Extracellular Vesicles Long Non-Coding RNA AGAP2-AS1 Contributes to Cervical Cancer Cell Proliferation Through Regulating the miR-3064-5p/SIRT1 Axis

Abstract

Cervical cancer is one of the most severe and prevalent female malignancies and a global health issue. The molecular mechanisms underlying cervical cancer development are poorly investigated. As a type of extracellular membrane vesicles, EVs from cancer cells are involved in cancer progression by delivering regulatory factors, such as proteins, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs). In this study, we identified an innovative function of extracellular vesicle (EV) lncRNA AGAP2-AS1 in regulating cervical cancer cell proliferation. The EVs were isolated from the cervical cancer cells and were observed by transmission electron microscopy (TEM) and were confirmed by analyzing exosome markers. The depletion of AGAP2-AS1 by siRNA significantly reduced its expression in the exosomes from cervical cancer and in the cervical cancer treated with AGAP2-AS1-knockdown exosomes. The expression of AGAP2-AS1 was elevated in the clinical cervical cancer tissues compared with the adjacent normal tissues. The depletion of EV AGAP2-AS1 reduced cell viabilities and Edu-positive cervical cancer cells, while it enhanced cervical cancer cell apoptosis. Tumorigenicity analysis in nude mice showed that the silencing of EV AGAP2-AS1 attenuated cervical cancer cell growth in vivo. Regarding the mechanism, we identified that AGAP2-AS1 increased SIRT1 expression by sponging miR-3064-5p in cervical cancer cells. The overexpression of SIRT1 or the inhibition of miR-3064-5p reversed EV AGAP2-AS1 depletion-inhibited cancer cell proliferation in vitro. Consequently, we concluded that EV lncRNA AGAP2-AS1 contributed to cervical cancer cell proliferation through regulating the miR-3064-5p/SIRT1 axis. The clinical values of EV lncRNA AGAP2-AS1 and miR-3064-5p deserve to be explored in cervical cancer diagnosis and treatments.

Keywords: cervical cancer; extracellular vesicles; lncRNA AGAP2-AS1; miR-3064-5p; proliferation; sirtuin 1 (SIRT1).

Figure 1

Cervical cancer transferees AGAP2-AS1 by EVs. (A) The EVs from Hela and C33A cells were observed by TEM. (B) The expression of CD9 and CD63 was detected by western blot analysis in the EVs from Hela and C33A cells. (C) The size distribution of EVs was detected by using Nanoparticle Tracking Analysis (NTA). (D) The expression of AGAP2-AS1 was measured by qPCR in EVs from Hela and C33A cells’ culture medium treated with RNase A (1 μg/ml) or co-treated with RNase A (1 μg/ml) and Triton X100 (0.1%). (E) Immunofluorescence image of internalized EVs. Green: PKH67-labelled EVs; Red: Rhodamine staining of cytoskeleton; Blue: DAPI staining of nuclei. (F) The expression of AGAP2-AS1 was measured by qPCR in EVs from Hela and C33A cells treated with AGAP2-AS1 siRNA. (G) The Hela and C33A cells were treated with EVs from Hela and C33A cells transfected with AGAP2-AS1 siRNA or negative control (sicontrol). The expression of AGAP2-AS1 was measured by qPCR in Hela and C33A cells. (H) The Hela and C33A cells were treated with EVs from Hela and C33A cells transfected with siRNA control or PBS. The expression of AGAP2-AS1 was measured by qPCR. mean ± SD, **P < 0.01 vs Control, sicontrol, or PBS.

Figure 2

EV AGAP2-AS1 contributes to cervical cancer cell proliferation in vitro and in vivo. (A) The expression of AGAP2-AS1 was measured by qPCR in EVs from clinical cervical cancer tissues (n = 50) and the adjacent normal tissues (n = 50). (B–G) The Hela and C33A cells were treated with EVs from Hela and C33A cells transfected with AGAP2-AS1 siRNA. (B, C) The cell viability was detected by the MTT assays. (D, E) The cell proliferation was measured by Edu assays. (F, G) The cell apoptosis was evaluated by flow cytometry analysis. (H, I) The tumorigenicity analysis was performed in the nude mice. The nude mice (n = 5) were injected with Hela cells and treated with EVs from Hela cells transfected with AGAP2-AS1 siRNA. The tumor growth curve (H), tumor volume and tumor weight (I) were shown. (J) Relative levels of AGAP2-AS1 were detected in mice tumors by qPCR. mean ± SD, **P < 0.01.

Figure 3

AGAP2-AS1 interacts with miR-3064-5p. (A) The binding site between AGAP2-AS1 and miR-3064-5p was analyzed in ENCORI database. (B, C) The Hela and C33A cells were treated with miR-3064-5p mimic. The expression of miR-3064-5p was detected by qPCR (B) and the luciferase activity was measured by dual luciferase reporter assays (C). (D) The Hela and C33A cells were transfected with AGAP2-AS1 siRNA, and the expression of miR-3064-5p was examined by qPCR. (E, F) Ago2 RIP (E) and RNA pulldown (F) assay were performed to identify AGAP2-AS1 directly combined with miR-3064-5p. mean ± SD, **P < 0.01 vs Control or sicontrol.

Figure 4

SIRT1 is a target of miR-3064-5p. (A) The binding site between SIRT1 and miR-3064-5p was analyzed in ENCORI database. (B, C) The Hela and C33A cells were treated with miR-3064-5p mimic for 48 h. The expression of SIRT1 was detected by qPCR, (B) and the luciferase activity was measured by dual luciferase reporter assays (C). (D) The expression of SIRT1 was detected by western blot analysis in the Hela and C33A cells transfected with AGAP2-AS1 siRNA or co-treated with AGAP2-AS1 siRNA and miR-3064-5p inhibitor. mean ± SD, **P < 0.01 vs Control.

Figure 5

MiR-3064-5p/SIRT1 axis is involved in EV AGAP2-AS1-induced cervical cancer cell proliferation in vitro. (A–D) The Hela and C33A cells were treated with EVs from Hela and C33A cells transfected with AGAP2-AS1 siRNA or co-treated with the EVs and miR-3064-5p inhibitor or SIRT1 overexpression vectors. (A, B) The cell proliferation was measured by Edu assays. (C, D) The cell apoptosis was evaluated by flow cytometry analysis. mean ± SD, **P < 0.01.