Silencing Exosomal circ102927 Inhibits Foot Melanoma Metastasis via Regulating Invasiveness, Epithelial-Mesenchymal Transition and Apoptosis

Abstract

Background: Exosomes contain abundant circular RNAs (circRNAs), playing an important role in intercellular communication. However, the function and underlying molecular mechanism of exosomal circRNAs in foot metastatic melanoma remain unclear.

Methods: Twelve differentially expressed exosomal circRNAs between patients with metastatic and primary foot melanoma were screened through high-throughput sequencing, and their expression levels were detected by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). CircRNA102927 silencing and overexpression A2058 cell line was constructed, and the effects of circRNA102927 on cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) were assessed using cell counting kit-8 (CCK-8), flow cytometry, wound healing, Transwell, and Western blot assays, respectively.

Results: Twelve differentially expressed exosomal circRNAs were screened and ROC curve showed that six circRNAs could be used as the diagnostic biomarkers for metastatic melanoma. Melanoma-secreted exosomes induced the differentiation of CD4+ T cells into Treg cells. CircRNA102927 was highly expressed in metastatic melanomas. Functionally, circRNA102927 silencing inhibited proliferation, EMT, migration, and invasion in metastatic melanoma cells, while promoting apoptosis. Meanwhile, overexpression of circRNA102927 had the opposite effects.

Conclusion: Our investigation suggests that silencing exosomal circRNA102927 may suppress foot melanoma metastasis by inhibiting invasiveness, EMT and promoting apoptosis.

Keywords: CircRNA102927; exosomal circRNAs; foot metastatic melanoma; metastasis.

Figure 1

Identification of differentially expressed exosomal circRNAs between foot metastatic melanoma (ZY) and primary foot melanoma (YF). (A) Boxplot of chip background correction. (B) Distribution of circRNA host gene chromosomes. (C) Heatmap for cluster analysis of 12 differentially expressed circRNAs. (D) Volcano map for 12 differentially expressed circRNAs.

Figure 2

Schematic representation of six differentially expressed circRNAs binding to their downstream miRNAs.

Figure 3

Bubble plots of GO enrichment analysis of up- and down-regulated circRNAs. The color change in the legend indicates the size of −log10 (p-value), and the circle size represents the number of genes enriched in the corresponding pathway.

Figure 4

Six circRNAs could be used as a biomarker to distinguish foot metastatic melanoma (ZY) and primary foot melanoma (YF). (A) Western blot was used to detect the expression of exosomal marker proteins CD9 and Tsg101 in the ZY, ZY-Exo, YF, and YF-Exo groups. (B) Protein levels of key regulators of EMT, including E-cadherin, N-cadherin, and Vimentin, which were measured by Western blot in the ZY and YF groups. (C) The expression of circRNA102927 in ZY and YF exosomes was determined by RT-qPCR. (D) The ROC curve profile illustrated the diagnostic efficacy of six circRNAs in ZY and YF. *P < 0.05, **P < 0.01 vs ZY group; ^#P < 0.05 vs YF group; ^&P < 0.05 vs ZY-Exo group.

Figure 5

Exosomes derived from melanoma promoted the differentiation of CD4+ T cells into T Treg cells. (A) The concentrations of Treg cell marker IL-10, Th1 cell marker IL-4, and Th17 marker IL-17 were assessed using ELISA. (B) Western blotting was carried out to assess the expression levels of Treg regulatory proteins, including FOXP3 and TGF-β, along with the Th1 regulatory protein GATA-3. *P < 0.05, ***P < 0.001 vs Control group; ^##P < 0. 01, ^###P < 0.001 vs ZY or YF group; ^&P < 0.05, ^&&P < 0. 01, ^&&&P < 0. 001 vs YF-Exo group.

Figure 6

RT-qPCR was used to assess the expression levels of circRNAs in human primary melanocytes, human malignant melanoma cells A375 (primary) and A2058 (metastatic). *P < 0.05, **P < 0.01, ***P < 0.001 vs Control group; ^#P < 0.05, ^##P < 0. 01 vs A375 group.

Figure 7

Silencing circ102927 inhibited the proliferation, migration, invasion, and EMT of foot metastatic melanoma (ZY) cells and promotes apoptosis. (A) The levels of circRNA102927 expression in A375 and A2058 cells were assessed using RT-qPCR. (B) RT-qPCR was used to determine the circRNA102927 silencing efficacy in A2058 cells. (C) A2058 cell proliferation was assessed by CCK8 after sh-NC and sh-circ102927 treatment. (D) The apoptosis of A2058 cells was assessed using Annexin V-FITC in the sh-NC and sh-circ102927 groups. (E) A2058 cell migration was detected by wound healing assay after 24 h transfection with sh-NC and sh-circ102927. (F) Transwell was used to determine cell invasion. (G) EMT-related protein expressions (E-cadherin, N-cadherin, and Vimentin) were measured by Western blot. ***P < 0.001 vs Control group; ^###P < 0.001 vs A375 group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs sh-NC group.

Figure 8

Upregulation of circRNA102927 led to enhanced proliferation, migration, invasion, and EMT in foot metastatic melanoma (ZY) cells, along with reduced apoptosis. (A) After A2058 cells were transfected with oe-circRNA102927, overexpression efficiency was assessed by RT-qPCR. (B) The proliferation of A2058 cells was measured by CCK8 after treatment with oe-NC or oe-circRNA102927. (C) Flow cytometry was used to assess the effect of circRNA102927 overexpression on apoptosis in metastatic melanoma cells. (D) A2058 cell migration was detected after circRNA102927 overexpression by wound healing assay. (E) The Transwell assay was employed to determine cell invasion upon circRNA102927 upregulation. (F) Western blot was performed to detect the effect of circRNA102927 overexpression on the expression levels of EMT-related proteins (E-cadherin, N-cadherin, and Vimentin) in A2058B cells. ^#P < 0.05, ^##P < 0.01,^###P < 0.001 vs oe-NC group.