lncRNA CEBPA-AS1 Overexpression Inhibits Proliferation and Migration and Stimulates Apoptosis of OS Cells via Notch Signaling

Peng Xia; Rui Gu; Wei Zhang; Yi-Fu Sun

doi:10.1016/j.omtn.2019.10.017

This article has been retracted.

Retraction in: Mol Ther Nucleic Acids. 2022 Jun 22;29:75 See also: PMC Retraction Policy

. 2019 Oct 24;19:1470–1481. doi: 10.1016/j.omtn.2019.10.017

lncRNA CEBPA-AS1 Overexpression Inhibits Proliferation and Migration and Stimulates Apoptosis of OS Cells via Notch Signaling

Peng Xia ¹, Rui Gu ², Wei Zhang ¹, Yi-Fu Sun ^2,^∗

PMCID: PMC7056609 PMID: 32160715

Abstract

Osteosarcoma (OS) is the most common primary bone malignancy derived from primitive bone-forming mesenchymal cells. Long noncoding RNA (lncRNA) expression profiles have been intensively studied for their involvement in OS. Herein, we clarify whether lncRNA CEBPA-AS1 is a regulator of NCOR2 in OS cells. Microarray-based expression analysis identified OS-related differentially expressed lncRNA and predicted microRNAs (miRs) binding to lncRNA and mRNA. lncRNA CEBPA-AS1 and NCOR2 were found to be weakly expressed in OS tissues and cells. Next, functional investigation revealed that lncRNAs CEBPA-AS1 bound to miR-10b-5p to upregulate NCOR2. Following that, gene-targeted knockdown and overexpressed recombinant vectors of lncRNA CEBPA-AS1 and NCOR2 were constructed to explore the effects of lncRNA CEBPA-AS1 and NCOR2 on cell proliferation, differentiation, migration, and apoptosis. Finally, tumor formation was measured in nude mice. lncRNA CEBPA-AS1 overexpression or NCOR2 elevation inhibited cell proliferation and migration, and alkaline phosphatase (ALP) and bone gla protein (BGP) activity, while enhancing apoptosis and tumor formation. Furthermore, NCOR2 was elevated in response to lncRNA CEBPA-AS1 overexpression, thus repressing the Notch signaling pathway. Taken together, lncRNA CEBPA-AS1 overexpression inhibits OS progression through diminishing activation of the Notch signaling pathway via upregulating NCOR2. Therefore, lncRNA CEBPA-AS1 may serve as a molecular target for treating OS.

Keywords: long noncoding RNA, CEBPA-AS1, Notch signaling pathway, NCOR2, osteosarcoma, proliferation, migration, apoptosis

Graphical Abstract

Introduction

Osteosarcoma (OS) is the most predominant malignant skeletal tumor occurring in children and young adults, with approximately 400–900 people diagnosed with OS in the United States.¹ Although children represent the primary victims of OS, its incidence has been reported to be characterized by a bimodal age distribution, with two peaks: the age from 10 to 14 years and that beyond 60 years.² The therapeutic regimens for OS include surgery, chemotherapy, and radiotherapy, all of which contribute remarkably to a 5-year survival rate ranging from 60% to 70%; however, approximately 30%–40% of patients still suffer from pulmonary metastasis or death posttreatment.³ More recently, accumulating investigations have been conducted to identify novel targets for treatment of OS, with the hope of attaining greater curative efficacy by identifying tumor pathways and mediators associated with the progression of OS.³^,⁴

Long noncoding RNAs (lncRNAs) are longer than 200 nt that do not possess protein-coding functions due to the absence of an open reading frame of significant length.⁵ lncRNAs have been reported to play a pivotal role in multiple cellular processes including cell growth, migration, and metastasis.⁶^,⁷ lncRNA CEBPA-AS1 is a recently discovered gene, as per the GEO database, which is downregulated in the GEO: GSE16088 expression profile of human OS. Ke et al.⁸ implicated decreased lncRNA CEBPA-AS1 expression in gastric cancer (GC) tissues by means of microarray analysis, highlighting its potential cancer-suppressive abilities. lncRNAs have been reported to serve as competing endogenous RNAs (ceRNAs) of microRNAs (miRNAs or miRs), and thus influence tumorigenesis.⁹ Notably, miR-10b-5p has been demonstrated to be involved in the tumorigenesis in renal cell carcinoma.¹⁰ Additionally, nuclear receptor corepressor 2 (NCOR2) has been verified as a direct target of miR-10a/b during neuroblastoma cell differentiation.¹¹ NCOR2, also known as silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), mediates transcription repression in diverse differentiation-relevant pathways, and it has been highlighted as a promising therapeutic target in the treatment of human malignancies.¹²^,¹³ It is important to note that the Notch pathway has been identified as a critical therapeutic target for the treatment of various cancers, with its activity implicated in the occurrence and development of numerous malignancies.¹⁴ For instance, existing literature suggests that dysregulation of the Notch signaling pathway plays a stimulatory functional role in OS.¹⁵ Hence, the present study set out to investigate the mechanism by which lncRNA CEBPA-AS1 influences OS cell proliferation, differentiation, and apoptosis in connection with miR-10b-5p and NCOR2.

Results

lncRNA CEBPA-AS1 and NCOR2 Are Poorly Expressed in OS

Microarray-based gene expression profiling was initially analyzed to further identify the expression of lncRNA CEBPA-AS1 in OS. Microarray analysis of the dataset GSE41445 revealed that the lncRNA CEBPA-AS1 was poorly expressed in three OS specimens (Figure 1A). The Multi Experiment Matrix (MEM) website predicted that lncRNA CEBPA-AS1 and NCOR2 were co-expressed in OS and participated in the Notch signaling pathway (Table 1). In order to verify this prediction, clinical samples were collected, with qRT-PCR and western blot analysis performed to determine the expression of lncRNA CEBPA-AS1, as well as the mRNA and protein expression levels of NCOR2 in normal tissues and OS tissues. The results revealed that compared with the normal tissues, lncRNA CEBPA-AS1 expression and the mRNA and protein expression of NCOR2 were reduced among the OS tissues (Figures 1B and 1C). qRT-PCR was performed again to determine lncRNA CEBPA-AS1 expression, as well as the mRNA expression of NCOR2 in four OS cell lines (MG63, HOS, G292, and SAOS-2). The results revealed that relative to cell lines HOS and SAOS-2, lncRNA CEBPA-AS1 expression and the mRNA expression of NCOR2 were lower in cell lines MG63 and G292 (Figure 1D). Thus, cells lines MG63 and SAOS-2 were selected for subsequent experiments.

Table 1.

Co-expression Gene of lncRNA CEBPA-AS1 and Its Regulatory Pathway

Gene	Pathway
ADCY1; TACR1	calcium signaling pathway
NCOR2	Notch signaling pathway
ADCY1; CAMK2B	aldosterone synthesis and secretion
ADCY1; LMNA	dilated cardiomyopathy (DCM)
ADCY1; CAMK2B	circadian entrainment
ADCY1; CAMK2B	Cushing syndrome
CNGB1; GRK1	phototransduction
ADCY1; ASIC4	inflammatory mediator regulation of TRP channels
CACNG4; LMNA	hypertrophic cardiomyopathy (HCM)

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ADCY1, adenylate cyclase 1; ASIC4, acid sensing ion channel subunit family member 4; CACNG4, calcium voltage-gated channel auxiliary subunit gamma 4; CAMK2B, calcium/calmodulin-dependent protein kinase II beta; CNGB1, cyclic nucleotide gated channel beta 1; GRK1, G protein-coupled receptor kinase 1; LMNA, lamin A; NCOR2, nuclear receptor corepressors 2; TACR1, tachykinin receptor 1.

lncRNA CEBPA-AS1 Overexpression Inhibits OS Cell Migration by Elevating NCOR2

In order to investigate the effect associated with lncRNA CEBPA-AS1 and NCOR2 on OS cell migration, the expression of lncRNA CEBPA-AS1 and NCOR2 was elevated in MG63 cells and silenced in SAOS-2 cells (Figure S1).

A Transwell assay was conducted to detect the migration of the MG63 and SAOS-2 cells (Figures 2A and 2D), the results of which revealed that compared with the blank group, the migration rate of the SAOS-2 cells did not differ in the empty vector group (p > 0.05), whereas an elevated migration rate was observed in the small interfering (si) RNA (siRNA) NCOR2 and siLncRNA CEBPA-AS1 groups (p < 0.05). In contrast with the siLncRNA CEBPA-AS1 group, the migration rate of the SAOS-2 cells decreased in the siLncRNA CEBPA-AS1 + oeNCOR2 group. However, in the MG63 cells, overexpressed lncRNA CEBPA-AS1 and elevated NCOR2 were identified to have an inhibitory effect on the migration of the MG63 cells, whereas silenced NCOR2 was determined to reverse the inhibitory effect of overexpressed lncRNA CEBPA-AS1. qRT-PCR and western blot analysis were subsequently performed to detect the mRNA and protein expression of matrix metallopeptidase-2 (MMP-2; Figures 2B, 2C, 2E, and 2F). The results obtained revealed that silenced lncRNA CEBPA-AS1 and NCOR2 upregulated mRNA and protein expression of MMP-2, whereas overexpressed lncRNA CEBPA-AS1 and elevated NCOR2 led to the downregulation of the mRNA and protein expression of MMP-2 (p < 0.05). Silencing NCOR2 were observed to reverse the inhibitory effects of lncRNA CEBPA-AS1 on MMP-2. Thus, based on our results, we concluded that lncRNA CEBPA-AS1 overexpression could repress OS cell migration through regulation of NCOR2.

lncRNA CEBPA-AS1 Overexpression Inhibits OS Cell Migration by Elevating NCOR2

(A and D) Representative images and quantitative analysis of cell migration in (D) MG63 and (A) SAOS-2 cells detected by transwell assay (original magnification ×200). (B and E) qRT-PCR analysis of the mRNA expression of MMP-2 in (E) MG63 and (B) SAOS-2 cells. (C and F) Western blot analysis of MMP-2 protein expression and bands in (F) MG63 and (C) SAOS-2 cells. The above data are measurement data and are expressed as the mean ± SD. Comparisons among multiple groups are analyzed using one-way ANOVA. All experiments were repeated three times. *p < 0.05 versus the blank group; ^#p < 0.05 versus the siLncRNA CEBPA-AS1 group or oelncRNA CEBPA-AS1 group.

lncRNA CEBPA-AS1 Overexpression Inhibits Cell Proliferation and Differentiation in OS by Elevating NCOR2

Cell viability was evaluated by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which permitted a further investigation into the effects of lncRNA CEBPA-AS1 and NCOR2 on cell proliferation (Figures 3A and 3D). The results obtained indicated there to be no significant difference between the blank and empty vector groups regarding SAOS-2 cell viability (p > 0.05). Besides, relative to the blank group, SAOS-2 cell viability was increased in the siNCOR2 and siLncRNA CEBPA-AS1 groups. In contrast with the siLncRNA CEBPA-AS1 group, SAOS-2 cell viability decreased in the siLncRNA CEBPA-AS1 + oeNCOR2 group (p < 0.05). Meanwhile, elevation of both lncRNA CEBPA-AS1 and NCOR2 resulted in reduced MG63 cell viability, whereas silenced NCOR2 was noted to reverse the repressive effect of lncRNA CEBPA-AS1 on MG63 cell viability. Next, ELISA was employed to detect the effects of lncRNA CEBPA-AS1 and NCOR2 on alkaline phosphatase (ALP) and bone gla protein (BGP) activity in MG63 and SAOS-2 cells at different time points (0, 3, 6, 9, and 12 days) (Figures 3B, 3C, 3E, and 3F). The results revealed that starting with the cell culture, the ALP activity of SAOS-2 cells in each group gradually increased before peaking on the 9^th day, followed by an initiation of a decline in the mineralization period. However, the BGP activity of SAOS-2 cells in each group exhibited a continual rise without decline at a slower rising speed. The ALP and BGP activities of SAOS-2 cells did not differ between the blank and empty vector groups (p > 0.05). In comparison with the blank group, the ALP and BGP activities of the SAOS-2 cells in the siNCOR2 and siLncRNA CEBPA-AS1 groups were notably strengthened (p < 0.05). When compared with the siLncRNA CEBPA-AS1 group, the ALP and BGP activities of the SAOS-2 cells were weakened in the siLncRNA CEBPA-AS1 + oeNCOR2 group (p < 0.05). Furthermore, the overexpression of both lncRNA CEBPA-AS1 and NCOR2 led to diminished ALP and BGP activities of the MG63 cells, yet NCOR2 silencing was determined to reverse the inhibitory effect of lncRNA CEBPA-AS1 on ALP and BGP activities. Based on the aforementioned results, we suggested that overexpression of lncRNA CEBPA-AS1 and elevation of NCOR2 could repress the cell proliferation and differentiation processes in OS.

Overexpression of lncRNA CEBPA-AS1 Represses OS Cell Proliferation and Differentiation by Increasing NCOR2

(A and D) Cell viability of (D) MG63 and (A) SAOS-2 cells assessed by MTT assay. (B and E) The ALP activity of (E) MG63 and (B) SAOS-2 cells at various time points. (C and F) The BGP activity of (F) MG63 and (C) SAOS-2 cells at various time points. The above data are measurement data and are expressed as the mean ± SD. Comparisons among multiple groups are analyzed using one-way ANOVA. All experiments were repeated three times. *p < 0.05 versus the blank group; ^#p < 0.05 versus the siLncRNA CEBPA-AS1 group or oelncRNA CEBPA-AS1 group.

lncRNA CEBPA-AS1 Overexpression Promotes OS Cell Apoptosis by Elevating NCOR2

Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) staining was applied to measure the effects of lncRNA CEBPA-AS1 and NCOR2 on MG63 and SAOS-2 cell apoptosis (Figures 4A, 4B, and 4E). The results revealed there to be no significant difference between the blank and empty vector groups regarding SAOS-2 cell apoptosis (p > 0.05). Besides, in comparison with the blank group, the siNCOR2 and siLncRNA CEBPA-AS1 groups displayed a diminished rate of SAOS-2 cell apoptosis (p < 0.05). When compared with the siLncRNA CEBPA-AS1 group, the siLncRNA CEBPA-AS1 + oeNCOR2 group displayed an increased rate of SAOS-2 cell apoptosis (p < 0.05). Moreover, the overexpression of both lncRNA CEBPA-AS1 and NCOR2 was identified to accelerate MG63 cell apoptosis, whereas the promotive effect of lncRNA CEBPA-AS1 on MG63 cell apoptosis was reversed by NCOR2 silencing. qRT-PCR and western blot analysis were subsequently performed to determine the mRNA and protein expression of Bax and Bcl-2 (Figures 4C–4F). The results obtained revealed that silencing of NCOR2 or knockdown of lncRNA CEBPA-AS1 resulted in decreased mRNA and protein expression of Bax, and increased mRNA and protein expression of Bcl-2 (p < 0.05). In addition, PI single staining was conducted using a flow cytometer to explore the roles of lncRNA CEBPA-AS1 and NCOR2 in the MG63 and SAOS-2 cell-cycle distribution (Figure S2). The results demonstrated that silenced lncRNA CEBPA-AS1 or absent NCOR2 led to an increase in the proportion of cells at the G2 phase and elevated expression of Cyclin D1, which was accompanied by a reduced proportion of cells at the G0/G1 phase. The aforementioned findings suggested that cell apoptosis was promoted, whereas cell-cycle entry was decelerated, when lncRNA CEBPA-AS1 and NCOR2 were elevated.

Overexpressed lncRNA CEBPA-AS1 Promotes OS Cell Apoptosis by Elevating NCOR2

(A) Representative images of cell apoptosis in SAOS-2 and MG63 cells detected by Annexin V-FITC/PI staining. (B) Apoptosis rate in SAOS-2 cells (%). (C) The mRNA expression of Bax and Bcl-2 expression in SAOS-2 cells determined by qRT-PCR. (D) The protein expression of Bax and Bcl-2 expression in SAOS-2 cells measured by Western blot analysis. (E) Apoptosis rate in MG63 cells (%). (F) The mRNA expression of Bax and Bcl-2 expression in MG63 cells determined by qRT-PCR. (G) The protein expression of Bax and Bcl-2 expression in MG63 cells measured by Western blot analysis.

lncRNA CEBPA-AS1 Overexpression Inhibits the Notch Signaling Pathway by Elevating NCOR2

In an attempt to investigate the regulatory effect of lncRNA CEBPA-AS1 on the Notch signaling pathway via NCOR2, we analyzed the mRNA and protein expression of HES1 and RBPJ by means of qRT-PCR and western blot analysis (Figure 5). The results demonstrated that, compared with the blank group, the mRNA and protein expression of HES1 and RBPJ of SAOS-2 cells did not differ in the empty vector group (p > 0.05), whereas upregulated levels were identified in the siNCOR2 and siLncRNA CEBPA-AS1 groups (p < 0.05). In comparison with the siLncRNA CEBPA-AS1 group, the mRNA and protein expression of HES1 and RBPJ of SAOS-2 cells were downregulated in the siLncRNA CEBPA-AS1 + oeNCOR2 group. Regarding the MG63 cells, overexpression of both lncRNA CEBPA-AS1 and NCOR2 was found to inhibit the mRNA and protein expression of HES1 and RBPJ of MG63 cells, whereas the knockdown of NCOR2 was observed to reverse the inhibitory effect elicited by overexpressed lncRNA CEBPA-AS1. Hence, based on the results obtained, we suggested that lncRNA CEBPA-AS1 elevation inhibits the Notch signaling pathway by increasing NCOR2.

Overexpressed lncRNA CEBPA-AS1 Inhibits Activation of the Notch Signaling Pathway by Increasing NCOR2

(A) The mRNA expression of HES1 and RBPJ expression in SAOS-2 cells determined by qRT-PCR. (B) The protein expression of HES1 and RBPJ expression in SAOS-2 cells measured by Western blot analysis. (C) The mRNA expression of HES1 and RBPJ expression in MG63 cells determined by qRT-PCR. (D) The protein expression of HES1 and RBPJ expression in MG63 cells measured by Western blot analysis.

lncRNA CEBPA-AS1 Overexpression Reduces Tumor Formation by Elevating NCOR2

Moreover, after 4 weeks of xenograft tumor inoculation, the tumor weight (Figures 6A and 6C) and tumor volume (Figures 6B and 6D) in nude mice injected with transfected MG63 and SAOS-2 cells were determined accordingly. The results revealed there to be no distinct difference between the blank and empty vector groups in terms of the tumor volume and weight in nude mice injected with transfected SAOS-2 cells (p > 0.05). Meanwhile, compared with the blank group, the siNCOR2 and siLncRNA CEBPA-AS1 groups displayed an elevated tumor volume and weight among the nude mice injected with transfected SAOS-2 cells (p < 0.05). When compared with the siLncRNA CEBPA-AS1 group, the siLncRNA CEBPA-AS1 + oeNCOR2 group exhibited reduced tumor volume and weight in nude mice injected with transfected SAOS-2 cells (p < 0.05). In addition, in nude mice injected with transfected MG63 cells, elevation of both lncRNA CEBPA-AS1 and NCOR2 led to increased tumor volume and weight, yet the promotive effect of lncRNA CEBPA-AS1 on increased tumor volume and weight could be reversed by NCOR2 silencing. Based on the above findings, we suggested that tumor formation in nude mice was repressed in response to elevated lncRNA CEBPA-AS1 and NCOR2 either together or alone.

Elevation of lncRNA CEBPA-AS1 Represses Tumor Formation by Elevating NCOR2

(A) Tumor weight (g) in nude mice injected with transfected SAOS-2 cells. (B) Tumor volume (cm³) in nude mice injected with transfected SAOS-2 cells. (C) Tumor weight (g) in nude mice injected with transfected MG63cells. (B) Tumor volume (cm³) in nude mice injected with transfected MG63 cells.

lncRNA CEBPA-AS1 Binds to miR-10b-5p to Regulate the Expression of NCOR2

The RNA22, starBase, and miRDB websites provided data predicting that four miRNAs (hsa-miR-4756-5p, hsa-miR-3918, hsa-miR-4739, and hsa-miR-10b-5p) could bind to lncRNA CEBPA-AS1 and NCOR2 (Figure 7A). Yet, except that hsa-miR-10b-5p has been confirmed to be differentially expressed in OS, rest miRNAs have been seldom reported. We speculated that lncRNA CEBPA-AS1 could potentially bind to miR-10b-5p to participate in the progression of OS by regulating NCOR2. Next, a dual-luciferase reporter gene assay was employed to verify the regulatory effect of miR-10b-5p on NCOR2, the results of which revealed that the luciferase activity of NCOR2-WT (wild-type) and lncRNA CEBPA-AS1-WT was reduced following co-transfection with miR-10b-5p mimic (Figure 7B). Besides, the binding of lncRNA CEBPA-AS1 and miR-10b-5p to AGO2 in SAOS-2 cells was determined using an RNA immunoprecipitation (RIP) assay. As depicted in Figure 7C, compared with immunoglobulin G (IgG), lncRNA CEBPA-AS1 and miR-10b-5p binding to AGO2 were significantly enriched (p < 0.05). Furthermore, an RNA pull-down was employed to help evaluate the correlation between lncRNA CEBPA-AS1 and miR-10b-5p in SAOS-2 cells (Figure 7D). The results obtained demonstrated that there was increased Bio-miR-10b-5p-WT that bound to lncRNA CEBPA-AS1 relative to Bio-miR-10b-5p-MUT (mutant), which suggested that miR-10b-5p could directly bind to lncRNA CEBPA-AS1.

lncRNA CEBPA-AS1 Binds to miR-10b-5p to Regulate NCOR2 Expression

(A) Four miRNAs that could bind to lncRNA CEBPA-AS1 and NCOR2 predicted by the RNA22, starBase, and miRDB websites. (B) The luciferase activity of NCOR2-WT and lncRNA CEBPA-AS1-WT verified by a dual-luciferase reporter gene assay. (C) The binding of lncRNA CEBPA-AS1 and miR-10b-5p to AGO2 in SAOS-2 cells determined by the RIP assay. (D) The correlation between lncRNA CEBPA-AS1 and miR-10b-5p in SAOS-2 cells detected by RNA pull-down. (E) qRT-PCR analysis of the mRNA expression of NCOR2 in SAOS-2 cells. (F) The subcellular location of lncRNA CEBPA-AS1 assessed by FISH. Scale bars, 25 μm. The above data are measurement data and are expressed as the mean ± SD. Comparisons between two groups are analyzed using non-paired t test. All experiments were repeated three times. *p < 0.05 versus the IgG, NC, or siLncRNA CEBPA-AS1 + miR-10b-5p inhibitor NC groups.

As previously discussed, lncRNA CEBPA-AS1 can competitively bind to miR-10b-5p to regulate NCOR2. The SAOS-2 cells were transfected with siLncRNA CEBPA-AS1 and miR-10b-5p inhibitor. qRT-PCR was performed to determine the mRNA expression of NCOR2 following transfection (Figure 7E). The results revealed that the mRNA expression of NCOR2 was clearly higher in the siLncRNA CEBPA-AS1 + miR-10b-5p inhibitor group than in the siLncRNA CEBPA-AS1 + miR-10b-5p inhibitor NC group. Silenced lncRNA CEBPA-AS1 decreased mRNA expression of NCOR2, whereas downregulated miR-10b-5p elevated its expression. Meanwhile, the results of fluorescence in situ hybridization (FISH) revealed that lncRNA CEBPA-AS1 was distributed in the nucleus and cytoplasm (Figure 7F). Taken together, the aforementioned results provided evidence suggesting that lncRNA CEBPA-AS1 that bound to miR-10b-5p can regulate the activity of NCOR2 in the cytoplasm.

Discussion

lncRNAs play a critical role in various biological processes and exert their oncogenic or tumor-suppressor effect on cancers, including OS.¹⁶^,¹⁷ The present study was implemented to assess the capability of lncRNA CEBPA-AS1 in proliferation, migration, and apoptosis of OS cells through the Notch signaling pathway by modulating NCOR2. The results obtained indicated that overexpression of lncRNA CEBPA-AS1 inhibited the proliferation and migration, and enhanced the apoptosis of OS cells by repressing the Notch signaling pathway via upregulating the expression of miR-10b-5p-mediated NCOR2 (Figure S3).

Initially, our study demonstrated that lncRNA CEBPA-AS1 was poorly expressed in OS. lncRNA CEBPA-AS1 has been reported to exhibit low levels of expression in GC tissues.⁸ Additionally, our key findings revealed that silencing of lncRNA CEBPA-AS1 could result in the inhibition of NCOR2 expression. Furthermore, our results demonstrated that silenced lncRNA CEBPA-AS1 could activate the Notch signaling pathway via the inhibition of NCOR2. The Notch signaling pathway has been reported to play a critical role in the regulation of both GC cells and GC stem cells via different Notch receptors and ligands.¹⁸ lncRNA HOTAIR has been shown to activate the Notch signaling pathway and subsequently regulate retinoblastoma proliferation and invasion.¹⁹ Dual-luciferase reporter gene assay provided confirmation suggesting that NCOR2 is a target gene of miR-10b-5p. A previous study suggested that miR-10b directly targets NCOR2 in neuroblastoma cell differentiation.¹¹ Moreover, our findings revealed that lncRNA CEBPA-AS1 bound to miR-10b-5p. According to Zhou et al.²⁰, SMRT has been reported to be an alias of NCOR2. Jepsen et al.²¹ revealed that SMRT (NCOR2) exerts a regulatory effect on the Notch signaling pathway, which helps to maintain the neural stem cell state. Additionally, Ghoshal et al.²² suggested that SMRT (NCOR2) is poorly expressed in multiple myeloma and combines with histone deacetylases (HDACs) to negatively regulate Notch, the overexpression of which increases the rate of multiple myeloma cell apoptosis. Furthermore, the ligand JAG2, as a Notch ligand, when overexpressed results in the activation of Notch receptors.²³ Moreover, from an epigenetic perspective, the acetylation of the Notch ligand promoter region is predominately regulated by HDAC, which is characteristically recruited via the interaction with nuclear corepressors such as SMRT.²⁴ The aforementioned studies were all consistent with the findings of our study, while providing additional support for our conclusions.

Additionally, a key observation of the current study revealed that overexpressed lncRNA CEBPA-AS1 inhibited cell proliferation and migration while promoting cell apoptosis through the Notch signaling pathway by mediating NCOR2, corresponding to diminished expression of Cyclin D1, MMP-2, and Bcl-2/Bax. Dong et al.²⁵ argued that attenuation of Notch-1, as well as the downregulation of its downstream genes, such as MMP-2, MMP-9, Hes-1, and Cyclin D1, leads to the inhibition of OS cell growth, invasion through the Matrigel, and the subsequent G2 phase cell-cycle arrest. MMPs have been highlighted as critical elements involved in the stimulation of metastasis and invasion in OS,²⁶ with recent evidence indicating the close association of the Notch-1 signaling pathway and MMPs in curcumin-induced cell invasion inhibition.²⁵ In prostate cancer, silencing of Notch-1 upregulates the pro-apoptotic protein Bax, which is accompanied by the downregulation of the anti-apoptotic protein Bcl-2, inducing cell apoptosis and restraining proliferation.²⁷

In conclusion, the present findings elucidate that lncRNA CEBPA-AS1 represses proliferation and migration, and expedites apoptosis of OS cells through inhibiting the Notch signaling pathway via upregulating miR-10b-5p-mediated NCOR2, which may offer new insights into future therapeutic research on OS. However, because many factors that affect lncRNA CEBPA-AS1 and the Notch signaling pathway remain unknown, further efforts are still needed to identify effective therapeutic regimens for patients with OS.

Materials and Methods

Ethics Statement

The protocols of the study were approved by the Ethics Committee of The China-Japan Union Hospital of Jilin University. All experimental procedures were performed in strict accordance with the Declaration of Helsinki. Signed written informed consent was obtained from all participants. All animal experiments were performed in strict adherence with the recommendations of the Guide for the Care and Use of Laboratory Animals of the NIH and under the approval of the Animal Ethics Committee of The China-Japan Union Hospital of Jilin University.

Microarray-Based Gene Expression Profiling

OS-related microarray expression data and gene probe annotation files were downloaded from the GEO DataSets database, and background correction and normalization processing were subsequently conducted using the Affy package in R language.²⁸ Next, linear model-empirical Bayesian statistics combined with traditional t tests were employed to specifically screen out differentially expressed genes.²⁹ MEM was employed to predict differential lncRNAs.³⁰ Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted using the WEB-based GEne SeT AnaLysis Toolkit³¹ in an attempt to elucidate the major biochemical metabolic pathways and signaling pathways.³² Next, miRNAs that had binding sites for both lncRNAs and mRNAs were obtained from RNA22,³³ starBase,³⁴ and miRDB.³⁵

Study Subjects

OS tissues were surgically resected from 36 patients who had been pathologically diagnosed with OS from June 2016 to June 2017. The patient population consisted of 20 males and 16 females ranging in age from 16 to 72 years with a median age of 19 years. As per the Enneking classification, there were 9 stage I/II A cases and 27 stage II B/III cases. Based on the pathological type, there were 21 cases of the osteoblastic type, 6 cases of the chondroblastic type, 5 cases of the fibroblastic type, and 4 cases of the mixed type. Moreover, 13 of the enrolled patients were diagnosed with distant metastasis. The adjacent normal tissues (4 cm away from the tumor tissues) were collected as a control.³⁶^,³⁷ All patients enrolled were confirmed to have complete clinical data and were yet to receive radiotherapy or chemotherapy prior to the study.

Cell Culture and Grouping

The MG63 (Art. No. XG-3061), G292, HOS, and SAOS-2 cells (American Type Culture Collection [ATCC], Manassas, VA, USA), as well as the collected tissue specimens, were cultured in DMEM containing 10% fetal bovine serum (FBS; supplemented with β-glycerophosphate [β-GP]). The SAOS-2 cells were assigned to the blank (without transfection), empty vector (transfected with empty vector), siLncRNA CEBPA-AS1 (transfected with LentiCRISOR-v2-CEBPA-AS1 and Cas9 plasmids), siNCOR2 (transfected with LentiCRISOR-v2-NCOR2 and Cas9 plasmids), and siLncRNA CEBPA-AS1 + oeNCOR2 groups. Next, MG63 cells were classified into the blank (without transfection), empty vector (transfected with empty vector), oeLncRNA CEBPA-AS1 (transfected with LentiCRISOR-v2-CEBPA-AS1 plasmids), oeNCOR2 (transfected with LentiCRISOR-v2-NCOR2 plasmids), and oeLncRNA CEBPA-AS1 + siNCOR2 groups. Moreover, G292, HOS, and SAOS-2 cells (ATCC, Manassas, VA, USA) were subjected to induction culture in DMEM supplemented with 10% FBS and β-GP, which were utilized for subsequent experiments and not subject to any further treatment.

RNA Pull-Down Assay

The complete sequence of lncRNA CEBPA-AS1 was synthesized by Invitrogen (Carlsbad, CA, USA) and inserted into the pSPT19 vector using the XbaI and EcoRI digestion sites. The positive clone was selected and referred to as pSPT19-lncRNA CEBPA-AS1.

The lncRNA CEBPA-AS1 plus strand was marked by biotin for future use: after pSPT19-lncRNA CEBPA-AS1 had been treated with XbaI restriction enzyme, the plasmid was purified as per the instructions provided by the QIAquick Nucleotide Removal Kit (28304; TIANGEN Biotechnology, Beijing, China). Next, in accordance with the instructions of the DIG RNA Labeling Kit (SP6-T7, 11175025910; Sigma, Chemical, St. Louis, MO, USA) and Biotin RNA Labeling Mix (Art. No. 11685597910; Sigma, Chemical, St. Louis, MO, USA), the reaction was conducted for 2 h at 37°C under the following conditions: 2 μL Transcription, 2 μL Biotin RNA Labeling Mix, 1 μg pSPT19-lncRNA CEBPA-AS1 (XbaI, single restriction enzyme digest), 1 μL SP6 RNA polymerase, 1 μL RNase inhibitor, and moderate RNase Free Water up to a total reaction system volume of 20 μL. After DNA restriction enzyme digestion, EDTA was applied to terminate the reaction, after which the RNeasy Mini Kit (Art. No. 74104; Shanghai Haoran Biological Technology, Shanghai, China) was utilized to purify the biotin-marked RNA products.

The lncRNA CEBPA-AS1 minus strand was marked by biotin for later use: after the pSPT19-lncRNA CEBPA-AS1 had been detached using an EcoRI restriction enzyme, the plasmid was purified using the QIAquick Nucleotide Removal Kit as per the manufacturer’s instructions. Next, based on the instructions supplied by the DIG RNA Labeling Kit (SP6-T7) and Biotin RNA Labeling Mix, the reaction was conducted for 2 h at 37°C under the following conditions: 2 μL Transcription, 2 μL Biotin RNA Labeling Mix, 1 μg pSPT19-lncRNA CEBPA-AS1 (EcoRI single restriction enzyme digestion), 1 μL T7 RNA polymerase, 1 μL RNase inhibitor, and moderate RNase Free Water up to 20 μL. After DNA restriction enzyme digestion, EDTA was applied to terminate the reaction, after which the RNeasy Mini Kit was employed to purify the biotin-marked RNA products.

Radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B; Beyotime Biotechnology, Shanghai, China) was applied to extract the total proteins of MG63 cells. The plus and minus strands of the biotin-marked lncRNA CEBPA-AS1 were permitted to react separately under conditions void of light with total cellular proteins for 1 h and magnetic beads (88816; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. The beads were then collected by magnetic support, with the supernatant removed accordingly. The mixture of lncRNA and protein was acquired after the protein had been rinsed three times with PBS and SDS loading buffer had been added. The protein was identified in connection with the combination of SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

RIP

The SAOS-2 cells were washed twice with cold PBS, followed by the addition of 10 mL PBS and transfer into a centrifuge tube. Next, the cells were centrifuged at 4°C and 1,000 × g for 2 min, with the supernatant discarded accordingly. The cells were subsequently permitted to react with RIP lysis buffer (N653-100 mL; Shanghai Haoran Biological Technology, Shanghai, China). Each tube was added with 50 μL magnetic beads, which was placed upside down for a greater degree of mixture followed by the addition of 0.5 mL RIP wash buffer (EHJ-BVIS08102; Xiamen Huijia Biotechnology, Fujian, China). After transient vortex, the tube was placed on the magnetic separator for the aggregation of the magnetic beads. After the supernatant had been discarded, the magnetic beads were washed once again, resuspended by 100 μL RIP wash buffer, and added with 5 μg Ago2 antibody (P10502500; Shenzhen Otwo Biological Technology, Shenzhen, Guangdong, China). The IgG of normal mice was regarded as the NC. The magnetic beads were incubated at room temperature for 30 min and rinsed with 0.5 mL RIP wash buffer for subsequent use. The magnetic bead-antibody mixture was added with 900 μL RIP immunoprecipitation buffer (P10403138; Shenzhen Otwo Biological Technology, Shenzhen, Guangdong, China), with the lysate centrifuged at 4°C and 14,000 × g for 10 min. The supernatant was subsequently transferred into a new centrifuge tube, with 100 μL of the supernatant then transferred into a magnetic bead-antibody tube; 1 mL was finally considered to be the final volume of RIP. The magnetic beads were then incubated at 4°C overnight, rinsed with 0.5 mL RIP wash six times with buffer, and finally incubated with 150 μL Proteinase K buffer at 55°C for 30 min. Finally, RNA was purified and extracted using the TRIzol method for qRT-PCR.

Dual-Luciferase Reporter Gene Assay

After the total RNA had been extracted from the MG63 cells, PCR was performed to amplify the potential target gene, followed by the addition of HindIII and PmeI endonuclease sites on both ends of the amplification products. Vectors carrying NCOR2-WT-Luc (luciferase) and NCOR2-MUT-Luc sequences were constructed using the pMIR-Report Luciferase vector (Biovector107902; Biovector, USA). As per the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), NCOR2-WT-Luc and NCOR2-MUT-Luc were cotransfected with miR-10b-5p mimic or mimic NC into cells. The medium was changed 6 h after transfection, followed by collection of the cells 24 h later. Luciferase activity was detected at an excitation wavelength of 560 nm on a microplate reader (MK3; Thermo Fisher Scientific, Waltham, MA, USA) using the Firefly Luciferase Reporter Gene Assay Kit (RG005; Beyotime Biotechnology, Shanghai, China). An identical method was subsequently applied to detect the interaction between lncRNA CEBPA-AS1 and miR-10b-5p.

qRT-PCR

The TRIzol method (15596026; Thermo Fisher Scientific, Waltham, MA, USA) was employed to extract total RNA from MG63 and SAOS-2 cells. The SuperScript III Platinum One-Step RT-qPCR Kit (11732088; Thermo Fisher Scientific, Waltham, MA, USA) was applied for one-step qRT-PCR purposes. The reaction system was as follows: 1 μL SuperScript III RT/Platinum Tag Mix, 25 μL 2× Reaction Mix, 10 μM forward primer, 10 μM reverse primer, 10 μM fluorogenic probe, and 1 μL template and diethyl pyrocarbonate (DEPC)-treated water up to a total reaction system volume of 50 μL. The reaction conditions were as follows: one cycle of 50°C for 15 min; one cycle of 95°C for 2 min; 40 cycles of 95°C for 15 s, and 60°C for 30 s. The 2^−ΔΔCT method was utilized for quantitative analyses with GAPDH as an internal reference (Table S1).

Western Blot Analysis

The MG63 and SAOS-2 cells were lysed using RIPA lysis buffer (P0013B; Beyotime Biotechnology, Shanghai, China) supplemented with PMSF. SDS-PAGE was conducted for protein separation purposes, with the separated protein subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then sealed with PBS (containing 5% skim milk powder [pH 7.2]) for 12 h and rinsed with Tris-buffered saline Tween 20 (TBST) once for 5 min. Membrane incubation was then performed with primary rabbit anti-human antibodies against NCOR2 (ab24551, 1:1,000), hes family bHLH transcription factor 1 (HES1) (ab71559, 1:1,000), recombination signal binding protein for immunoglobulin kappa J region (RBPJ) (ab25949, 1 μg/mL), Cyclin D1 (ab134175, 1:10,000–50,000), MMP-2 (ab37150, 1 μg/mL), B cell lymphoma-2 (Bcl-2) (ab59348, 1:1,000), Bcl-2-associated X protein (Bax) (ab32503, 1:1,000), and GAPDH (ab9485, 1:2,500) at room temperature for 1 h. All of the above antibodies were purchased from Abcam (Cambridge, MA, USA). After the cells had been rinsed with PBS, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary IgG antibody (goat anti-rabbit, ab6721, 1:10,000; Abcam, Cambridge, MA, USA) at room temperature for 1 h. The membrane was developed by enhanced chemiluminescence (ECL) (NCI4106; Shanghai Huiying Biological Technology, Shanghai, China) and photographed by SmartView Pro 2000 (UVCI-2100; Major Science, CA, USA). Quantity One software was employed to evaluate the gray value of the protein bands.

ELISA

Rat ALP ELISA kit (Shanghai EYSIN, Shanghai, China) was employed to assess the viability of both the MG63 and the SAOS-2 cells. Ten microliters of protein was subsequently incubated with 50 μL enzyme-labeled working fluid, followed by incubation in a water bath at 37°C for 30 min. Each well was then permitted to react with coloration liquids A and B under conditions void of light at 37°C for 15 min. In accordance with the instructions of the rat BGP ELISA kit (Shanghai Ximei Biological Technology, Shanghai, China), 50 μL protein was incubated with 50 μL biotin-labeled antibody at 37°C for 45 min. After incubation with 100 μL streptavidin-HRP for 30 min, the cells were permitted to react with substrates A and B (50 μL for each) at 37°C for 5 min. The optical density (OD) value was detected using a microplate reader (MK3; Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.

MTT Assay

The MG63 and SAOS-2 cells (1 × 10⁷ cells/mL) were seeded into a 96-well plate and incubated for 24 h. The cells were then subjected to incubation with 5 mg/mL MTT solution at 37°C for 3–6 h. Next, 200 μL DMSO was added for an additional 2-h period of incubation. A microplate reader (MK3; Thermo Fisher Scientific, Waltham, MA, USA) was applied to determine the OD value at a wavelength of 570 mm. The cells were added to the wells, with the culture medium regarded as the positive control, whereas the wells added only with the culture medium were considered to be the blank control. During colorimetric determination, the blank control wells were zeroed. The experiment was repeated three times. The cell proliferation rate = the OD value of the experimental wells/the OD value of the positive control wells × 100%.

Transwell Assay

The MG63 and SAOS-2 cells at the logarithmic growth phase were inoculated into a six-well plate. After 48 h of transfection, the cells were treated with 0.25% trypsin (200 μL/well) and re-suspended with serum-free DMEM after which cell density was adjusted to 3 × 10⁵ cells/mL. Next, a 100-μL cell suspension was added into the apical chamber of a 24-well transwell chamber, while 500 μL DMEM containing 10% FBS was added into the basolateral chamber. After incubation at 37°C and 5% CO₂ for 24 h, the chambers were withdrawn, washed twice with PBS, fixed by methyl alcohol for 10 min, and stained with crystal violet for 10 min. The cells in the apical chamber were wiped using cotton swabs, with the fibrous membranes of the chambers removed and sealed using neutral gum on the cover glasses. Six fields were randomly selected for cell counting purposes under the guidance of a microscope.

Annexin FITC/PI Double Staining

The MG63 and SAOS-2 cells were collected after a 48-h period of culturing had elapsed. The cell mixture (1 mL, containing 1 × 10⁶ cells/mL) was centrifuged at 178 × g at 4°C for 10 min with the supernatant aspirated, supplemented with 1 mL PBS followed by an additional round of centrifugation. The aforementioned procedures were repeated three times. The cells were cultured with 200 μL binding buffer, 10 μL Annexin V-FITC, and 5 μL PI under conditions void of light at 4°C for 30 min, followed by the addition of 300 μL binding buffer. Cell apoptosis was examined using a flow cytometer.

Statistical Analysis

All statistical analyses were performed using SPSS 21.0 software (IBM, Armonk, NY, USA). Data were presented as the mean ± SD. All experiments were repeated at least three times. Comparisons between two groups were analyzed using t tests. Comparisons among multiple groups were assessed by one-way ANOVA. A p value <0.05 was considered to be indicative of statistical significance, whereas p < 0.01 was considered to be exceptionally statistically significant.

Author Contributions

P.X. and R.G. designed the study. W.Z. and Y.-F.S. collated the data, carried out data analyses, and produced the initial draft of the manuscript. P.X. and Y.-F.S. contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

Conflicts of Interest

The authors declare no competing interests.

Acknowledgments

We would like to thank our researchers for their hard work and reviewers for their valuable advice.

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.omtn.2019.10.017.

Supplemental Information

Document S1. Figures S1–S3, Tables S1 and S2, and Supplemental Materials and Methods

mmc1.pdf^{(449.8KB, pdf)}

Document S2. Article plus Supplemental Information

mmc2.pdf^{(2.3MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S3, Tables S1 and S2, and Supplemental Materials and Methods

mmc1.pdf^{(449.8KB, pdf)}

Document S2. Article plus Supplemental Information

mmc2.pdf^{(2.3MB, pdf)}

[bib1] 1.Li Y., Nakka M., Kelly A.J., Lau C.C., Krailo M., Barkauskas D.A., Hicks J.M., Man T.K. p27 Is a Candidate Prognostic Biomarker and Metastatic Promoter in Osteosarcoma. Cancer Res. 2016;76:4002–4011. doi: 10.1158/0008-5472.CAN-15-3189. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib2] 2.Durfee R.A., Mohammed M., Luu H.H. Review of Osteosarcoma and Current Management. Rheumatol. Ther. 2016;3:221–243. doi: 10.1007/s40744-016-0046-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Broadhead M.L., Clark J.C., Myers D.E., Dass C.R., Choong P.F. The molecular pathogenesis of osteosarcoma: a review. Sarcoma. 2011;2011:959248. doi: 10.1155/2011/959248. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Rickel K., Fang F., Tao J. Molecular genetics of osteosarcoma. Bone. 2017;102:69–79. doi: 10.1016/j.bone.2016.10.017. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Xiang J., Guo S., Jiang S., Xu Y., Li J., Li L., Xiang J. Silencing of Long Non-Coding RNA MALAT1 Promotes Apoptosis of Glioma Cells. J. Korean Med. Sci. 2016;31:688–694. doi: 10.3346/jkms.2016.31.5.688. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6.Leone S., Santoro R. Challenges in the analysis of long noncoding RNA functionality. FEBS Lett. 2016;590:2342–2353. doi: 10.1002/1873-3468.12308. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Fang Y., Fullwood M.J. Roles, Functions, and Mechanisms of Long Non-coding RNAs in Cancer. Genomics Proteomics Bioinformatics. 2016;14:42–54. doi: 10.1016/j.gpb.2015.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Ke D., Li H., Zhang Y., An Y., Fu H., Fang X., Zheng X. The combination of circulating long noncoding RNAs AK001058, INHBA-AS1, MIR4435-2HG, and CEBPA-AS1 fragments in plasma serve as diagnostic markers for gastric cancer. Oncotarget. 2017;8:21516–21525. doi: 10.18632/oncotarget.15628. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9.Wang Y., Hou J., He D., Sun M., Zhang P., Yu Y., Chen Y. The Emerging Function and Mechanism of ceRNAs in Cancer. Trends Genet. 2016;32:211–224. doi: 10.1016/j.tig.2016.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Li Y., Chen D., Li Y., Jin L., Liu J., Su Z., Qi Z., Shi M., Jiang Z., Ni L. Oncogenic cAMP responsive element binding protein 1 is overexpressed upon loss of tumor suppressive miR-10b-5p and miR-363-3p in renal cancer. Oncol. Rep. 2016;35:1967–1978. doi: 10.3892/or.2016.4579. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Foley N.H., Bray I., Watters K.M., Das S., Bryan K., Bernas T., Prehn J.H., Stallings R.L. MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2. Cell Death Differ. 2011;18:1089–1098. doi: 10.1038/cdd.2010.172. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Kumar S., Cunningham T.J., Duester G. Nuclear receptor corepressors Ncor1 and Ncor2 (Smrt) are required for retinoic acid-dependent repression of Fgf8 during somitogenesis. Dev. Biol. 2016;418:204–215. doi: 10.1016/j.ydbio.2016.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

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This article has been retracted.

lncRNA CEBPA-AS1 Overexpression Inhibits Proliferation and Migration and Stimulates Apoptosis of OS Cells via Notch Signaling

Peng Xia

Rui Gu

Wei Zhang

Yi-Fu Sun

Abstract

Graphical Abstract

Introduction

Results

lncRNA CEBPA-AS1 and NCOR2 Are Poorly Expressed in OS

Figure 1.

Table 1.

lncRNA CEBPA-AS1 Overexpression Inhibits OS Cell Migration by Elevating NCOR2

Figure 2.

lncRNA CEBPA-AS1 Overexpression Inhibits Cell Proliferation and Differentiation in OS by Elevating NCOR2

Figure 3.

lncRNA CEBPA-AS1 Overexpression Promotes OS Cell Apoptosis by Elevating NCOR2

Figure 4.

lncRNA CEBPA-AS1 Overexpression Inhibits the Notch Signaling Pathway by Elevating NCOR2

Figure 5.

lncRNA CEBPA-AS1 Overexpression Reduces Tumor Formation by Elevating NCOR2

Figure 6.

lncRNA CEBPA-AS1 Binds to miR-10b-5p to Regulate the Expression of NCOR2

Figure 7.

Discussion

Materials and Methods

Ethics Statement

Microarray-Based Gene Expression Profiling

Study Subjects

Cell Culture and Grouping

RNA Pull-Down Assay

RIP

Dual-Luciferase Reporter Gene Assay

qRT-PCR

Western Blot Analysis

ELISA

MTT Assay

Transwell Assay

Annexin FITC/PI Double Staining

Statistical Analysis

Author Contributions

Conflicts of Interest

Acknowledgments

Footnotes

Supplemental Information

References

Associated Data

Supplementary Materials

ACTIONS

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RESOURCES

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