Long non-coding RNA AGAP2-AS1 exerts oncogenic properties in glioblastoma by epigenetically silencing TFPI2 through EZH2 and LSD1

AGAP2-AS1 expression was up-regulated in GBM and positively correlated with poor prognosis

According to the data from bioinformatics tool GEPIA (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=boxplot), AGAP2-AS1 expression was higher in 163 GBM tissues than that in 207 normal tissues (Figure 1A). In order to validate this result, qRT-PCR was performed to measured AGAP2-AS1 expression in 58 paired GBM tumor tissues and adjacent normal tissues. The results showed an increased AGAP2-AS1 expression in tumor tissues versus matched histologically non-cancerous tissues (Figure 1B). Also, we detected endogenous expression of AGAP2-AS1 in various GBM cell lines (A172, U87/MG, U251/MG, LN229, SHG44) and normal human astrocytes (NHA). As expected, AGAP2-AS1 expression was significantly up-regulated in GBM cells when compared with NHA (Figure 1C). Moreover, GEPIA data displayed that patients with high AGAP2-AS1 expression had a shorter overall survival than those with low AGAP2-AS1 level (Figure 1D). All these results indicated that AGAP2-AS1 was up-regulated and associated with poor prognosis in GBM.

Figure 1. AGAP2-AS1 expression is overexpressed in GBM and correlated with poor prognosis of GBM patients. (A) AGAP2-AS1 expression in GBM tissues (n=163) and normal tissues (n=207) in from GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene=&clicktag=boxplot). (B) qRT-PCR analysis of AGAP2-AS1 expression in tumor tissues and matched surrounding tissues from 58 patients with GBM. (C) qRT-PCR analysis of AGAP2-AS1 enrichment in five GBM cells (A172, U87/MG, U251/MG, LN229, SHG44) and normal human astrocytes (NHA). (D) Kaplan-Meier analysis of overall survival in GBM patients according to AGAP2-AS1 expression levels. *P < 0.05, **P < 0.01, ***P < 0.001.

Knockdown of AGAP2-AS1 suppressed proliferation and invasion, and facilitated apoptosis in GBM cells

To explore the functional relevance of AGAP2-AS1 in GBM cells, we interfered endogenous AGAP2-AS1 expression in U87/MG and U251/MG cells by transfection with specific siRNA, and increased AGAP2-AS1 expression in A172 cells by transfection with one overexpression plasmid (Figure 2A). CCK-8 assays showed that knockdown of AGAP2-AS1 impaired the growth ability of U87/MG and U251/MG cells, whereas overexpression of AGAP2-AS1 promoted the proliferation capability of A172 cells (Figure 2B). EdU staining assay manifested that the proliferation potential was suppressed in U87/MG and U251/MG cells following down-regulation of AGAP2-AS1 (Figure 2C and 2D), while AGAP2-AS1 up-regulation increased A172 cell proliferation (Figure 2E). Colony formation assay demonstrated that the clonogenic ability was reduced in U87/MG and U251/MG cells after inhibiting AGAP2-AS1 expression (Figure 2F and 2G), but was enhanced in AGAP2-AS1-overexpressing A172 cells (Figure 2H). Transwell assays presented that depletion of AGAP2-AS1 resulted in a suppression of invasive ability in U87/MG and U251/MG cells (Figure 2I and 2J), while a promotion of invasiveness in AGAP2-AS1-transfected A172 cells (Figure 2K). Flow cytometry assays also revealed that silencing of AGAP2-AS1 greatly induced apoptosis in U87/MG and U251/MG cells (Figure 2L and 2M). All these data suggested the oncogenic role of AGAP2-AS1 in GBM progression in vitro.

Figure 2. Silencing of AGAP2-AS1 represses proliferation and invasion, and promoted apoptosis in GBM cells. U87/MG and U251/MG cells were transfected with si-NC or si-AGAP2-AS1, while A172 cells were introduced with empty vector or pcDNA-AGAP2-AS1. (A) qRT-PCR analysis of transfection efficiency in U87/MG, U251/MG and A172 cells. (B) CCK-8 analysis was conducted to detect the viability in transfected U87/MG, U251/MG and A172 cells. (C–E) (d) EdU staining assay was performed to evaluate the proliferation ability in transfected U87/MG, U251/MG and A172 cells. Red, EdU staining for dividing cell; blue, DAPI staining for nuclear. (F–H) Colony-forming assay was used to examine the cloning ability in transfected U87/MG, U251/MG and A172 cells. (I–K) Transwell assay was carried out to assess the invasiveness in transfected U87/MG, U251/MG and A172 cells. (L and M) Flow cytometry analysis was applied to determine the apoptotic rate in transfected U87/MG, U251/MG and A172 cells. *P < 0.05, **P < 0.01.

AGAP2-AS1 inhibited TFPI2 transcription by binding with LSD1 and EZH2 in GBM cells.

It is well known that lncRNAs are able to regulate cell phenotypes through interacting with specific RNA-binding proteins. To examine the potential biological mechanisms of AGAP2-AS1 involved in GBM cells, subcellular fractionation assays were performed to determine the distribution of AGAP2-AS1 in nuclear and cytoplasmic fractions in GBM cells. Results revealed that AGAP2-AS1 was mainly located in the nucleus of U87/MG and U251/MG cells (Figure 3A), indicating that AGAP2-AS1 may exert regulatory effects at transcriptional levels. Then, RIP assays were used to analyze the possible RNA-binding proteins of AGAP2-AS1 in GBM cells. As presented in Figure 3B, AGAP2-AS1 could directly bind with EZH2 and LSD1 in U87/MG and U251/MG cells. Moreover, RNA pull-down assays showed that EZH2 and LSD1 in the nuclear extract fraction of U87/MG and U251/MG cells were pulled down by labeled AGAP2-AS1 (Figure 3C), further confirming the binding between AGAP2-AS1 and EZH2 or LSD1.

Figure 3. AGAP2-AS1 recruits EZH2 and LSD1 to suppress TFPI2 expression. (A) qRT-PCR analysis of AGAP2-AS1 level in the nuclear and cytoplasmic fraction of U87/MG and U251/MG cells. GAPDH and U6 were used as the cytoplasm marker and nucleus marker, respectively. (B) RIP experiments were conducted in U87/MG and U251/MG cells using antibodies against EZH2, SUZ12, WDR5, LSD1, DNMT1, and CoREST, followed by qRT-PCR assay of AGAP2-AS1 levels in immunoprecipitates. The fold enrichment was relative to IgG immunoprecipitate. (C) HuR, EZH2, and LSD1 protein levels in immunoprecipitates with AGAP2-AS1 were determined in U87/MG and U251/MG cells by western blot. Androgen receptor (AR) RNA was used as a positive control for HuR protein. (D) qRT-PCR analysis was performed to evaluate the levels of P15, P21, KLF2, PTEN, TFPI2, LATS2, E-cadherin and RND3 in si-NC- or si-AGAP2-AS1-transfected U87/MG and U251/MG cells. (E) Western blot analysis of TFPI2 expression in U87/MG and U251/MG cells following knockdown of AGAP2-AS1. (F and G) TFPI2 expression level at mRNA and protein levels were detected in U87/MG and U251/MG cells after transfection with si-EZH2 or si-LSD1. (H) ChIP assay of EZH2/LSD1 occupancy and H3K27me3/H3K4me2 binding in the TFPI2 promoter in U87/MG and U251/MG cells after transfection with si-NC or si-AGAP2-AS1. Enrichment was quantified relative to input control. IgG was used as a negative control. *P < 0.05, **P < 0.01.

Next, we selected several EZH2 or LSD1 potential targets (P15, P21, KLF2, PTEN, TFPI2, LATS2, E-cadherin, and RND3) with tumor-suppressive role, and hypothesized that some of which may be associated with AGAP2-AS1-mediated carcinogenicity. qRT-PCR results disclosed that TFPI2 expression was significantly up-regulated in both U87/MG and U251/MG cells when AGAP2-AS1 was knocked down (Figure 3D). Western blot analysis also clarified an increase of TFPI2 protein level in AGAP2- AS1-depleted U87/MG and U251/MG cells (Figure 3E). Meanwhile, knockdown of EZH2 or LSD1 resulted in en evident up-regulation of TFPI2 expression at mRNA and protein levels (Figure 3F and 3G). These results implied that TFPI2 might be a downstream mediator of AGAP2-AS1.

To further address whether AGAP2-AS1 inhibited TFPI2 expression through interacting with EZH2 and LSD1, ChIP analysis was conducted in U87/MG and U251/MG cells. Results illuminated that AGAP2-AS1 could recruit EZH2 and LSD1 to the TFPI2 promoter region, resulting in trimethylation of H3K27 or demethylation of H3K4 at this region (Figure 3H). However, silencing of AGAP2- AS1 lowered their binding ability and H3K27me3 or H3K4me2 modification (Figure 3H). In summary, AGAP2-AS1 epigenetically suppressed TFPI2 expression by recruiting EZH2 and LSD1 to their promoter region in GBM cells.

Overexpression of TFPI2 decreased proliferation and invasion, and induced apoptosis in GBM cells

Subsequently, we further explored the functions of TFPI2 in GBM cells by transfection with TFPI2-overexpression plasmid (TFPI2). As displayed in Figure 4A and 4B, TFPI2 mRNA and protein levels were obviously increased in U87/MG and U251/MG cells after transfection with pcDNA-TFPI2. CCK-8 and EdU staining assays demonstrated that up-regulation of TFPI2 led to a remarkable decline of proliferation ability in U87/MG and U251/MG cells (Figure 4C and 4D). Colony formation assay confirmed the suppressive effect of TFPI2 overexpression on clonogenic ability (Figure 4E). Transwell assay declared that enforced expression of TFPI2 dramatically reduced the invasive ability of U87/MG and U251/MG cells (Figure 4F). Flow cytometry analysis highlighted a significant increase of apoptotic rate in U87/MG and U251/MG cells upon TFPI2 overexpression (Figure 4G). Together, TFPI2 inhibited cancer phenotype in GBM in vitro.

Figure 4. TFPI2 up-regulation represses proliferation and invasion, and enhanced apoptosis in GBM cells. (A and B) TFPI2 mRNA and protein levels were evaluated in U87/MG and U251/MG cells after transfection with empty vector or pcDNA-TFPI2. (C and D) CCK-8 and EdU staining assays were performed to examine the effects of TFPI2 overexpression on U87/MG and U251/MG cell proliferation. (E) Colony forming assay was conducted to detect the cloning ability in Vector- or pcDNA-TFPI2-transfected U87/MG and U251/MG cells. (F) Transwell assay of invasive ability in U87/MG and U251/MG cells following TFPI2 up-regulation. (G) Flow cytometry analysis of apoptotic rate in U87/MG and U251/MG cells introduced with empty vector or pcDNA-TFPI2. *P < 0.05, **P < 0.01.

AGAP2-AS1 exerts oncogenic activity by silencing TFPI2 in GBM cells

To gain insight into whether TFPI2 was involved in AGAP2-AS1-mediated oncogenic functions, rescue experiments were conducted in U87/MG cells by co-transfection with si-AGAP2-AS1 and si-TFPI2. Results revealed that si-AGAP2-AS1-induced increase of TFPI2 expression was greatly abrogated by TFPI2 knockdown in U87/MG cells (Figure 5A). Moreover, silencing of TFPI2 partly reversed the anti-proliferation (Figure 5B and 5C), anti-invasion (Figure 5D) and pro-apoptosis (Figure 5E) effects caused by AGAP2-AS1 knockdown. These data suggested that AGAP2-AS1 contributed to GBM progression possibly by down-regulating TFPI2.

Figure 5. AGAP2-AS1 promotes GBM progression partially through epigenetically inhibiting TFPI2 expression in vitro. U87/MG cells were transfected with si-NC, si-AGAP2-AS1, or co-transfected with si-AGAP2-AS1 and si-TFPI2. (A) Western blot analysis of TFPI2 protein level in transfected cells. (B) CCK-8 assay of proliferation ability in transfected cells. (C) Colony formation assay of cloning ability in transfected cells. (D) Transwell assay of invasiveness in transfected cells. (E) Flow cytometry analysis of apoptosis in transfected cells. *P < 0.05, **P < 0.01.

Knockdown of AGAP2-AS1 inhibited GBM tumorigenesis in vivo

To further address the biological significance of AGAP2-AS1 in tumor growth in vivo, sh-NC or sh-AGAP2-AS1 stably transfected U87/MG cells were subcutaneously injected into the left flank of nude mice. Results showed that silencing of AGAP2-AS1 obviously slowed down the tumor growth (Figure 6A). Moreover, the weights were decreased in tumors developed from sh-AGAP2-AS1-transfected U87/MG cells (Figure 6B). Additionally, knockdown of AGAP2-AS1 triggered a reduction of AGAP2-AS1 expression (Figure 6D), while an increase of TFPI2 protein level (Figure 6E) in excised tumor masses. In all, depletion of AGAP2-AS1 impaired tumor xenograft possibly by modulating TFPI2.

Figure 6. Silencing of AGAP2-AS1 suppresses GBM growth in vivo. U87/MG cells stably transfected with sh-NC or sh-AGAP2-AS1 were inoculated into nude mice (n=6). (A) Tumor volumes were monitored from the seventh day after cell injection. (B and C) At day 27 after cell inoculation, the tumors were removed, photographed and weighed. (D) qRT-PCR analysis of AGAP2-AS1 expression in tumors. (E) Western blot analysis of TFPI2 expression in tumors. *P < 0.05, **P < 0.01.

Patients and tissue samples

GBM tumor tissues and matched non-cancerous tissues were obtained from 58 GBM patients diagnosed by experienced pathologists at the Department of Neurosurgery at the First Affiliated Hospital of Zhengzhou University. No local or systemic treatment was administrated to patients before surgery. Fresh specimens were immediately snap frozen in liquid nitrogen and then stored at -80°C. The protocol used in this study was approved by the Research Ethic Committee of the First Affiliated Hospital of Zhengzhou University. All participants provide written informed consents prior to this study.

Cell lines and culture conditions

GBM cell lines (A172, U87/MG, U251/MG, LN229, and SHG44) and normal human astrocytes (NHA) were purchased from the Institute of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS at 37°C with 5% CO₂.

Transfection

To overexpress AGAP2-AS1 or TFPI2, the full length cDNA sequences of AGAP2-AS1 or TFPI2 were amplified and subcloned into pcDNA3.1 vector (Invitrogen) according to the manufacture’s guidelines. To suppress gene expression, small interfering RNAs (siRNAs) including si-AGAP2-AS1, si-EZH2, si-LSD1 and si-TFPI2 were purchased from RiboBio (Guangzhou, China). GBM cell were seeded into six-well plates and transfected with plasmids, siRNAs or corresponding negative control (Vector or si-NC) using Lipofectamine 2000 (Invitrogen).

RNA extraction and qRT-PCR

Total RNA was extracted from frozen tissues and cultured cells using TRIzol reagent (Invitrogen) according to the manufacture’s protocol. Then, 1 μg of RNA was reversely transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). qRT-PCR reaction was performed using SYBR Green Master Mix (TaKaRa Bio, Otsu, Japan) on an ABI Prism 7900 Sequence Detection System (Applied Biosystems). The primer sequences used were listed as follows: AGAP2-AS1, 5′-TACCTTGACCTTGCTGCTCTC-3′ (Forward) and 5′-TGTCCCTTAATGACCCCATCC-3′ (Reverse); TFPI2, 5′-CTGGGGCTGTCGATTCTGC-3′ (Forward) and 5′-TCTCCGCGTTATTTCCTGTTG-3′ (Reverse); GAPDH, 5′-CGCTCTCTGCTCCTCCTGTTC-3′ (Forward) and 5′-ATCCGTTGACTCCGACCTTCAC-3′ (Reverse). The expression levels of AGAP2-AS1 and TFPI2 mRNA were normalized to GAPDH.

Cell proliferation analysis

Cell proliferation was detected using a Cell Counting Kit-8 (CCK-8; Beyotime, Beijing, China) in accordance with the manufacture’s specification. GBM cells were plated into 96-well plates after transfection and counted every 24 h. The optical absorbance at 450 nm was measured. Cell viability was also determined with the 5-ethynyl-2′-deoxyuridine (EdU) assay using Cell-light EdU Apollo 567 In Vitro Imaging Kit (Ribobio, Guangzhou, China) following the manufacturer’s instructions. For the colony formation assay, cells were plated in 6-well plates and cultured in medium with 10% FBS to allow colony formation. The medium was replenished every 4 days. Two weeks later, cells were fixed with 96% ethanol and stained with 0.1% crystal violet. Colony formation ability was evaluated by counting the number of colonies consisting of more than 50 cells.

Transwell assay of cell invasion

Cell invasion assays were performed using a transwell chamber (Corning Incorporated, Corning, NY, USA) pre-coated with Matrigel (BD Biosciences, Franklin Lakes, CA, USA). Briefly, 1 × 10⁵ GBM cells in serum-free RPMI 1640 medium were seeded on the top side of upper chamber, while medium containing 10% FBS was added into the lower chamber. Following 24 h of incubation, cells on the upper membrane were removed with a cotton swab. Cells that passed through the membrane were fixed with methanol and stained with 0.1 % crystal violet.

Flow cytometric analysis of cell apoptosis

FITC Annexin V Apoptosis Detection Kit (BD Biosciences) was used to stain GBM cells according to the manufacturer’s recommendations. Then, cell apoptosis was measured by a FACScan flow cytometry (BD Biosicences).

Western blot analysis

Protein lysates were separated by 10% SDS-PAGE gel, transferred to 0.22 μm nitrocellulose (NC) membrane (Millipore, Bedford, MA, USA), and then incubated with primary antibodies against EZH2 (Cell Signaling Technology, Beverly, MA, USA), LSD1 (Cell Signaling Technology), TFPI2 (Abcam, Cambridge, MA, USA) and GAPDH (Cell Signaling Technology). Immunoreactive bands were visualized by chemiluminescence, and protein expression was normalized to that of GAPDH.

Subcellular fractionation location

PARIS Kit (Life Technologies, Carlsbad, CA, USA) was used to separate the nuclear and cytosolic fractions of GBM cells according to the manufacturer’s instructions. The levels of AGAP2-AS1, GAPDH and U6 RNA in cytoplasm and nuclear fraction were measured using qRT-PCR assay. GAPDH was used as the cytoplasm control, and U6 was used as the nuclear control.

RNA immunoprecipitation (RIP)

RIP assay was performed to investigate whether AGAP2-AS1 could interact with the potential binding protein (EZH2, LSD1, and SUZ12 et al.) in GBM cells using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore). Cells were scraped off and lysed in complete RIP lysis buffer. Then, the whole cell extract was incubated with magnetic beads coupled with specific antibodies or control IgG (Cell Signaling Technology). Subsequently, beads were washed and incubated with Proteinase K to remove proteins. Finally, the co-precipitated RNAs were purified and analyzed by qRT-PCR.

RNA pull down assays

AGAP2-AS1 and antisense-AGAP2-AS1 were in vitro transcribed with TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA), purified with RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and then treated with RNase-free DNase I (Qiagen). Purified RNAs were biotin-labeled using Biotin RNA Labeling Mix (Roche Diagnostics, Indianapolis, IN, USA). Subsequently, biotinylated RNAs were mixed with magnetic beads and incubated with cell lysates. Finally, the beads were washed, and the eluted proteins were analyzed by western blot.

Chromatin immunoprecipitation (ChIP)

ChIP assays were performed using the MagnaChIP Kit (Millipore) following the manufacturer's protocol. Cells were cross-linked with 1% formaldehyde, lysed and sonicated to acquire the DNA fragments of 200-500 bp. Then, the chromatin was immunoprecipitated with antibodies against EZH2, LSD1, H3K27me3, H3K4me2 or control IgG (Cell Signaling Technology). Precipitated chromatin DNA was purified and detected by qRT-PCR. The primer sequences for TFPI2 promoter region amplification are 5′-GGATGTTTGTTTTGTATAAAGTG-3′ (Forward) and 5′-AAACATCCAAAAAAACACCTAAC-3′ (Reverse). ChIP data is shown as a percentage relative to input DNA.

Tumor xenograft model

Five-week-old male BALB/C nude mice were purchased from the Shanghai Sippr/BK Laboratory Animal Co. (Shanghai, China). U87/MG cells with nontarget sh-NC or sh-AGAP2-AS1 stable transfection were subcutaneously injected into the left flank of mice. Tumor growth was assessed every 4 days, and tumor volumes were calculated using the following formula: 0.5 × length × width². After 27 days, the mice were sacrificed, and the tumors were removed, photographed and weighed. Tissue masses were also harvested for qRT-PCR, and western blot. This study complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Ethics Committee of Animal experiments of the First Affiliated Hospital of Zhengzhou University.

Statistical analysis

All experiments were conducted at least three times. All experimental data were presented as mean ± SD. The Student’s t-test or one-way ANOVA was performed to examine the difference between groups by using SPSS 20.0 software (SPSS, Chicago, IL, USA). P < 0.05 denotes a significant difference.