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Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer.

ABSTRACT: Long noncoding ribonucleic acids (LncRNAs) have been found to be involved in the proliferation, apoptosis, invasion, migration, and other pathological processes of triple-negative breast cancer (TNBC). Expression of the lncRNA actin filament-associated protein 1 antisense RNA1 (AFAP1-AS1) has been found to be significantly higher in TNBC than in other subtypes or in normal tissue samples, but the specific mechanism by which AFAP1-AS1 affects the occurrence and development of TNBC is yet to be revealed. In this study, we used Cell Counting Kit-8 (CCK-8), colony formation, wound healing migration, Transwell invasion, and nude mouse xenograft assays to confirm the role of AFAP1-AS1 in the proliferation, migration of TNBC cells in vitro and in vivo. In addition, we performed bioinformatics analyses, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and dual-luciferase reporter assays (dual-LRA) to confirm interaction among AFAP1-AS1, micro-RNA 2110 (miR-2110), and Sp1 transcription factor (Sp1). We found that silencing AFAP1-AS1 and Sp1 or upregulating miR-2110 suppressed the proliferation, migration, and invasion of MDA-MB-231 and MDA-MB-468 cells in vitro as well as tumor growth in vivo. Mechanistically, the dual-LRA highlighted that miR-2110 was an inhibitory target of AFAP1-AS1, and that AFAP1-AS1 functioned as a miR-2110 sponge to increase Sp1 expression. AFAP1-AS1 silencing led to a reduction in Sp1 mRNA and protein levels, which could be reversed by joint transfection with miR-2110 inhibitor. Our findings demonstrated that AFAP1-AS1 could modulate the progression of breast cancer cells and affect tumorigenesis in mice by acting as a miR-2110 sponge, resulting in regulation of Sp1 expression. Therefore, AFAP1-AS1 could play a pivotal role in the treatment of TNBC.

SUBMITTER: Zhang X

PROVIDER: S-EPMC8213778 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer.

Zhang Xiaohui X Li Fangyuan F Zhou Yidong Y Mao Feng F Lin Yan Y Shen Songjie S Li Yuntao Y Zhang Sheng S Sun Qiang Q

Cell death & disease 20210618 7

Long noncoding ribonucleic acids (LncRNAs) have been found to be involved in the proliferation, apoptosis, invasion, migration, and other pathological processes of triple-negative breast cancer (TNBC). Expression of the lncRNA actin filament-associated protein 1 antisense RNA1 (AFAP1-AS1) has been found to be significantly higher in TNBC than in other subtypes or in normal tissue samples, but the specific mechanism by which AFAP1-AS1 affects the occurrence and development of TNBC is yet to be re ...[more]

PMID: 34145213

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Project description:BackgroundVasculogenic mimicry (VM) is an alternative intratumoral microcirculation system that depends on the capacity of tumor cells to reorganize and grow in three-dimensional (3D) channel architectures like the capillaries formed by endothelial cells. Both VM and angiogenesis may coordinately function to feed cancer cells, allowing tumor growth. Long noncoding RNAs (lncRNAs) regulate critical cellular functions in cancer cells, including cell proliferation, apoptosis, angiogenesis, invasion, and metastasis. The lncRNA, known as actin filament-associated protein 1-antisense RNA 1 (AFAP1-AS1), has been described as an oncogene in diverse types of cancers. However, its role in VM and metastasis in triple-negative breast cancer (TNBC) is unknown.MethodsReverse transcription and quantitative polymerase chain reaction (RT‒qPCR) experiments were performed to evaluate the expression of 10 selected lncRNAs from literature in metastatic and nonmetastatic biopsies from TNBC patients. The expression of AFAP1-AS1 was analyzed in Genotype-Tissue Expression Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. The AFAP1-AS1 expression was knocked in TNBC Hs578T cells by transfection of specific siRNAs. Channel-like formation assays were performed using 3D cultures over Matrigel in hypoxia-treated Hs578T cancer cells with diminished expression of AFAP1-AS1. The angiogenesis tests were conducted using human umbilical vein endothelial cells (HUVECs) and AFAP1-AS1- silenced Hs578T cells on 3D cell cultures. The presence of VM (CD31-/PAS+) in tumor tissues from TNBC patients with and without metastasis was assessed through immunohistochemistry using endothelial marker CD31 antibodies and periodic acid-Schiff (PAS) staining.ResultsCompared with normal mammary tissues, AFAP1-AS1 expression was higher in breast cancer tissues. Moreover, AFAP1-AS1 expression was upregulated in the TNBC subtype compared to receptor-positive breast tumors. In addition, the expression of AFAP1-AS1 was correlated with the expression of the thirteen genes characteristic of a previously reported hypoxia signature. Interestingly, AFAP1-AS1 was upregulated in primary TNBC tumors from patients who developed metastasis compared with the nonmetastatic group. Functional analysis revealed that the knockdown of AFAP1-AS1 in Hs578T cells significantly impaired the hypoxia-induced VM, accompanied by a decrease in the development of 3D channel networks. Similarly, AFAP1-AS1 knockdown counteracts the angiogenic potential of cancer cells, as indicated by a reduction in the number of polygons, sprouting cells, and nodes in HUVEC cells. Remarkably, an increase in CD31-/PAS + staining of 3D channel networks in primary breast tumors from metastatic patients was found compared with the nonmetastatic group. Finally, we found that the number of blood vessels increased in the nonmetastatic group more than in the metastatic cohort.ConclusionsOur data suggested that AFAP1-AS1 controls both VM and angiogenesis in Hs578T breast cancer cells and that increased metastasis is associated with VM in TNBC patients.

Dataset Information

Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer.

Publications

Long noncoding RNA AFAP1-AS1 promotes tumor progression and invasion by regulating the miR-2110/Sp1 axis in triple-negative breast cancer.

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