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

Abstract

Background: Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer associated with a high mortality. Long non-coding RNAs (lncRNAs) have recently emerged as regulators in the development and progression of several cancers, and therefore represent an opportunity to uncover new targets for therapy. In the present study, we aimed to investigate the potential effect of lncRNA BZRAP1-AS1 on the angiogenesis of HCC.

Methods: Microarray-based data analysis was initially employed to screen genes and lncRNAs that are differentially expressed in HCC and the candidate BZRAP1-AS1 was identified as a hit. The expression of BZRAP1-AS1 and thrombospondin-1 (THBS1) in HCC tissues and cells were then determined using RT-qPCR. The gene methylation level was measured by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) assays. Next, the interactions between BZRAP1-AS1, DNA methyltransferase 3B (DNMT3b), and THBS1 were assessed by RIP, RNA pull-down and ChIP assays. Finally, the roles of BZRAP1-AS1, DNMT3b and THBS1 in angiogenesis in vitro as well as tumorigenesis in vivo were evaluated by a battery of the gain- and loss-of function experiments.

Results: BZRAP1-AS1 was identified as a highly expressed lncRNA in HCC tissues and cells. Down-regulation of BZRAP1-AS1 in HCC cells inhibited HUVEC proliferation, migration and angiogenesis. By interacting with DNMT3b, BZRAP1-AS1 induced methylation of the THBS1 promoter and inhibited the transcription of THBS1, resulting in promoted angiogenesis of HUVECs. Moreover, silencing of BZRAP1-AS1 repressed the angiogenesis as well as the tumor growth of HCC in vivo via up-regulating THBS1.

Conclusion: This study provides evidence that angiogenesis in HCC is hindered by silencing of BZRAP1-AS1. Thus, BZRAP1-AS1 may be a promising marker for the treatment of HCC.

Keywords: Angiogenesis; BZRAP1-AS1; DNA methylation; DNA methyltransferase 3B; Hepatocellular carcinoma; Long non-coding RNA; Thrombospondin-1.

Fig. 1

BZRAP1-AS1 is highly expressed in HCC tissues and cells. a, b Differentially expressed lncRNAs screened from HCC-related microarrays GSE33006 and GSE49515. The abscissa shows the number of sample and the ordinate shows differentially expressed genes. The histogram in the upper right is the color gradation, and each rectangle in the figure corresponds to the expression of one sample. c Expression of BZRAP1-AS1 relative to GAPDH in HCC tissues and the adjacent normal tissues. d Expression of BZRAP1-AS1 relative to GAPDH in HCC cells and the normal liver cell line. e Localization of BZRAP1-AS1 in HCC cells analyzed by lncATLAS online site. f Subcellular localization (×200) of BZRAP1-AS1 in HuH-7 cells detected by FISH. g Nuclear expression of BZRAP1-AS1 in HuH-7 cells determined by RT-qPCR. *p < 0.05, vs. adjacent normal tissues; ^#p < 0.05 vs. L-02 cell line. The data (mean ± S.D.) obeying the normal distribution 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, HCC hepatocellular carcinoma, FISH fluorescence in situ hybridization, ANOVA analysis of variance

Fig. 2

BZRAP1-AS1 silencing suppresses HUVEC proliferation and angiogenesis. a The expression of BZRAP1-AS1 in HuH7 cells infected with lentivirus expressing sh-NC or sh-BZRAP1-AS1 determined by RT-qPCR. b The effect of BZRAP1-AS1 on the proliferation of HUVECs co-cultured with HuH7 cells revealed by EdU assay (×200). c The effect of BZRAP1-AS1 on the migration of HUVECs evaluated by Transwell assay (×40). d The effect of BZRAP1-AS1 on angiogenesis of HCC cells assessed by tube formation assay (×50). e The level of VEGFA in HuH-7 concentrated supernatant measured by ELISA. f The effect of BZRAP1-AS1 on angiogenesis evaluated by CAM assay (×20). *p < 0.05 vs. HuH-7 cells infected with lentivirus expressing sh-NC. The data (mean ± S.D.) obeying the normal distribution and homogeneity of variance among multiple groups were analyzed using one-way ANOVA, followed with Tukey’s post hoc test. The experiment was repeated 3 times independently. HCC hepatocellular carcinoma, BZRAP1-AS1 BZRAP1-antisense RNA 1, RT-qPCR reverse transcription quantitative polymerase chain reaction, EdU 5-ethynyl-2′-deoxyuridine, VEGFA vascular endothelial growth factor, ELISA enzyme-linked immunosorbent assay, CAM chicken chorioallantoic membrane, ANOVA analysis of variance, NC negative control, HUVECs human umbilical vein endothelial cells

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

Fig. 4

BZRAP1-AS1 regulates the development of HCC by negatively regulating THBS1 via DNMT3b. a Transfection efficiency of sh-DNMT3b assessed by RT-qPCR. b Transfection efficiency of oe-THBS1 assessed by RT-qPCR. c Effects of BZRAP1-AS1, DNMT3b and THBS1 on the proliferation of HUVECs evaluated by EdU assay (×200). d Effects of BZRAP1-AS1, DNMT3b and THBS1 on the HUVEC migration evaluated by Transwell assay (×40). e Effects of BZRAP1-AS1, DNMT3b and THBS1 on angiogenesis evaluated by tube formation assay (×50). f The level of VEGFA in HuH-7 concentrated supernatant analyzed by ELISA. g Effects of BZRAP1-AS1, DNMT3b and THBS1 on angiogenesis detected by CAM assay (×20). *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. 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, DNMT DNA methyltransferase, HCC hepatocellular carcinoma, THBS1 thrombospondin1, RT-qPCR reverse transcription quantitative polymerase chain reaction, DNMT3b DNA methyltransferase 3B, EdU 5-ethynyl-2′-deoxyuridine, VEGFA vascular endothelial growth factor, ELISA enzyme-linked immunosorbent assay, CAM chicken chorioallantoic membrane, ANOVA analysis of variance, NC negative control

Fig. 5

BZRAP1-AS1 silencing represses the angiogenesis of HCC in vivo. a Xenograft tumor growth curve. b Tumor volume and weight at the 35th day after injection. c MVD and the level of CD31 determined by immunohistochemical staining (×200). d Tumor angiogenesis in vivo determined by immunofluorescence staining (×500). *p < 0.05 vs. mice injected with oe-NC-treated cells; ^#p < 0.05 vs. mice injected with oe-THBS1-treated cells; ^&p < 0.05 vs. mice injected with sh-NC-treated cells. The data (mean ± S.D.) obeying the normal distribution homogeneity of variance among multiple groups were assessed by one-way ANOVA and repeated measures ANOVA was performed for proliferation ability at different time points followed with Tukey’s post hoc test. The experiment was repeated 3 times independently. n = 15. HCC hepatocellular carcinoma, MVD microvessel density, ANOVA analysis of variance

Fig. 6

The mechanism map depicting that BZRAP1-AS1 promotes the progression of HCC through down-regulation of THBS1 via recruiting DNMT3b. BZRAP1-AS1, highly expressed in HCC, binds to the promoter of THBS1, recruits the DNMT3b, and enhances the methylation of THBS1 promoter, thus inhibiting the transcription of THBS1. THBS1 depletion promotes angiogenesis via increasing VEGFA and CD31 expression. HCC hepatocellular carcinoma, BZRAP1-AS1 BZRAP1-antisense RNA 1, THBS1 thrombospondin1