Hsa_circ_0103232 promotes melanoma cells proliferation and invasion via targeting miR-661/RAB3D

Abstract

Little is known about the role of hsa_circ_0103232 in melanoma. This study researched the role of hsa_circ_0103232 in melanoma progression. Hsa_circ_0103232 expression in clinical tissues of melanoma patients and melanoma cells was detected by qRT-PCR. Hsa_circ_0103232 localization in melanoma cells was visualized by fluorescence in situ hybridization. Hsa_circ_0103232 effect on melanoma cells viability, proliferation, migration, and invasion was explored by cell counting kit-8 (CCK-8) assay, Edu experiment, wound healing assay, and Transwell experiment. RNA pull-down assay and dual-luciferase reporter gene assay were performed to verify the binding of hsa_circ_0103232 with miR-661, and the binding of miR-661 and RAB3D. Xenograft tumor models were constructed. Western blot and immunohistochemistry were used for protein expression detection. Hsa_circ_0103232 expression was increased in melanoma patients, indicating lower overall survival. Hsa_circ_0103232 was mainly expressed in the cytoplasm of melanoma cells. Silencing hsa_circ_0103232 suppressed melanoma cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) (P < 0.01). Hsa_circ_0103232 functioned as a sponge of miR-661 to increase RAB3D expression. miR-661 overexpression partially reversed hsa_circ_0103232 promoting effect on melanoma cells viability, proliferation, migration, invasion, and EMT (P < 0.01). In melanoma patients, hsa_circ_0103232 expression was negatively correlated with miR-661 and positively correlated with RAB3D. Silencing hsa_circ_0103232 suppressed melanoma cell growth in vivo and Ki67 and RAB3D expression in xenograft tumors (P < 0.01). Hsa_circ_0103232 is a tumor promoter in melanoma to enhance malignant phenotype and growth in vivo via sponging miR-661/RAB3D. Hsa_circ_0103232 may be a novel target for melanoma treatment.

Keywords: Hsa_circ_0103232; Melanoma; RAB3D; miR-661; progression.

Figure 1.

Hsa_circ_0103232 expression was increased in melanoma. (a) qRT-PCR revealed that hsa_circ_0103232 expression was increased in melanoma tissues. (b) Kaplan–Meier curve indicated that high hsa_circ_0103232 expression was associated with poor survival of melanoma patients. (c) qRT-PCR illustrated that hsa_circ_0103232 expression was increased in melanoma cell lines (SKMel1, A375 and A875). (d) FISH verified that hsa_circ_0103232 was mainly expressed in the cytoplasm, and highly expressed in the melanoma cells (SKMel1 and A375). * P < 0.05. ** P < 0.01.

Figure 2.

Silencing hsa_circ_0103232 suppressed melanoma cells proliferation, migration, invasion and EMT. (a) qRT-PCR revealed that hsa_circ_0103232 expression in SKMel1 and A375 cells was effectively silenced by shRNA. (b) CCK-8 assay verified that silencing hsa_circ_0103232 suppressed melanoma cells viability. (c) Edu experiment indicated that silencing hsa_circ_0103232 inhibited melanoma cells proliferation. (d) Wound healing assay illustrated that silencing hsa_circ_0103232 attenuated melanoma cells migration. (e) Transwell experiment demonstrated that silencing hsa_circ_0103232 inhibited melanoma cells invasion. (f) Western blot revealed that silencing hsa_circ_0103232 suppressed melanoma cells EMT. ** P < 0.01.

Figure 3.

Hsa_circ_0103232 functioned as a sponge of miR-661 to increase RAB3D expression. (a) CircInteractome predicted the miRNAs containing the binding site of hsa_circ_0103232. (b) CircInteractome predicted the binding site between hsa_circ_0103232 and miR-661. (c) RNA pull down assay indicated that miR-661 was effectively enriched by hsa_circ_0103232. (d) Dual luciferase reporter gene assay exhibited that miR-661 was a target gene of hsa_circ_0103232. (e) Targetscan showed the binding site between RAB3D and miR-661. (f) RNA pull down assay revealed that miR-661 was obviously enriched by RAB3D. (g) Dual luciferase reporter gene assay verified that RAB3D was target gene of miR-661. (h) Western blot illustrated that hsa_circ_0103232 silencing decreased RAB3D protein expression in SKMel1 and A375 cells. * P < 0.05. ** P < 0.01.

Figure 4.

miR-661 partially reversed hsa_circ_0103232 promoting effect on melanoma cells proliferation, migration, invasion and EMT. (a) CCK-8 assay illustrated that miR-661 partially reversed hsa_circ_0103232 promoting effect on melanoma cells viability. (b) Edu experiment revealed miR-661 partially reversed hsa_circ_0103232 promoting effect on melanoma cells proliferation. (c) Wound healing assay indicated that miR-661 partially reversed hsa_circ_0103232 promoting effect on melanoma cells migration. (d) Transwell experiment demonstrated that miR-661 partially reversed hsa_circ_0103232 promoting effect on melanoma cells invasion. (e) Western blot reveal that miR-661 partially reversed hsa_circ_0103232 promoting effect on melanoma cells EMT. ** P < 0.01 compared with oe-NC + miR-NC group. ## P < 0.01 compared with oe-hsa_circ_0103232 + miR-NC group.

Figure 5.

miR-661 acted as a tumor suppressor and RAB3D was an oncogene in melanoma (a) CCK-8 assay implied that miR-661 suppressed the viability of melanoma cells and RAB3D had the opposite function. (b) EdU experiment indicated that miR-661 inhibited the proliferation ability of melanoma cells and RAB3D showed the opposite function. (c) Wound healing assay revealed that miR-661 possessed inhibitory effect on the migration ability of melanoma cells and RAB3D exhibited the opposite function. (d) Transwell experiment suggested that miR-661 could inhibit the invasion capacity of melanoma cells and RAB3D presented the opposite function. (e) Western blot indicated that miR-661 attenuated the EMT in melanoma cells and RAB3D possessed the opposite function. ** P < 0.01 compared with miR-NC group. ** P < 0.01 compared with siNC group.

Figure 6.

Hsa_circ_0103232 expression in melanoma was negatively correlated with miR-661 and positively correlated with RAB3D. (a) qRT-PCR indicated that miR-661 expression was reduced in melanoma tissues. (b) qRT-PCR revealed that RAB3D mRNA expression was increased in melanoma tissues. (c) Pearson’s correlation analysis illustrated that hsa_circ_0103232 expression was negatively correlated with miR-661 expression in melanoma tissues. (d) Pearson’s correlation analysis revealed that RAB3D mRNA expression was negatively correlated with miR-661 expression in melanoma tissues. (e) Pearson’s correlation analysis indicated that RAB3D mRNA expression was positively correlated with hsa_circ_0103232 expression in melanoma tissues. ** P < 0.01.

Figure 7.

Silencing hsa_circ_0103232 suppressed melanoma cells growth in vivo. (a) The xenograft tumors in nude mice were obtained after 28 days of subcutaneous injection. (b and c) Silencing hsa_circ_0103232 reduced A375 cells growth in vivo.(d) qRT-PCR revealed the decreased hsa_circ_0103232 expression and increased miR-661 expression in xenograft tumors. (e) IHC indicated that silencing hsa_circ_0103232 reduced Ki67 and RAB3D proteins expression in xenograft tumors. * P < 0.05. ** P < 0.01.