Long non-coding RNA CASC15 promotes melanoma progression by epigenetically regulating PDCD4

Abstract

Background: Long non-coding RNAs (LncRNAs) have been identified as critical regulators in a variety of cancer types. Cancer susceptibility candidate 15 (CASC15), a lncRNA located at chromosome 6p22.3, has been discovered to participate in melanoma progression and phenotype switching. Nevertheless, the roles and molecular mechanisms of CASC15 in melanoma are far from being understood.

Results: We found that CASC15 expression was up-regulated in melanoma tissues and associated with advanced pathological stages. Function experiments displayed that CASC15 knockdown hindered proliferation, facilitated apoptosis and suppressed invasion, while CASC15 overexpression facilitated proliferation and invasion in melanoma cells. Further mechanistic analysis showed that CASC15 epigenetically silenced the expression of programmed cell death 4 (PDCD4) by recruiting EZH2 and increasing H3K27me3 level at the promoter region of PDCD4. Additionally, PDCD4 overexpression inhibited proliferation, enhanced apoptosis and decreased invasion of melanoma cells. Moreover, CASC15-knockdown-induced anti-cancer effects were abated by PDCD4 down-regulation. Furthermore, depletion of CASC15 blocked tumor growth of melanoma by up-regulating PDCD4 in vivo.

Conclusions: CASC15 acts as an oncogene by negatively regulating PDCD4 expression via recruiting EZH2 and subsequently increasing H3K27me3 level. Together, our study indicates that CASC15/EZH2/PDCD4 may serve as a promising therapeutic target for melanoma intervention.

Keywords: CASC15; EZH2; LncRNA; Melanoma; PDCD4.

Fig. 1

CASC15 expression is increased in melanoma tumor tissues and correlated with melanoma development. a CASC15 expression in melanoma tissues (n = 461) and normal tissues (n = 558) was analyzed by GEPIA tool (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=boxplot). b CASC15 expression in different pathological stages of melanoma from GEPIA database. Relative expression levels of CASC15 in melanoma tissues (n = 42) and adjacent non-cancerous tissues (n = 42) (c), different clinical pathological stages (stage I (n = 11), stage II (n = 17), stage III (n = 14) (d), primary (n = 24) and metastatic melanoma tissues (n = 18) (e). f CASC15 expression in melanoma cells (A375, SK-MEL-2, M21, MEL-RM, B16 and SK-MEL-1) and normal human epidermal melanocytes (HEMa-LP). Results are represented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Knockdown of CASC15 suppresses proliferation, enhanced apoptosis and decreased invasion in melanoma cells. A375 and M21 cells were transfected with si-con, si-CASC15 #1, or si-CASC15 #2. a, b qRT-PCR analysis was used to determine the relative expression of CASC15 in transfected A375 and M21 cells. c, d CCK-8 analysis was performed to measure the proliferation ability in transfected A375 and M21 cells. e, f Flow cytometry analysis was applied to detect cell cycle distribution in transfected A375 and M21 cells. The bar chart represents the percentage of cells in G1/G0, S, and G2/M phases. g, h The apoptotic rate of transfected A375 and M21 cells was analyzed by flow cytometry. i, j Transwell invasion assay was conducted to assess the invasive capability of transfected A375 and M21 cells. Results are represented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

Fig. 3

CASC15 overexpression facilitates proliferation and invasion in melanoma cells. A375 and M21 cells were transfected with pcDNA empty vector or pcDNA-CASC15, followed by the evaluation of CASC15 expression (a), cell proliferation (b), cell cycle distribution (c), apoptosis (d) and invasion (e)

Fig. 4

CASC15 epigenetically silences PDCD4 expression by binding to EZH2. a Western blot assay was carried out to evaluate the protein level of PDCD4 in A375 and M21 cells transfected with si-con or si-CASC15 #2. b, c qRT-PCR assay was used to measure CASC15 expression in cytoplasm and nucleus of A375 and M21 cells. GAPDH was used as a cytoplasm marker and U6 was used as a nucleus marker. d The probability of interaction of CASC15 and EZH2 was predicted by RNA–protein interaction prediction website (http://pridb.gdcb.iastate.edu/RPISeq/). Predictions with probabilities > 0.5 were considered positive. RPISeq predictions are based on Random Forest (RF) or Support Vector Machine (SVM). e Western blot assay of PDCD4 protein expression in A375 and M21 cells after transfection with si-con or si-EZH2. f Luciferase reporter assay was performed in both A375 and M21 cells after co-transfection with PDCD4 promoter reporter and si-con or si-CASC15. g, h RIP experiments were conducted in A375 and M21 cells, followed by qRT-PCR analysis of CASC15 in the coprecipitated RNA. The fold enrichment of CASC15 in EZH2 RIP is relative to its matched IgG control. i, j In vitro transcribed biotin-labeled CASC15 and anti-CASC15 were incubated with A375 and M21 cell lysates, and the extracted protein was analyzed by western blot using an antibody against EZH2. A nonspecific protein (β-actin) is shown as the control. k, l ChIP of EZH2 occupancy and H3K27me3 binding in the PDCD4 promoter in A375 and M21 cells after transfection with si-con or si-CASC15 #2. Enrichment was quantified relative to input control. Antibody against IgG was used as a negative control. m PDCD4 mRNA expression in melanoma tissues (n = 461) and normal tissues (n = 558) from GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=boxplot). n Relative expression levels of PDCD4 mRNA in melanoma tissues (n = 42) and adjacent normal tissues (n = 42). o Spearman correlation analysis of CASC9 and PDCD4 expression in 42 melanoma tissues. Results are represented as mean ± SD of three independent experiments. **P < 0.01

Fig. 5

PDCD4 overexpression inhibits proliferation, induced apoptosis and attenuate invasion in melanoma cells. A375 and M21 cells were transfected with pcDNA empty vector or pcDNA-PDCD4, followed by the assessment of PDCD4 mRNA and protein level (a and b), cell viability (c), cell cycle distribution (d), apoptosis (e) and invasion (f). Results are represented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

Fig. 6

Silencing PDCD4 is partly involved in the oncogenic function of CASC15 in melanoma cells. A375 cells were transfected with si-con, si-CASC15, or co-transfected with si-CASC15 and si-PDCD4, followed by the measurement of PDCD4 protein level (a), cell proliferation (b), cell cycle distribution (c), apoptosis (d) and invasion (e)

Fig. 7

Down-regulation of CASC15 represses melanoma tumorigenesis in vivo. A375 cells stably transfected with sh-con or sh-CASC15 were injected into nude mice (n = 6). a Tumor volumes of mice in sh-con and sh-CASC15 groups were detected every 4 days. b Representative images of excised tumor masses. c Tumor weights were calculated in sh-con and sh-CASC15 groups. d–f The expression patterns of Ki-67, CASC15 and PDCD4 were assessed in tumors derived from sh-con- and sh-CASC15-transfected A375 cells. Results are represented as mean ± SD of three independent experiments. **P < 0.01