Circular RNA circRNA_0082835 promotes progression and lymphatic metastasis of primary melanoma by sponging microRNA miRNA-429

Figures & data

Table 1. TNM stage and lymphatic metastasis of malignant melanoma

Age	Gender	Tumor size (mm)	TNM stage	Lymphatic metastasis
47	Male	1	IIA	No
63	Female	0.1	IA	No
44	Female	0.3	IA	No
67	Male	0.4	IA	No
63	Female	0.2	IA	No
56	Male	0.1	IV	No
64	Female	0.1	IIIC	Yes
49	Female	0.3	IIIA	Yes
53	Male	0.1	IIIA	Yes
58	Male	0.2	IV	Yes

Figure 1. Increased circRNA_0082835 expression and decreased miR-429 expression in clinical melanoma samples. (a-b) Relative expressions of circRNA_0082835 and miR-429 in metastatic melanoma, detected by qPCR. ***P < 0.001 vs. No metastasis group. (c) correlation analysis between circRNA_0082835 and miR-429

Figure 2. Inhibiting circRNA_0082835 and/or EZH2 suppresses melanoma cell proliferation. (a-b) Relative expressions of circRNA_0082835 and EZH2 in different melanoma cell strains, detected by qPCR. *P < 0.05 and ***P < 0.001 vs. HM group. (c-d) Relative expressions of circRNA_0082835 and EZH2 in WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by qPCR. (e) OD value of WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by CCK-8. *P < 0.05, **P < 0.01 and ***P < 0.01 vs. Control group. ^#P < 0.05 and ^###P < 0.001 vs. shRNA-NC group. ^$$$P < 0.001 vs. shRNA-circ group. ^&&&p < 0.001 vs. shRNA-EZH2 group

Figure 3. Inhibiting circRNA_0082835 and/or EZH2 regulates cell cycle and suppresses the invasion and migration of melanoma cells. (a) WM451 cell cycle distribution before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by flow cytometry. (b) Relative invasive WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by transwell. (c) Relative wound width of WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by wound healing. **P < 0.01 and ***P < 0.01 vs. Control group. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs. shRNA-NC group. ^$$P < 0.01 and ^$$$P < 0.001 vs. shRNA-circ group. ^&&&p < 0.001 vs. shRNA-EZH2 group

Figure 4. Inhibiting circRNA_0082835 and/or EZH2 blocks Wnt/β-catenin, suppresses cell cycle-related protein expression and attenuates EMT in melanoma cells. (a) Relative protein expressions of Wnt/β-catenin signaling-related Wnt5a and β-catenin, cell cycle-related CyclinD1 and CyclinE1 in WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by western blot. (b) Relative protein expressions of EMT-related CRKL, E-cad and N-cad in WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by western blot. *P < 0.05, **P < 0.01 and ***P < 0.01 vs. Control group. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs. shRNA-NC group. ^$P < 0.05, ^$$P < 0.01 and ^$$$P < 0.001 vs. shRNA-circ group. ^&&p < 0.01 and ^&&&p < 0.001 vs. shRNA-EZH2 group

Figure 5. MiRNA-429 binds to circRNA_0082835 and its inhibition reverses the anti-proliferation effect of cicrRNA_0082835 knockdown. (a) Relative expression of miR-429 in WM451 cells before and after transfection of shRNA-circ and/or shRNA-EZH2, detected by qPCR. ***P < 0.001 vs. Control group. ^###P < 0.001 vs. shRNA-NC group. ^&&&P < 0.001 vs. shRNA-EZH2 group. (b-c) Relative luciferase activity in wt-circ + mimic NC, mut-circ + mimic NC, wt-circ + miR-429 mimic, wt-EZH2 + mimic NC, mut-EZH2 + mimic NC, wt-EZH2 + miR-429 mimic and mut-EZH2 + miR-429 mimic, detected by dual-luciferase reporter assay. ***P < 0.001 vs. wt-circ group. (d-e) Relative expression of miR-429 in WM451 cells before and after the transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups was detected by qPCR. **P < 0.01 and ***P < 0.001 vs. shRNA-circ group. ^##P < 0.01 and ^###P < 0.001 vs. shRNA-circ +miR-429 inhibitor group. (f) OD values of WM451 cells before and after the transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups were detected by CCK-8. ***P < 0.001 vs. shRNA-circ group

Figure 6. MiR-429 inhibitor affects cell cycle regulation and reverses the anti-invasion and anti-migration effects of circRNA_0082835 knockdown. (a) WM451 cell cycle distribution before and after transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups, detected by flow cytometry. (b) Relative invasive WM451 cells before and after transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups, detected by transwell. (c) Relative wound width of WM451 cells before and after transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups, detected by wound healing. ***P < 0.001 vs. shRNA-circ group. ^##P < 0.01 and ^###P < 0.001 vs. shRNA-circ +miR-429 inhibitor group. ^$$P < 0.01 vs. shRNA-EZH2 group. ^&&&P < 0.001 vs. shRNA-EZH2 + miR-429 inhibitor group

Figure 7. MiR-429 inhibitor reverses the effects of circRNA_0082835 knockdown on Wnt/β-catenin, cell cycle-related protein expressions and EMT in melanoma cells. (a) Relative expressions of Wnt/β-catenin signaling-related proteins and cell cycle-related proteins in WM451 cells before and after transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups, detected by western blot. (b) Relative expressions of and EMT-related proteins in WM451 cells before and after transfection of miR-429 inhibitor in shRNA-circ groups and shRNA-EZH2 groups, detected by western blot. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. shRNA-circ group. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 vs. shRNA-circ +miR-429 inhibitor group. ^$$P < 0.01 vs. shRNA-EZH2 group. ^&P < 0.05 and ^&&P < 0.01 vs. shRNA-EZH2 + miR-429 inhibitor group

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.