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Fig. 3 | Journal of Translational Medicine

Fig. 3

From: Long non-coding RNA BZRAP1-AS1 silencing suppresses tumor angiogenesis in hepatocellular carcinoma by mediating THBS1 methylation

Fig. 3

BZRAP1-AS1 inhibits the transcription of THBS1 through mediating the methylation of the THBS1 promoter by interacting with DNMT3b. a Venn map of down-regulated genes in HCC in three microarray expression profiles (GSE45267, GSE49515, and GSE89377). b Correlation analysis of the expression of BZRAP1-AS1 and THBS1 (GSE49515). c mRNA expression of THBS1 in HuH-7 and L-02 cells measured by RT-qPCR. d The effect of BZRAP1-AS1 on the transcription of THBS1 assessed by RT-qPCR. e Analysis of CpG island enrichment in THBS1 promoter region. f mRNA expression of THBS1 when the methylation was inhibited by 5-aza-dc in the presence of BZRAP1-AS1 measured by RT-qPCR. g Methylation level of THBS1 promoter measured by the MSP assay. H2O was used as double negative control, in vitro methylated DNA (IVD) as positive methylation control, and normal lymphocyte DNA (NL) as unmethylated positive control. U means unmethylation while M means methylation. h Methylation level of THBS1 promoter measured by the BSP assay. Black circle stands for methylation site and white circle stands for un-methylated site. i Interaction between DNMT3b and BZRAP1-AS1 confirmed by RIP assay. j Interaction between BZRAP1-AS1 and DNMT3b verified by RNA pull-down assay. k Blast alignment of BZRAP1-AS1 and THBS1 promoter sequences. l Interaction between BZRAP1-AS1 and THBS1 promoter region analyzed by dual luciferase reporter gene assay. m THTS1 promoter region directly bound to DNMT3b, as detected by the ChIP assay. * p < 0.05 vs. L-02 cell line; #p < 0.05 vs. cells infected with lentivirus expressing sh-NC; &p < 0.05 vs. cells infected with lentivirus expressing oe-NC; @p < 0.05 vs. cells infected with lentivirus expressing oe-BZRAP1-AS1 and treated with DMSO; %p < 0.05 vs. cells introduced with IgG. The data (mean ± S.D.) obeying the normal distribution and homogeneity of variance between two groups were analyzed using unpaired t test while those among multiple groups were assessed by one-way ANOVA, followed with Tukey’s post hoc test. The experiment was repeated 3 times independently. BZRAP1-AS1 BZRAP1-antisense RNA 1, THBS1 thrombospondin1, DNMT3b DNA methyltransferase 3B, HCC hepatocellular carcinoma, RT-qPCR reverse transcription quantitative polymerase chain reaction, MSP methylation specific PCR, BSP bisulfite sequencing PCR, RIP RNA immunoprecipitation, ChIP chromatin immunoprecipitation, DMSO dimethyl sulfoxide, IgG immunoglobulin G, ANOVA analysis of variance

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