Exosomal lncRNA SNHG12 promotes angiogenesis and breast cancer progression

Abstract

Objective: Breast cancer is one of the most prevalent malignancies in women. Exosomes are important mediators of intercellular communication; however, their regulatory mechanisms in human umbilical vein endothelial cells (HUVECs) angiogenesis in breast cancer remain unknown.

Methods: We isolated and characterized breast cancer cell-derived exosomes and investigated their functions. Exosomal sequencing and the TCGA database were used to screen long non-coding RNA (lncRNA). In vitro and in vivo experiments were performed to investigate the role of exosomal lncRNA in HUVEC angiogenesis and tumor growth. Molecular methods were used to demonstrate the molecular mechanism of lncRNA.

Results: We demonstrated that breast cancer cell-derived exosomes promoted HUVEC proliferation, tube formation, and migration. Combining exosomal sequencing results with The Cancer Genome Atlas Breast Cancer database, we screened lncRNA small nucleolar RNA host gene 12 (SNHG12), which was highly expressed in breast cancer cells. SNHG12 was also upregulated in HUVECs co-cultured with exosome-overexpressed SNHG12. Moreover, overexpression of SNHG12 in exosomes increased HUVEC proliferation and migration, whereas deletion of SNHG12 in exosomes showed the opposite effects. In vivo experiments showed that SNHG12 knockdown in exosomes inhibited breast cancer tumor growth. Transcriptome sequencing identified MMP10 as the target gene of SNHG12. Functional experiments revealed that MMP10 overexpression promoted HUVEC angiogenesis. Mechanistically, SNHG12 blocked the interaction between PBRM1 and MMP10 by directly binding to PBRM1. Moreover, exosomal SNHG12 promoted HUVEC angiogenesis via PBRM1 and MMP10.

Conclusions: In summary, our findings confirmed that exosomal SNHG12 promoted HUVEC angiogenesis via the PBRM1-MMP10 axis, leading to enhanced malignancy of breast cancer. Exosomal SNHG12 may be a novel therapeutic target for breast cancer.

Keywords: Angiogenesis; Breast cancer; Exosomes; Progression; lncRNA SNHG12.

Fig. 1

Breast cancer cell-derived exosomes enhanced HUVEC angiogenesis. Breast cancer cell-derived exosomes were isolated and characterized by transmission electron microscope (A) and western blot (B). Scale bar: 100 nm. C The protein concentration of exosomes was determined. D DiI-labeled exosomes were internalized by HUVECs. Scale bar = 50 μm. The role of breast cancer cell-derived exosomes in HUVECs proliferation (E), migration (F), and tube formation (G) was assayed by CCK-8, wound healing, and tube formation assay, respectively. MCF-10A exo represents MCF-10A cell-derived exosomes. MDA-MB-231 exo represents MDA-MB-231 cell-derived exosomes. Magnification: ×4. n = 3, * p < 0.05, ** p < 0.01

Fig. 2

Screening and identification of lncRNAs associated with angiogenesis in exosomes. A Upregulated lncRNAs co-expressed genes in TCGA-Breast Cancer data. B The volcano plot showing differentially expressed genes between breast cancer and normal tissues using the TCGA database. C Venn diagram of co-expressed genes of exo-up-lncRNA and differentially expressed genes in breast cancer. GO (D) and KEGG (E) enrichment analysis of intersection genes. F The network diagram of lncRNAs-mRNAs-GO/KEGG. G The expression of lncRNA SNHG12 and UBE2SP1 was detected in breast cancer cells by qRT-PCR. H The expression of lncRNA SNHG12 and UBE2SP1 was detected in breast cancer cell-derived exosomes by qRT-PCR. I qRT-PCR was used to evaluate the expression of SNHG12 in exosomes from breast cancer cell lines MCF-7 and MDA-MB-468. J Breast cancer cell-derived exosomes increased SNHG12 levels in HUVECs. n = 3, ** p < 0.01

Fig. 3

Exosomal SNHG12 enhanced HUVEC angiogenesis. A Transfection efficiency of SNHG12 overexpression and knockdown was determined by qRT-PCR. B qRT-PCR was used to detect the expression of SNHG12 in exosomes-overexpressed or deleted SNHG12. C qRT-PCR was used to detect the expression of SNHG12 in HUVECs co-cultured with exosomes-overexpressed or deleted SNHG12. D CCK-8 assay was performed to measure the effect of exosomal SNHG12 overexpression or silencing on HUVEC proliferation. E Wound healing assay was performed to determine the effect of exosomal SNHG12 overexpression or silencing on HUVEC migration. Magnification: ×4. F Transwell assay was performed to determine the effect of exosomal SNHG12 overexpression or silencing on HUVEC migration. Magnification: ×20. n = 3, * p < 0.05, ** p < 0.01

Fig. 4

Exosomal with downregulated SNHG12 inhibited breast cancer tumorigenesis in vivo. A,B Representative pictures of tumors in each group. Exosomal with downregulated SNHG12 suppressed tumor volume (C) and weight (D), and decreased SNHG12 levels in tumor (E). n = 5. F Immunofluorescence was used to detect the expression of CD31 and CD34 in tumor tissue sections. Magnification: ×20. n = 3, * p < 0.05, ** p < 0.01

Fig. 5

Screening of SNHG12 target genes. A Differentially expressed target genes of SNHG12 are shown in the heatmap. The differentially expressed SNHG12 target genes were subject to GO (B) and KEGG (C) analysis. D The network diagram of the SNHG12-mRNAs-pathway. E The selected target genes of SNHG12 were verified by qRT-PCR. n = 3, ** p < 0.01

Fig. 6

MMP10 enhanced HUVEC angiogenesis. A, B Transfection efficiency of MMP10 overexpression and knockdown was determined by qRT-PCR and western blot. C CCK-8 assay was conducted to determine the effect of MMP10 overexpression or silencing on HUVEC proliferation. D Wound healing assay was performed to determine the effects of MMP10 overexpression or silencing on HUVEC migration. Magnification: ×4. E Tube formation assay was performed to determine the effects of MMP10 overexpression or silencing on HUVEC tube formation. Magnification: ×4. F Transwell assay was performed to examine the effect of MMP10 overexpression or silencing on HUVEC migration. Magnification: ×20. n = 3, * p < 0.05, ** p < 0.01

Fig. 7

SNHG12 directly interacts with PBRM1 to upregulate MMP10. A Silver staining of SNHG12 pull down. B Western blot was used to detect SNHG12-binding proteins. C RIP-PCR was applied for the interaction between SNHG12 and PBRM1 in HUVECs. D The presumptive sites between PBRM1 and MMP10. E ChIP-PCR was applied for the interaction between MMP10 and PBRM1 in HUVECs. F SNHG12 overexpression weakens the interaction between MMP10 and PBRM1. G The MMP10 mRNA expression was verified by qRT-PCR. n = 3, * p < 0.05, ** p < 0.01

Fig. 8

Exosomal SNHG12 facilitates HUVEC angiogenesis via MMP10 and PBRM1. A CCK-8 assay was used to evaluate the effect of MMP10 overexpression or PBRM1 silencing on exosomal SNHG12-mediated HUVEC proliferation. B Tube formation assay was performed to determine the effects of MMP10 overexpression or PBRM1 silencing on exosomal SNHG12-mediated HUVEC tube formation. Magnification: ×4. C Wound healing assay and D transwell assay were applied to determine the effects of MMP10 overexpression or PBRM1 silencing on exosomal SNHG12-mediated HUVEC migration. n = 3, * p < 0.05, ** p < 0.01