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Fig. 4 | Cell & Bioscience

Fig. 4

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

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

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