Circ_0084043-miR-134-5p axis regulates PCDH9 to suppress melanoma

Abstract

The low survival rates, poor responses, and drug resistance of patients with melanoma make it urgent to find new therapeutic targets. This study investigated whether the circ_0084043-miR-134-5p axis regulates the antitumor effect of protocadherin 9 (PCDH9) in melanoma. Ectopic expression or knock down (KD) of PCDH9 with a lentivirus vector, we explored its effects on the proliferation, invasion, and apoptosis of melanoma and verified its regulatory effect on ras-related C3 botulinum toxin substrate 1 (RAC1), proline-rich tyrosine kinase 2 (Pyk2), Cyclin D1, matrix metalloproteinase 2 (MMP2), and MMP9. We further observed the effect of KD circ_0084043 on the malignant behavior of melanoma and studied whether circ_0084043 sponged miR-134-5p and regulated PCDH9. We found that circ_0084043 was overexpressed in melanoma and associated with the malignant phenotype. PCDH9 was poorly expressed in human melanoma tissues, and overexpression of PCDH9 inhibited melanoma progression. Quantitative real-time PCR and Western blotting results showed that overexpression of PCDH9 could downregulate RAC1, MMP2, and MMP9 and upregulate Pyk2 and Cyclin D1. Circ_0084043 KD inhibited invasion and promoted apoptosis in melanoma cells. Circ_0084043 could sponge miR-134-5p and thus indirectly regulate PCDH9. Furthermore, we discovered that inhibiting circ_0084043 had an anti-PD-Ll effect. In vivo, PCDH9 overexpression inhibited melanoma tumor growth, but PCDH9 KD promoted it. In conclusion, PCDH9, which is regulated by the circ 0084043-miR-134-5p axis, can suppress malignant biological behavior in melanoma and influence the expression levels of Pyk2, RAC1, Cyclin D1, MMP2, and MMP9.

Keywords: PCDH9; Pyk2; RAC1; apoptosis; circRNAs; malignant melanoma.

Figure 1

Effects of PCDH9 overexpression in melanoma cells measured by qRT-PCR. The expressions of PCDH9 were significantly upregulated by lentivirus infection (A, B). Overexpressed PCDH9 significantly upregulated the gene expressions of Pyk2 and Cyclin D1 and downregulated RAC1, MMP2, and MMP9 except for FAK in A375 (C) and G361 (D) cells. *p < 0.05 and ***p < 0.001 compared groups using one-way ANOVA followed by least significant difference post-hoc tests. The results cited from the preprint doi: https://doi.org/10.21203/rs.3.rs-95026/v1.

Figure 2

Effects of PCDH9 overexpression and KD in melanoma cells measured by Western blot. Overexpressing PCDH9 significantly upregulated levels of Pyk2 and Cyclin D1 and downregulated RAC1, MMP2, and MMP9 in A375 (A) and G361 (B) cells. KD of PCDH9 downregulated levels of Pyk2 and Cyclin D1 and upregulated RAC1, MMP2, and MMP9 in A375 (C) and G361 (D) cells. ^ns P > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 compared groups using one-way ANOVA followed by least significant difference post-hoc tests.

Figure 3

The viability of melanoma cells was significantly reduced by PCDH9 overexpression in A375 (A, B) and G361 (C, D) cells. *p < 0.05, **p < 0.01, and ***p < 0.001 compared groups using one-way ANOVA followed by least significant difference post-hoc tests. The alteration of PCDH9 expression significantly affected the apoptosis of melanoma cells. The overexpression of PCDH9 significantly promoted apoptosis in G361 cells and a more modest manner in A375 cells, whereas PCDH9 KD reduced apoptosis in G361 cells and a more modest manner in A375 cells (E, F). ^ns P > 0.05, *p < 0.05, **p < 0.01, and ****p < 0.0001 compared with blank group by using Student’s t-tests.

Figure 4

The alteration of PCDH9 expression did not affect the melanoma cell cycle. The cell percentage of A375 (A) and G361 (B) cells in G1, S, and G2 phases when PCDH9 was overexpressed. The cell percentage of A375 (C) and G361 (D) cells in G1, S, and G2 phases when PCDH9 interfered. *p < 0.05, **p < 0.01, and ***p < 0.001 compared groups using one-way ANOVA followed by least significant difference post-hoc tests. Overexpression of PCDH9 inhibits the migration of melanoma cells. Scratch width of A375 (E) and G361 (F) cells at 0, 24, and 48 h. The scratch area of A375 (G) and G361 (H) cells overexpressing PCDH9 did not alter at 24 and 48 h. After 24 and 48 h, the relative scratch area of the PCDH9 overexpression group was larger than that of the blank group and negative control group in A375 (I) and G361 (J) cells. ^nsP > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 compared groups using one-way ANOVA followed by least significant difference post hoc tests.

Figure 5

qRT-PCR results showed the expression of circ_0084043 in human melanoma cell lines (A375, SK-MEL-28, and A875) and normal human epidermal melanocytes cell line (PIG1) (A). Circ_0084043 was knocked down in A375 and A875 cells by siRNA (B). Data represent mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with control groups by using Student’s t-tests. Immunohistochemical analyses of PCDH9 and RAC1 expression in normal skin, pigmented nevus, and melanoma tissue. The positive percentage of PCDH9 expression was lower in human melanoma tissue than in normal skin or/and pigmented nevus tissue and the positive percentage of RAC1 expression was higher in human melanoma tissue than normal skin or/and pigmented nevus tissue. PCDH9 is mainly localized in the cytoplasm, with a small amount localized in the nuclei. S100 and HMB45 are melanoma markers. The scale bar represents 200 μm (C). The volcano map of differential miRNA showed that three miRNAs are upregulated and five are downregulated after knocking down circ_0084043 (the red dot indicates upregulation, the green dot indicates downregulation, and the blue dot indicates no significant difference) (D). Differential miRNA cluster analysis showed that miR-134-5p, miR-4659b-5p, and miR-760 were upregulated, and miR-202-5p, miR-33a-3p, miR-181b-3p, miR-217-5p, and miR-3143 were downregulated after knocking down circ_0084043 (P < 0.05) (red indicates high expression of miRNA, and blue indicates low expression of miRNA) (E). Dual-luciferase reporter assays revealed that the luciferase activity of circ_0084043 luciferase reporters was reduced by miR-134-5p mimic (P < 0.01) (F). Predicted binding sites of circ_0084043 and miR-134-5p (G).

Figure 6

The cell migration ability of A375 cells was detected by scratch wound assay (A–C). Apoptosis was determined by flow cytometry after transfection for 48 h (D, E). Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared groups using one-way ANOVA followed by least significant difference post-hoc tests.

Figure 7

Photographs illustrated tumors in the subcutaneous xenograft tumor model. Tumors in the KD PCDH9 group were significantly heavier than those in the control and blank groups. The nude mice with overexpression of PCDH9 did not grow tumors of significant size.

Figure 8

Western blot was used to detect the expression levels of PCDH9, Pyk2, RAC1, Cyclin D1, MMP2, c-Jun, XIAP, and PD-L1 in melanoma cells after transfection for 48 h (A–C). Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared groups using one-way ANOVA followed by least significant difference post-hoc tests.