the treatment with hsa-miR-4679 mimics, GNG10 overexpression, and hsa-miR-4679 mimics+GNG10 overexpression. (c) The statistical result of (b). (d) WB was performed to detect the levels of proliferation markers. (e) Transwell migration and invasion assays were performed to access the migration and invasion abilities after the treatment with hsa-miR-4679 mimics, GNG10 overexpression, and hsa-miR-4679 mimics+GNG10 overexpression. (f) FACS was performed to analyze the apoptosis rate after the treatment with hsa-miR-4679 mimics, GNG10 overexpression, and hsa-miR-4679 mimics+GNG10 overexpression. (g) The statistical results of (e). (h) The statistical results of (f). ^∗∗p < 0.01 and ^∗∗∗p < 0.001.

Figure 6

sh-CCAT1 inhibited the CRC progression in vivo via the miR-4679/GNG10 axis. (a) The size of tumor tissues was significantly decreased after sh-CCAT1 using HT-29 and HCT-116 cells. (b) The statistical result of the tumor volume. (c) H&E staining was performed to detect the damages after the treatment with sh-CCAT1. (d) Ki-67 staining was performed to detect the proliferation status of the tissues after sh-CCAT1. (e) The ratio of Ki-67-positive cells. (f) WB was performed to detect the levels of proliferation markers. (g) WB was performed to detect the levels of pro- and antiapoptotic proteins. (h) Relative expression of hsa-miR-4679 after sh-CCAT1 in vivo. (i) Relative expression of GNG10 after sh-CCAT1 in vivo. ^∗∗p < 0.01 and ^∗∗∗p < 0.001.