lncRNA HOXA-AS2 functions as an oncogene by binding to EZH2 and suppressing LATS2 in Acute myeloid leukemia (AML)


 BackgroundLong noncoding RNAs (lncRNAs) plays an important role in the development of physiology and pathology. Many reports have shown that lncRNA HOXA cluster antisense RNA 2 (HOXA-AS2) is a carcinogen and plays an important role in many tumors, but little is known about its role in Acute myeloid leukemia (AML). MethodsThe expression of HOXA-AS2 in AML cell line was detected by qRT-PCR. AML cases from the public database (GEPIA) were also included in this study. Cell counting kit-8 (CCK-8) assay, flow cytometry, immunofluorescence and Western blot were used to detect the role of HOXA-AS2 in AML cells. Luciferase reporter gene detection, RIP, RNA pull-down and RNA-ChIP detection were used to demonstrate the molecular biological mechanism of HOXA-AS2 in AML.ResultsHOXA-AS2 was upregulated in AML cell lines and tissues, and the overexpression of HOXA-AS2 is negatively correlated with the survival of patients. Silencing HOXA-AS2 can inhibit the proliferation and induce differentiation of AML cells in vitro and in vivo. Overexpressing HOXA-AS2 showed the opposite result. Moreover, more in-depth mechanism studies showed that carcinogenicity of HOXA-AS2 exerted mainly through binding with the epigenetic inhibitor Enhancer of zeste homolog 2 (EZH2) and then inhibiting the expression of Large Tumor Suppressor 2 (LATS2). ConclusionsTaken together, our findings highlight the important role of HOXA-AS2 in AML, suggesting that HOXA-AS2 may be an effective therapeutic target for patients with AML.

lncRNA is likely to be a carcinogen gene [18,19] or a tumor suppressor gene [18] in the occurrence and development of tumors, and has increasingly become a biomarker of tumor diagnosis, treatment or prognosis, including AML [15,36]. Therefore, it is very important to identify more new cancer related lncRNAs and explore their biological functions and molecular mechanisms in order to explore new therapeutic methods of AML.
However, little is known about the role of HOXA-AS2 in the pathogenesis and development of AML. Our results showed that HOXA-AS2 was up-regulated in AML cells. TCGA database also showed that HOXA-AS2 expression was also up-regulated in AML tissues and was associated with poor prognosis in AML patients. Moreover, silencing of HOXA-AS2 not only inhibited the proliferation and induced differentiation of AML cells, but also reduced the tumorigenicity of nude mice. We further proved that HOXA-AS2 acts as a modular scaffold for histone modi ed complexes by binding with EZH2 to silence LATS2 expression. In conclusion, our results suggest that HOXA-AS2-EZH2-LATS2 axis may provide a new strategy for the diagnosis and treatment of AML.

Materials And Methods
Cell Lines and Culture NB4 and THP-1 cell lines were provided by Shanghai Gecko gene in 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) containing penicillin (100 U/ml) and streptomycin (100 g/ml) (Gibco, Grand Island, NY, USA). All cells were cultured in a humidi ed atmosphere, at 37 °C with 5% CO 2 . We changed the cell culture medium every 24 hours, and also subcultured the cells for 48 hours.
RNA extraction and qRT-PCR assays Trizol (Invitrogen, USA) was used to extract total RNA from tissues and cell lines, and then reverse transcripted using Primer-Script One Step RTPCR kit (TaKaRa, China). Finally, SYBR premix dimming eraser Kit (TaKaRa, China) was used for real-time RT-PCR detection. GAPDH was used as a standardized control. All measurements were repeated three times. The primer sequences are shown in Table 1.

Gene knockdown
Small interfering RNA (siRNA) and short hairpin RNA (shRNA) were used for knockdown of genes. Lipofectamine 2000 (Invitrogen) was used for transient transfection, and the correlation was performed 48 hours after transfection. We purchased HOXA-AS2 siRNA (siHOXA-AS2) and negative control siRNA (siNC) from Bioujing Bio (Shanghai, China). The primer interference sequences are shown in Table 1. HOXA-AS2 short-hairpin RNA (LV-shHOXA-AS2) and respective negative control (LV-shNC) were obtained from Bioujing Bio (Shanghai, China). First, NB4 and THP-1 cells were seeded in 24-well plates before being transfected. Then, 30 ml shRNA was added to each well, allowed to stand at room temperature for 15 min, and placed in a cell culture incubator; and the medium was changed after 24 h. The primer interference sequences are shown in Table 1.
Cell counting kit-8 (CCK-8) assays CCK-8 kit was used for detection (Beyotime Institute of Biotechnology, China). The transformed cells were inoculated into 96 well plate (1×10 3 cells/well), and then detected at 450 nm absorbance every 24 hours for 96 hours. Five multiple holes were taken for testing each time, and all the assays were conducted in triplicate.

Cell Cycle Analysis
The transfected cells were collected and xed with 70% precooled ethanol. Then according to the instructions of Shanghai Beibo biological cell cycle detection kit. Finally, cell cycle was detected by CytoFLEX (Becton Dickinson, USA) and analyzed by ModFit software.

Differentiation Marker Analysis
The detection methods of differentiation indicators (CD11b and CD14) has been presented in our previously published articles [11].

Western blotting
We rst used 8% SDS-PAGE to isolate the protein lysate, and then transferred the protein to 0.22um PDVF membrane. PDVF membrane was incubated with the corresponding primary antibody at 4 °C overnight, and then incubated with horseradish peroxidase labeled secondary antibody at room temperature. All the experiments were repeated three times

RNA immunoprecipitation (RIP)
The treated cells were collected and their nuclear proteins were extracted and then resuspended in RIP buffer. Then divide the resuspended RIP buffer into input group, IgG group and EZH2 group. Then collect the supernatant after centrifugation, add IgG (Abcam) or human anti-EZH2 antibody (Abcam), and then incubate at 4°C for 2 hours. Subsequently, protein A beads were added and incubated at 4°C for 1 hour. After centrifugation, the cells were washed 3 times with RIP buffer and then once with PBS, and the beads were resuspended in Trizol. Finally, quantitative detection was performed by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Luciferase Reporter Assay
Human LATS2 luciferase reporter gene plasmid was prepared and synthesized by Bioujing bio. The luciferase activities of re y and marine kidney were measured (Promega, Shanghai, China) consecutively 40~44 h after transfection. Finally, the luciferase activity of re y was quanti ed according to the luciferase standardization.

RNA Pull-Down Assay
Biotin-labeled RNA pull-down was performed as described previously (Xiang et al., 2014). In brief, 1 × 10 7 AML cells were harvested, lysed, and sonicated. Then, nuclear proteins were extracted using a nuclearplasma-isolation extraction kit and incubated with biotin-labeled HOXA-AS2 truncated probes and streptavidin agarose beads (Invitrogen) at 4 °C overnight. After washing with the wash buffer, the protein was recovered and detected by Western blot.

Chromatin Immunoprecipitation (ChIP)
ChIP analysis was carried out according to the manufacturer of EZ-ChIP kit (Upstate Biotechnology, Lake Placid, NY, USA). The nal results were determined by RT-qPCR. The primers were designed by the Premier Fluorescence in situ hybridization (FISH) was used to detect the location of HOXA-AS2 in the cells. The experimental method was similar to that reported before [11]. The cells were rst xed with 4% paraformaldehyde, then apply the cells on the anti-dropping glass slides to make droplets, then x and permeabilize the cells, then incubate with FISH probes, stain with DAPI, and nally mount the slides. Fluorescence imaging was analyzed by laser scanning confocal microscope. The probe sequence of HOXA-AS2 for the FISH analysis have been provided.

Tumor Xenografts
All animal care and experiments were carried out in accordance with the guidance of the National Institutes of Health and approved by the Ethics Committee of Anhui Medical University. We selected 4- week-old NCG mice with severe immunode ciency for tumor xenotransplantation experiments to study the effect of HOXA-AS2 on tumor proliferation and differentiation. In tumor growth assay in vivo, NB4 cells stably transfected with HOXA-AS2 short-hairpin RNA (LV-shHOXA-AS2) and respective negative control (LV-shNC) were subcutaneously injected into the upper back of the nude mice (1 × 10 7 , 200 μl). Eight weeks after inoculation, the cells were killed. Finally, mice were sacri ced, and subcutaneous tumor tissues were detected for tumor weight, WB and IHC staining.

Immunohistochemistry
Tumor tissue was xed in 4% paraformaldehyde and embedded in para n. After sectioning, it was incubated with CD11b antibody (Bioss, China), CD14 antibody (Bioss, China), Ki67 antibody (Bioss, China) and LATS2 antibody (CST, USA) overnight at 4 ° C. Then they were incubated with the second antibody and stained with diaminobenzidine. Photographs were taken with a light microscope.
Nitroblue tetrazolium (NBT) assay NB4 (1×10 5 cells/ml) was inoculated in a 6-well plate and treated with ATPR. A 10 μl aliquot of NBT solution, composed of 10 mg/ml NBT (Sigma-Aldrich) and 2 ug/ml PMA (Sigma-Aldrich), was added to each well, and then cells were incubated for 30 min at 37°C. The positive cells ratio was analysed by light light microscopy.

Statistical Analysis
Data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and Duncan test were used for comparison between multiple groups. If the p-value was less than 0.05, the difference is considered statistically signi cant. The experimental results were represented at least three independent experiments.

HOXA-AS2 levels were increased in AML
In order to explore the role of HOXA-AS2 in AML, we rst used GEPIA database to analyze the expression of HOXA-AS2 in AML patients. The results showed that the expression of HOXA-AS2 in AML tissues (left bar) was signi cantly higher than that in normal tissues (right bar) (Fig. 1a) (http://gepia.cancerpku.cn/detail.php). Furthermore, Kaplan Meier survival analysis showed that the overall survival rate of AML patients with high expression of HOXA-AS2 (n = 53) was signi cantly lower than the patients with low expression of HOXA-AS2 (n = 53) (Fig. 1b). qRT-PCR results showed that HOXA-AS2 was highly expressed in many leukemia cell lines (KG-1, NB4, U937, HL-60, and THP-1 cells) compared to the normal human monocytes (Fig. 1c). Based on the above results, we conducted a series of in vitro and in vitro studies to determine the function of HOXA-AS2 in AML.

HOXA-AS2 regulates AML cell proliferation
In order to further explore the biological function of HOXA-AS2 in AML cells, we choose the NB4 and THP-1 cell lines for further studies. The e ciency of silencing and overexpression was veri ed by qRT-PCR ( Fig. 1d, e). si-HOXA-AS2 1# and 2# demonstrated the better silencing capacity. HOXA-AS2 expression was increased in AML cell lines compared with the negative control following transfection with pCMV6-XL5-HOXA-AS2. Thus, we selected si-HOXA-AS2 1# and 2# and pCMV6-XL5-HOXA-AS2 in all following experiments. CCK8 results showed that silencing HOXA-AS2 could inhibit the proliferation of AML cells, but overexpression of HOXA-AS2 in AML cells had the opposite effect (Fig. 2a, b). Flow cytometry and Western blot were then used to demonstrate the above results. The results of ow cytometry showed that inhibition of HOXA-AS2 expression could induce G0 / G1 cycle arrest of AML cells, thus inhibit the proliferation of AML cells. On the contrary, overexpression of HOXA-AS2 had the opposite effect in AML cells (Fig. 2c, d). Overexpression of HOXA-AS2 in AML cells can increase the expression of G0/G1 marker protein (cyclin D3, cyclin A2, P-Rb and CDK4) (Fig. 2e). In contrast, our results also showed that silencing HOXA-AS2 could induce the decrease of G0/G1 marker protein (Fig. 2f). In order to further con rm the effect of HOXA-AS2 on the proliferation of AML cells, we used immuno uorescence to analyze the expression of Ki67 in the transfected AML cells (Fig. 2g, h). Western blot showed that silencing HOXA-AS2 inhibited Ki67 expression compared with that in control tumors (Fig. 2i). These results demonstrated that HOXA-AS2 expression levels were associated with AML proliferation.

HOXA-AS2 regulates AML cell differentiation
Our results have demonstrated that HOXA-AS2 can regulate the proliferation of AML. As we all known, AML is mainly caused by blocked differentiation. We used a series of experiments to prove the effect of HOXA-AS2 on differentiation. Many studies have shown that CD11b and CD14 are classic differentiation markers of leukemia. Flow cytometry showed that knockdown of HOXA-AS2 could induce the differentiation of AML cells (Fig. 3b). On the contrary, overexpression of HOXA-AS2 decreased the proportion of differentiated cells in AML cells compared with control cells (Fig. 3a). Subsequently, Western blotting showed that silencing HOXA-AS2 promoted the expression of CD11b and CD14 in AML cells (Fig. 3d), whereas overexpression of HOXA-AS2 resulted in decreased expression of CD11b and CD14 (Fig. 3c). The reduction of NBT showed that HOXA-AS2 knockdown signi cantly increased the NBT reduction in AML cells compared with NC group (Fig. 3e). In addition, shRNA knockdown of HOXA-AS2 could promote the expression of CD11b and CD14 (Fig. 3f). In conclusion, these data suggested that HOXA-AS2 can not only regulate the proliferation of AML, but also regulate the differentiation of AML.

HOXA-AS2 regulated LATS2 transcription in AML Cells
In order to study the role of LATS2 in AML, we next explored the relationship between HOXA-AS2 and LATS2. As shown in Figure 4a, silencing HOXA-AS2 signi cantly increased LATS2 protein levels compared with negative controls. On the contrary, overexpression of HOXA-AS2 signi cantly decreased the expression of LATS2 (Fig. 4b). The results of gene level showed that inhibition of HOXA-AS2 could promote the expression of LATS2 mRNA (Fig. 4c), and overexpression of HOXA-AS2 could inhibit the expression of LATS2 mRNA in AML cells (Fig. 4d). To further elucidate the speci c mechanism of HOXA-AS2-regulating LATS2 expression, we used a human LATS2 luciferase reporter gene plasmid for the luciferase reporter gene analysis. Knocking down of HOXA-AS2 can increase LATS2 promoter activity in AML cells (Fig. 4e), whereas overexpression of HOXA-AS2 had the opposite effect (Fig. 4f). These results suggested that the transcription of LATS2 in AML cells can be regulated by HOXA-AS2.

HOXA-AS2 regulated transcriptional expression of LATS2 through recruitment of EZH2 in AML cells
The expression of HOXA-AS2 was further con rmed by FISH. The nucleus was labeled with DAPI, and HOXA-AS2 was labeled with Cy3 and 18S rRNA (cytoplasmic positive). The results showed that HOXA-AS2 was located in the nuclei of AML cells (Fig. 5a). The next CHIP analysis results showed that overexpression of HOXA-AS2 can improve its binding ability to EZH2 (Fig. 5b). Many literatures have shown that EZH2 may be a key regulator of AML, therefore we silenced EZH2 expression using EZH2 siRNA. RT-qPCR and western blot results showed that the expression of EZH2 and LATS2 was negatively correlated, and silencing EZH2 could promote the expression of LATS2 ( Fig. 5c and d). Subsequently, RNA immunoprecipitation (RIP) analysis showed that EZH2 protein could bind to HOXA-AS2 (Fig. 5e).
The direct interaction between EZH2 and HOXA-AS2 was further demonstrated by applying the total protein (Fig. 5f) to the HOXA-AS2 pull-down assay. Taken together, we concluded that HOXA-AS2 regulated transcriptional expression of LATS2 through recruitment of EZH2 in AML cells.
LATS2 silencing potentially involves the oncogenic function of HOXA-AS2 Furthermore, to validate whether HOXA-AS2 regulates AML cell proliferation by silencing LATS2 expression, rescue assays were performed. AML cells were co-transfected with si-HOXA-AS2 and si-LATS2, and the CCK8 assay results indicated that co-transfection partially rescued si-HOXA-AS2damaged proliferation ability (Fig. 6a). In addition, the ki67 was determined by immuno uorescence staining analysis also con rmed this result (Figure 5b). Furthermore, ow cytometry analysis showed that the decreased LATS2 expression reversed the G0 / G1 arrest induced by HOXA-AS2 silencing (Fig. 6c).
Moreover, the western blot results indicated that co-transfection increased the downregulated expression of cyclin D3, cyclin A2, P-Rb and CDK4 triggered by the knockdown of HOXA-AS2 (Fig. 6d). Flow cytometry analysis showed that the decreased LATS2 expression reversed the higher level of differentiation resulted from HOXA-AS2 silencing (Fig. 6e). Moreover, the western blot results indicated that co-transfection decreased the upregulated expression of CD11b and CD14 induced by the knockdown of HOXA-AS2 (Fig. 6f). These results indicated that the effect of HOXA-AS2 on AML partially involves targeting LATS2.

Knockdown of HOXA-AS2 inhibits AML tumorigenesis in vivo
We have studied the in vitro study of HOXA-AS2 in AML cells, and the biological function of HOXA-AS2 in vivo. To further study the carcinogenic effect of HOXA-AS2 in AML, the control cells and shRNA HOXA-AS2 NB4 cells were injected into nude mice. Tumor volume was measured every week after injection, and the tumor tissue was removed 4 weeks later. The results showed that compared with the control group, silencing HOXA-AS2 signi cantly inhibited the tumor growth ( Fig. 7a and b). The tumor weight of HOXA-AS2 knockout group was signi cantly lower than that of control group (Fig. 7c). Moreover, RT-PCR analysis showed that the expression level of HOXA-AS2 in tumor was lower than that after shRNA transfection (Fig. 7d). In addition, immunohistochemistry and Western blot showed that HOXA-AS2 upregulated LATS2 protein ( Fig. 7e and f). In conclusion, these data suggested that HOXA-AS2 may affect the proliferation and differentiation of AML tumors in vivo, as well as in vitro experiments.

Discussion
With the continuous improvement and progress of experimental technology, more and more new lncRNAs have been discovered by RNA sequencing technology, which play a main role in the occurrence and development of diseases and malignant progress [2,25,38]. Many studies have shown that many lncRNAs express differently in AML and can regulate their functions [11,12,21,30]. However, whether the function and molecular mechanism of many lncRNAs are associated with AML is still unclear.
HOXA-AS2, as a new 1048 bp lncRNA, was rst reported to act as an inhibitor of apoptosis in NB4 promyelocytic leukemia cells treated with all trans retinoic acid [40], and has been reported to be associated with many types of malignant tumors [33]. Studies have shown that the high expression of HOXA-AS2 may be related to various biological processes of malignant tumors, such as apoptosis, invasion, migration, proliferation and so on [7,32,35,39]. We found that HOXA-AS2 is highly expressed in AML tissues and cell lines. The predicted results from the database website (GEPIA) showed that the expression level of HOXA-AS2 was negatively related to the survival time of AML patients. In the current study, we found that silencing HOXA-AS2 in AML cells inhibited cell proliferation and induced differentiation. Overexpression of HOXA-AS2 had the opposite biological function. In vivo experiments also showed that knockout of HOXA-AS2 can inhibit tumor growth.
As a highly conserved histone methyltransferase, EZH2 is a subunit of PRC2, which plays a regulatory role mainly by triggering H3K27me3 trimethylation [reference]. It can inhibit the translation of many target genes and regulate cell cycle regulation, aging, cell proliferation, differentiation, apoptosis and tumorigenesis [20,29]. Many studies have shown that lncRNA binds with EZH2 to exert its biological functions by regulating the expression of downstream genes [5,34]. Our recent results showed that ATPR can up-regulate the expression of lncRNA NR-104098 and inhibit the differentiation and proliferation of AML [11]. The in-depth exploration revealed that lncRNA NR-104098 played a role by enhancing the binding of E2F1 to EZH2 promoter [11]. Our results indicated that HOXA-AS2 can directly bind to EZH2 and inhibit the proliferation and induce differentiation of AML by regulating the expression of LATS2.
As a con rmed tumor suppressor [31], LATS2 plays an anti-tumor role through different signaling pathways [14,16,17]. Our ChIP results con rmed that EZH2 can directly bind to LATS2 promoter region in AML to regulate the expression of XXX. In addition, further rescue experiments showed that HOXA-AS2 played its biological function through LATS2. Silence of LATS2 can reverse this behavior.

Conclusions
In conclusion, the up-regulation of HOXA-AS2 may be associated with the negative prognosis of AML patients. This study rst reported the high expression of HOXA-AS2 in AML tissues and cells. Silencing HOXA-AS2 can inhibit the tumorigenesis of nude mice, inhibit its proliferation and induce its differentiation in AML cells. Moreover, HOXA-AS2 inhibited the expression of LATS2 by binding with EZH2. Our ndings suggested that HOXA-AS2 may be a new target for AML therapy, and provide a new hope for the diagnosis and treatment of AML targeting lncRNA. HOXA-AS2 downregulated transcriptional expression of LATS2 through recruitment of EZH2 in AML cells.
a HOXA-AS2 distribution in NB4 and THP-1 cells was detected by FISH. b HOXA-AS2 plasmid was transfected into NB4 and THP-1 cells, and chrome immunoprecipitations were performed by using speci c anti-EZH2 antibodies. c NB4 and THP-1 cells transfected with EZH2 siRNAs or the control siRNA for 72 h were collected, and EZH2 and LATS2 protein levels were detected by Western-blot assay. d EZH2 siRNAs (EZH2-1, 2) or the control siRNA were transfected into NB4 and THP-1 cells for 48 h, EZH2 and LATS2 mRNA levels were then assessed by RT-qPCR. e RNA immunoprecipitations were performed in NB4 and THP-1 cells, and the relative quantities of HOXA-AS2 were detected by RT-qPCR assay, normalized to the input groups. IgG and EZH2 represented for the groups coprecipitation with IgG protein and anti-EZH2 antibody respectively. f Total proteins were extracted from NB4 and THP-1 cells, and then HOXA-AS2 pull-down assay was performed. The EZH2 protein levels were evaluated by Western-blot. HOXA-AS2 probe represented the biotin-labeled HOXA-AS2 probe group and control stood for the oligo probe group.
*p < 0.05, **p < 0.01 Figure 6 Silencing LATS2 potentially involves the oncogenic function of HOXA-AS2. a CCK8 assay was performed to determine the cell viability for si-HOXA-AS2 and si-LATS2 co-transfected NB4 and THP-1 cells. b The cell cycle of transfected NB4 and THP-1 cells. c Western blot analysis of cyclin D3, cyclin A2, P-rb, and CDK4 after si-HOXA-AS2 and si-LATS2 co-transfection in NB4 and THP-1 cells. β-actin protein was used as an internal control. d The level of ki67 protein in NB4 and THP-1 cells co-transfection with si-HOXA-AS2 and si-LATS2 was detected by immuno uorescence. e The levels of CD11b and CD14 protein in NB4 and THP-1 cells co-transfection with si-HOXA-AS2 and si-LATS2 were detected by ow cytometry analysis. f Western blot analysis of CD11b and CD14 after si-HOXA-AS2 and si-LATS2 co-transfection in