# Long Noncoding RNA NR2F1-AS1 Enhances the Migration and Invasion of Hepatocellular Carcinoma via Modulating miR-642a/DEK Pathway

Authors: Yingxia Xu; Chunrong Han; Jing Sun; Jingjing Zhao; Qing Liu; Ping An
Journal: Journal of Oncology
 (2021)

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## Abstract

Purpose. Hepatocellular carcinoma (HCC), a malignant tumor that exists worldwide, has a high morbidity and mortality rate. Previous studies have reported that lncRNA NR2F1-AS1 plays a critical role in several cancers. Here, we aimed to investigate the biological function of NR2F1-AS1 and its molecular mechanism in the migration and invasion of HCC. Methods. Quantitative real-time PCR (qRT-PCR) was performed to analyze NR2F1-AS1 expression in HCC. The biological function was investigated by transwell invasion and migration assays. The protein level was identified by Western blot. In addition, the downstream targets of NR2F1-AS1 and miR-642a were confirmed by luciferase reporter assays. Results. NR2F1-AS1 was significantly upregulated in HCC and associated with the poor prognosis of HCC patients. Biological function experiments revealed that the silence of NR2F1-AS1 suppressed cell invasion and migration in HCC. More importantly, NR2F1-AS1 directly interacted with miR-642a and negatively regulated miR-642a. DEK was the target of miR-642a, and NR2F1-AS1 positively regulated DEK expression by suppressing miR-642a. Conclusion. Taken together, it is the first time we discovered the interaction of NR2F1-AS1 with miR-642a in modulating HCC cell invasion and migration.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, which initiates in hepatocytes and has a high mortality rate [1, 2]. Surgical resection has been considered the most effective therapy for HCC; however, it may produce new metastases [3]. Therefore, it is becoming more and more urgent to explore the molecular mechanism for the treatment of HCC.Long noncoding RNAs (lncRNAs), as a new epigenetic regulatory molecule, have received wide attention in various cellular and biological processes. And dysregulation of lncRNAs is closely related to tumorigenesis, metastasis, and apoptosis by competing for corresponding miRNAs [4]. Recently, lncRNAs have been found to be critical regulators in HCC [5, 6]. For instance, lncRNA OTUD6B-AS1 was highly expressed in HCC and associated with HCC patients’ prognosis. Moreover, silencing OTUD6B-AS1 inhibited HCC proliferation and invasion through the Wnt/β-catenin pathway [7]. lncRNA HCG18 was discovered to be increased in HCC and facilitated the progression of HCC via the miR-214-3p/CENPM axis [8]. Recently, a novel lncRNA, NR2F1 antisense RNA 1 (NR2F1-AS1), was functionally identified [9]. Mounting evidence has discovered that NR2F1-AS1 played a critical role in the development of various cancers, including colorectal cancer [10], breast cancer [11], and esophageal squamous cell carcinoma [12]. However, the biological role of NR2F1-AS1 and the detailed mechanism in HCC cell invasion and migration are yet to be explored.In this study, NR2F1-AS1 expression was detected in HCC tissues and its correlation with prognosis. Furthermore, the significance and molecular mechanism of NR2F1-AS1 were measured in HCC cell invasion and migration. The findings demonstrated that NR2F1-AS1 was highly expressed in HCC tissues and associated with the prognosis of HCC patients. Moreover, the results elucidated that NR2F1-AS1 enhanced HCC progression via modulating the miR-642a/DEK axis, which strengthened understanding of HCC pathogenesis and the development of effective therapies.


## 2. Methods

### 2.1. Clinical Samples

Pairs of HCC tumor tissues (n = 36) were taken from patients who received hepatectomy at Weifang People’s Hospital, Weifang, Shandong, China. All patients had not received any preoperative chemotherapy or radiotherapy before surgery. This study was approved by the Ethics Committee of the Weifang People’s Hospital, Weifang, Shandong, China, and all patients gave informed written consent in advance. The collected samples were stored at −80°C for further use.


### 2.2. Cell Culture and Cell Transfection

Human HCC cell lines (Huh7 and SK-Hep-1) and normal liver cell line HL-7702 were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Gibco, CA, USA) supplemented with 10% FBS with 5% CO2 at 37°C. The shRNAs targeting NR2F1-AS1 (sh-NR2F1-AS1) were used to decrease NR2F1-AS1 expression. miR-642a mimic or inhibitor, obtained from Shanghai R&S Biotechnology Co. (Shanghai, China), was used to increase or decrease miR-642 expression. Lipofectamine® 2000 (Invitrogen, CA, USA) was carried out to perform the transfections for 48 h.


### 2.3. Quantitative Real-Time PCR (qRT-PCR)

Isolation of total RNA from HCC tissues or cells was carried out with TRIzol reagent (Invitrogen, NY, USA). cDNA was synthesized using a specific reverse transcription kit (Thermo Fisher). QRT-PCR was performed with SYBR Green (ThermoFisher, MA, USA). U6 or GAPDH was applied as an internal control. The relative quantitation of gene expressions was performed using the 2 − ΔΔCt method. Primers used in this study are listed as follows: NR2F1-AS1-F: 5′-CAGCGGTGCAAACCATGTGC-3′; NR2F1-AS1-R: 5′-GTAAACCAAGTCGGTTGAACG-3′; miR-642a-F: 5′-GCGGTCCCTCT CCAAATGT-3′, miR-642a-R: 5′-AGTGCAGGGTCCGAGGTATT-3′; U6-F: 5′-CTC GCTTCGGCAGCACA-3′, U6-R: 5′-AACGCTTCACGAATTTGCGT-3′; DEK-F: 5′-TGTTAAGAAAGCAGATAGCAGGACC-3′; and DEK-R: 5′-ATTAAAGGTTCAT CATCTGAACTATCCTC-3′.


### 2.4. Western Blot Assay

50μg protein lysates were separated by SDS-PAGE and then transferred to PVDF membranes. Afterwards, the membranes were blocked with 5% skim milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. After incubating with a secondary antibody for 2 h at room temperature, the protein bands were visualized by an ECL kit. GAPDH was used as the loading control.


### 2.5. Transwell Assay

For the migration experiment, HCC cells were seeded into the upper chamber of a transwell insert, and the lower chamber was fixed with a medium with 20% FBS. For the invasion experiment, the upper chamber was coated with Matrigel, and the lower chamber contained a 20% FBS medium. After incubation for 48 h, the cells were fixed, stained, and then counted under a microscope.


### 2.6. RNA Immunoprecipitation (RIP) Assay

After transfection with miR-642a mimic or inhibitor for 48 h, Huh7 cells were lysed with RIP lysis buffer and incubated with RIP immunoprecipitation buffer mixed with magnetic beads conjugating human anti-Argonaute 2 (Ago2) antibody or negative control mouse IgG (Millipore, MA, USA). Acquired RNAs were analyzed by qRT-PCR. IgG served as control in the RIP assay.


### 2.7. Luciferase Reporter Assay

The wild and mutant types of NR2F1-AS1 or DEK containing the binding site for miR-642a were cloned into pGL3 reporter vectors, designed as pGL3-NR2F1-AS1-WT and pGL3-NR2F1-AS1-MuT or pGL3-DEK-WT and pGL3-DEK-MuT, and then, they were transfected into Huh7 cells with miR-642a mimic/inhibitor for 48 h using Lipofectamine 2000.


### 2.8. Statistical Analysis

Independent repetitions of experiments were performed three times. The data were presented as means ± SD. The differences were analyzed using two-tailed Student’st-tests or Turkey’s post hoc tests in one-way ANOVA. Statistical analysis was performed using SPSS Statistics 20.0 software. P<0.05 was considered statistically significant.


## 2.1. Clinical Samples

Pairs of HCC tumor tissues (n = 36) were taken from patients who received hepatectomy at Weifang People’s Hospital, Weifang, Shandong, China. All patients had not received any preoperative chemotherapy or radiotherapy before surgery. This study was approved by the Ethics Committee of the Weifang People’s Hospital, Weifang, Shandong, China, and all patients gave informed written consent in advance. The collected samples were stored at −80°C for further use.


## 2.2. Cell Culture and Cell Transfection

Human HCC cell lines (Huh7 and SK-Hep-1) and normal liver cell line HL-7702 were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Gibco, CA, USA) supplemented with 10% FBS with 5% CO2 at 37°C. The shRNAs targeting NR2F1-AS1 (sh-NR2F1-AS1) were used to decrease NR2F1-AS1 expression. miR-642a mimic or inhibitor, obtained from Shanghai R&S Biotechnology Co. (Shanghai, China), was used to increase or decrease miR-642 expression. Lipofectamine® 2000 (Invitrogen, CA, USA) was carried out to perform the transfections for 48 h.


## 2.3. Quantitative Real-Time PCR (qRT-PCR)

Isolation of total RNA from HCC tissues or cells was carried out with TRIzol reagent (Invitrogen, NY, USA). cDNA was synthesized using a specific reverse transcription kit (Thermo Fisher). QRT-PCR was performed with SYBR Green (ThermoFisher, MA, USA). U6 or GAPDH was applied as an internal control. The relative quantitation of gene expressions was performed using the 2 − ΔΔCt method. Primers used in this study are listed as follows: NR2F1-AS1-F: 5′-CAGCGGTGCAAACCATGTGC-3′; NR2F1-AS1-R: 5′-GTAAACCAAGTCGGTTGAACG-3′; miR-642a-F: 5′-GCGGTCCCTCT CCAAATGT-3′, miR-642a-R: 5′-AGTGCAGGGTCCGAGGTATT-3′; U6-F: 5′-CTC GCTTCGGCAGCACA-3′, U6-R: 5′-AACGCTTCACGAATTTGCGT-3′; DEK-F: 5′-TGTTAAGAAAGCAGATAGCAGGACC-3′; and DEK-R: 5′-ATTAAAGGTTCAT CATCTGAACTATCCTC-3′.


## 2.4. Western Blot Assay

50μg protein lysates were separated by SDS-PAGE and then transferred to PVDF membranes. Afterwards, the membranes were blocked with 5% skim milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. After incubating with a secondary antibody for 2 h at room temperature, the protein bands were visualized by an ECL kit. GAPDH was used as the loading control.


## 2.5. Transwell Assay

For the migration experiment, HCC cells were seeded into the upper chamber of a transwell insert, and the lower chamber was fixed with a medium with 20% FBS. For the invasion experiment, the upper chamber was coated with Matrigel, and the lower chamber contained a 20% FBS medium. After incubation for 48 h, the cells were fixed, stained, and then counted under a microscope.


## 2.6. RNA Immunoprecipitation (RIP) Assay

After transfection with miR-642a mimic or inhibitor for 48 h, Huh7 cells were lysed with RIP lysis buffer and incubated with RIP immunoprecipitation buffer mixed with magnetic beads conjugating human anti-Argonaute 2 (Ago2) antibody or negative control mouse IgG (Millipore, MA, USA). Acquired RNAs were analyzed by qRT-PCR. IgG served as control in the RIP assay.


## 2.7. Luciferase Reporter Assay

The wild and mutant types of NR2F1-AS1 or DEK containing the binding site for miR-642a were cloned into pGL3 reporter vectors, designed as pGL3-NR2F1-AS1-WT and pGL3-NR2F1-AS1-MuT or pGL3-DEK-WT and pGL3-DEK-MuT, and then, they were transfected into Huh7 cells with miR-642a mimic/inhibitor for 48 h using Lipofectamine 2000.


## 2.8. Statistical Analysis

Independent repetitions of experiments were performed three times. The data were presented as means ± SD. The differences were analyzed using two-tailed Student’st-tests or Turkey’s post hoc tests in one-way ANOVA. Statistical analysis was performed using SPSS Statistics 20.0 software. P<0.05 was considered statistically significant.


## 3. Results

### 3.1. NR2F1-AS1 Was Highly Expressed in HCC Tissues

To investigate the significance of NR2F1-AS1 in HCC, the expressional level of NR2F1-AS1 was first measured in 36 matched pairs of HCC tissues using the qRT-PCR assay. Results displayed that NR2F1-AS1 was highly expressed in HCC tumor tissues when compared to adjacent nontumor tissues (Figure1(a)). The median value of NR2F1-AS1 expression was used as the cutoff point to divide NR2F1-AS1 expression into high and low expression of NR2F1-AS1. HCC patients with low NR2F1-AS1 expression exhibited high overall survival, while those with high NR2F1-AS1 expression showed low overall survival (Figure 1(b)). Furthermore, NR2F1-AS1 was increased in HCC cells in comparison with normal HL-7702 cells (Figure 1(c)). These results demonstrated that NR2F1-AS1 was closely related to the prognosis of HCC patients and might take part in HCC progression.Figure 1
NR2F1-AS1 highly expressed in HCC. (a) High expression of NR2F1-AS1 detected in HCC tumor tissues (n = 36). (b) Lower survival of HCC patients with high NR2F1-AS1 expression than (n = 18) with low NR2F1-AS1 expression (n = 18). (c) High expression of NR2F1-AS1 measured in HCC cell lines. ∗∗P<0.01.
(a)(b)(c)


### 3.2. Knockdown of NR2F1-AS1 Inhibited HCC Cell Invasion and Migration

Next, the functional significance of NR2F1-AS1 was explored in Huh7 and SK-Hep-1 cells. NR2F1-AS1 expression was downregulated after transfection with shRNA-NR2F1-AS1 in both two HCC cells (Figure2(a)). Transwell migration assay manifested that HCC cells migration was decreased in the sh-NR2F1-AS1 group when compared to the control group (Figure 2(b)). Moreover, decreasing NR2F1-AS1 attenuated the invasion of HCC cells compared to the control group (Figure 2(c)). These results indicated that the silence of NR2F1-AS1 suppressed HCC cell invasion and migration.Figure 2
Knockdown of NR2F1-AS1 suppressing cell invasion and migration in HCC. (a) NR2F1-AS1 expression significantly inhibited by sh-NR2F1-AS1 in Huh7 and SK-Hep-1 cells. (b) HCC cells migration suppressed by transfection with sh-NR2F1-AS1. (c) HCC cells invasion repressed by transfection with sh-NR2F1-AS1.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)


### 3.3. NR2F1-AS1 Directly Interacted with miR-642a

Then, the underlying molecular mechanism of NR2F1-AS1 in Huh7 cells was further studied. The starBase database predicted miR-642a as a target of NR2F1-AS1 (Figure3(a)). MiR-642a expression was increased or decreased after transfection with miR-642a mimic or inhibitor in Huh7 cells by qRT-PCR (Figure 3(b)). Luciferase reporter assay was then performed to validate this prediction, followed by RIP assay. Luciferase reporter assay discovered that miR-642a mimic reduced, while miR-642a inhibitor increased NR2F1-AS1-WT’ luciferase activity. However, neither miR-642a mimic nor inhibitor showed any effect on NR2F1-AS1-MuT’ luciferase activity (Figure 3(c)). RIP results displayed that NR2F1-AS1 and miR-642a expressions were elevated in the anti-Ago2 group versus the control group (Figure 3(d)). Then, qRT-PCR was applied to investigate the correlation of NR2F1-AS1 with miR-642a, and the findings exhibited that overexpression of NR2F1-AS1 significantly decreased miR-642a expression, while knockdown of NR2F1-AS1 markedly increased miR-642a expression in both two HCC cells (Figure 3(e)), indicating a negative correlation of NR2F1-AS1 with miR-642a. Then, miR-642a expression was detected in HCC tissues, and the findings displayed that miR-642a was underexpressed in HCC tissues versus nontumor tissues (Figure 3(f)). Pearson’s correlation analysis discovered that NR2F1-AS1 was inversely associated with miR-642a in HCC tissues (Figure 3(g)). These results suggested that NR2F1-AS1 directly targets miR-642a in HCC.Figure 3
NR2F1-AS1 directly interacted with miR-642a. (a) The predicted binding site between NR2F1-AS1 and miR-642a. (b) qRT-PCR detection of the overexpression and knockdown efficiency for miR-642a. (c) The luciferase activity of the NR2F1-AS1-WT affected by miR-642a overexpression or knockdown. (d) Increased NR2F1-AS1 and miR-642a expression in the anti-Ago2 group compared to the LgG group. (e) NR2F1-AS1 negatively regulating miR-642a expression. (f) Downregulation of miR-642a in HCC tumor tissues versus nontumor tissues (n = 36). (g) The inverse correlation between NR2F1-AS1 and miR-642a in HCC tissues (n = 36). ∗∗P<0.01 and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. NR2F1-AS1 Sponged miR-642a to Modulate Cell Invasion and Migration in HCC

Next, the impact of miR-642a on NR2F1-AS1-regulated cell invasion and migration was investigated in HCC cells. Huh7 and SK-Hep-1 cells were transfected with sh-NR2F1-AS1 or combined with miR-642a inhibitors. Transwell migration results displayed that the migration of HCC cells reduced by sh-NR2F1-AS1 could be rescued by incorporating miR-642a inhibitor (Figures4(a) and 4(b)). Moreover, the transwell invasion assay manifested that the sh-NR2F1-AS1 effect on HCC cells invasion was also partly restored by combining with miR-642a inhibitor (Figures 4(c) and 4(d)). These results demonstrated that NR2F1-AS1 modulated HCC cell migration and invasion through miR-642a.Figure 4
NR2F1-AS1 modulating HCC cell migration and invasion by sponging miR-642a. (a) miR-642a inhibitor increasing the cell migration reduced by sh-NR2F1-AS1 in Huh7 and (b) SK-Hep-1 cells. (c) miR-642a inhibitor increasing the cell invasion reduced by sh-NR2F1-AS1 in Huh7 and (d) SK-Hep-1 cells.∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)


### 3.5. NR2F1-AS1-Regulated DEK through miR-642a in HCC Cells

TargetScan Human 7.2 was applied to predict the potential target genes of miR-642a, and DEK was found to own a binding site for miR-642a (Figure5(a)). Then, the luciferase assay was carried out to confirm this prediction further, and results discovered that DEK-WT’ luciferase activity was remarkably inhibited by miR-642a mimic in Huh7 cells, but not in the DEK-MuT group (Figure 5(b)), indicating that DEK was a downstream target of miR-642a. More importantly, DEK expression was inhibited by miR-642a mimic, while enhanced by miR-642a inhibitor using qRT-PCR and Western blot assays (Figures 5(c) and 5(d)). Afterwards, the relationship between NR2F1-AS1, miR-642a, and DEK was explored, and results showed that DEK expression increased by miR-642a inhibitor could be reduced by sh-NR2F1-AS1 (Figures 5(e) and 5(f)), indicating that NR2F1-AS1 facilitated DEK expression via suppressing miR-642a.Figure 5
NR2F1-AS1 regulating DEK expression through miR-642a. (a) The putative DEK binding site with miR-642a. (b) Luciferase activities of DEK-WT or -MuT detected after transfection with miR-642a mimic in Huh7 cells. (c) DEK mRNA expression and (d) protein level decreased by miR-642a mimic, while increased by miR-642a inhibitor in HCC cells. (e-f) Upregulation of DEK increased by miR-642a inhibitor reduced by NR2F1-AS1 knockdown.∗∗P<0.01.


## 3.1. NR2F1-AS1 Was Highly Expressed in HCC Tissues

To investigate the significance of NR2F1-AS1 in HCC, the expressional level of NR2F1-AS1 was first measured in 36 matched pairs of HCC tissues using the qRT-PCR assay. Results displayed that NR2F1-AS1 was highly expressed in HCC tumor tissues when compared to adjacent nontumor tissues (Figure1(a)). The median value of NR2F1-AS1 expression was used as the cutoff point to divide NR2F1-AS1 expression into high and low expression of NR2F1-AS1. HCC patients with low NR2F1-AS1 expression exhibited high overall survival, while those with high NR2F1-AS1 expression showed low overall survival (Figure 1(b)). Furthermore, NR2F1-AS1 was increased in HCC cells in comparison with normal HL-7702 cells (Figure 1(c)). These results demonstrated that NR2F1-AS1 was closely related to the prognosis of HCC patients and might take part in HCC progression.Figure 1
NR2F1-AS1 highly expressed in HCC. (a) High expression of NR2F1-AS1 detected in HCC tumor tissues (n = 36). (b) Lower survival of HCC patients with high NR2F1-AS1 expression than (n = 18) with low NR2F1-AS1 expression (n = 18). (c) High expression of NR2F1-AS1 measured in HCC cell lines. ∗∗P<0.01.
(a)(b)(c)


## 3.2. Knockdown of NR2F1-AS1 Inhibited HCC Cell Invasion and Migration

Next, the functional significance of NR2F1-AS1 was explored in Huh7 and SK-Hep-1 cells. NR2F1-AS1 expression was downregulated after transfection with shRNA-NR2F1-AS1 in both two HCC cells (Figure2(a)). Transwell migration assay manifested that HCC cells migration was decreased in the sh-NR2F1-AS1 group when compared to the control group (Figure 2(b)). Moreover, decreasing NR2F1-AS1 attenuated the invasion of HCC cells compared to the control group (Figure 2(c)). These results indicated that the silence of NR2F1-AS1 suppressed HCC cell invasion and migration.Figure 2
Knockdown of NR2F1-AS1 suppressing cell invasion and migration in HCC. (a) NR2F1-AS1 expression significantly inhibited by sh-NR2F1-AS1 in Huh7 and SK-Hep-1 cells. (b) HCC cells migration suppressed by transfection with sh-NR2F1-AS1. (c) HCC cells invasion repressed by transfection with sh-NR2F1-AS1.∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)


## 3.3. NR2F1-AS1 Directly Interacted with miR-642a

Then, the underlying molecular mechanism of NR2F1-AS1 in Huh7 cells was further studied. The starBase database predicted miR-642a as a target of NR2F1-AS1 (Figure3(a)). MiR-642a expression was increased or decreased after transfection with miR-642a mimic or inhibitor in Huh7 cells by qRT-PCR (Figure 3(b)). Luciferase reporter assay was then performed to validate this prediction, followed by RIP assay. Luciferase reporter assay discovered that miR-642a mimic reduced, while miR-642a inhibitor increased NR2F1-AS1-WT’ luciferase activity. However, neither miR-642a mimic nor inhibitor showed any effect on NR2F1-AS1-MuT’ luciferase activity (Figure 3(c)). RIP results displayed that NR2F1-AS1 and miR-642a expressions were elevated in the anti-Ago2 group versus the control group (Figure 3(d)). Then, qRT-PCR was applied to investigate the correlation of NR2F1-AS1 with miR-642a, and the findings exhibited that overexpression of NR2F1-AS1 significantly decreased miR-642a expression, while knockdown of NR2F1-AS1 markedly increased miR-642a expression in both two HCC cells (Figure 3(e)), indicating a negative correlation of NR2F1-AS1 with miR-642a. Then, miR-642a expression was detected in HCC tissues, and the findings displayed that miR-642a was underexpressed in HCC tissues versus nontumor tissues (Figure 3(f)). Pearson’s correlation analysis discovered that NR2F1-AS1 was inversely associated with miR-642a in HCC tissues (Figure 3(g)). These results suggested that NR2F1-AS1 directly targets miR-642a in HCC.Figure 3
NR2F1-AS1 directly interacted with miR-642a. (a) The predicted binding site between NR2F1-AS1 and miR-642a. (b) qRT-PCR detection of the overexpression and knockdown efficiency for miR-642a. (c) The luciferase activity of the NR2F1-AS1-WT affected by miR-642a overexpression or knockdown. (d) Increased NR2F1-AS1 and miR-642a expression in the anti-Ago2 group compared to the LgG group. (e) NR2F1-AS1 negatively regulating miR-642a expression. (f) Downregulation of miR-642a in HCC tumor tissues versus nontumor tissues (n = 36). (g) The inverse correlation between NR2F1-AS1 and miR-642a in HCC tissues (n = 36). ∗∗P<0.01 and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. NR2F1-AS1 Sponged miR-642a to Modulate Cell Invasion and Migration in HCC

Next, the impact of miR-642a on NR2F1-AS1-regulated cell invasion and migration was investigated in HCC cells. Huh7 and SK-Hep-1 cells were transfected with sh-NR2F1-AS1 or combined with miR-642a inhibitors. Transwell migration results displayed that the migration of HCC cells reduced by sh-NR2F1-AS1 could be rescued by incorporating miR-642a inhibitor (Figures4(a) and 4(b)). Moreover, the transwell invasion assay manifested that the sh-NR2F1-AS1 effect on HCC cells invasion was also partly restored by combining with miR-642a inhibitor (Figures 4(c) and 4(d)). These results demonstrated that NR2F1-AS1 modulated HCC cell migration and invasion through miR-642a.Figure 4
NR2F1-AS1 modulating HCC cell migration and invasion by sponging miR-642a. (a) miR-642a inhibitor increasing the cell migration reduced by sh-NR2F1-AS1 in Huh7 and (b) SK-Hep-1 cells. (c) miR-642a inhibitor increasing the cell invasion reduced by sh-NR2F1-AS1 in Huh7 and (d) SK-Hep-1 cells.∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)


## 3.5. NR2F1-AS1-Regulated DEK through miR-642a in HCC Cells

TargetScan Human 7.2 was applied to predict the potential target genes of miR-642a, and DEK was found to own a binding site for miR-642a (Figure5(a)). Then, the luciferase assay was carried out to confirm this prediction further, and results discovered that DEK-WT’ luciferase activity was remarkably inhibited by miR-642a mimic in Huh7 cells, but not in the DEK-MuT group (Figure 5(b)), indicating that DEK was a downstream target of miR-642a. More importantly, DEK expression was inhibited by miR-642a mimic, while enhanced by miR-642a inhibitor using qRT-PCR and Western blot assays (Figures 5(c) and 5(d)). Afterwards, the relationship between NR2F1-AS1, miR-642a, and DEK was explored, and results showed that DEK expression increased by miR-642a inhibitor could be reduced by sh-NR2F1-AS1 (Figures 5(e) and 5(f)), indicating that NR2F1-AS1 facilitated DEK expression via suppressing miR-642a.Figure 5
NR2F1-AS1 regulating DEK expression through miR-642a. (a) The putative DEK binding site with miR-642a. (b) Luciferase activities of DEK-WT or -MuT detected after transfection with miR-642a mimic in Huh7 cells. (c) DEK mRNA expression and (d) protein level decreased by miR-642a mimic, while increased by miR-642a inhibitor in HCC cells. (e-f) Upregulation of DEK increased by miR-642a inhibitor reduced by NR2F1-AS1 knockdown.∗∗P<0.01.


## 4. Discussion

Hepatocellular carcinoma is one of the most common causes of cancer-related death globally [13]. Recently, lncRNA profile analysis and functional analysis of various types of cancer have provided more and more evidence that lncRNA plays a critical role in tumor growth and development [14]. lncRNA is considered to be a new diagnostic biomarker, effective prognostic predictor, and attractive therapeutic target for HCC through interactions with cellular macromolecules, such as proteins and mRNAs [15, 16]. Thus, it is critically called for to understand the molecular mechanism of lncRNAs in HCC progression.Prior research confirmed that lncRNA NR2F1-AS1 modulated the pathological progressions of tumors. For instance, NR2F1-AS1 was overexpressed in tumor tissues and involved in endometrial cancer development [17]. NR2F1-AS1 knockdown inhibited thyroid cancer progression in vitro and in vivo [18]. Liet al. discovered that NR2F1-AS1 was highly expressed in osteosarcoma and exhibited an oncogene role in OS development [19]. In this study, we found that NR2F1-AS1 was increased in HCC tissues and associated with the prognosis of HCC patients. Moreover, HCC cell migration and invasion were inhibited distinctly by sh-NR2F1-AS1. These results suggested that NR2F1-AS1 may also play an oncogenic role in HCC.Accumulating evidence has discovered that lncRNAs exhibited key roles in tumors by sponging miRNAs [20, 21]. In HCC, lncRNA H19 was discovered to promote cell proliferation, invasion, and migration yet suppressed cell apoptosis through miR-520a/LIMK1 axis [22]. Upregulation of CASC15 enhanced the tumorigenicity and epithelial to mesenchymal transition of HCC by increasing TWIST1 gene expression via miR-33a-5p sponging [23]. Xiao et al. found that LINC01123 sponges miR-34a-5p to promote cell proliferation and invasion in HCC through modulating TUFT1 [24]. To identify NR2F1-AS1’s underlying mechanism in HCC, the bioinformatics analysis was applied to screen the potential targets of NR2F1-AS1 and found that miR-642a served as the candidate target. Luciferase reporter assay and RIP assay further confirmed miR-642a was a direct target of NR2F1-AS1. Moreover, we observed that NR2F1-AS1 negatively regulated miR-642a expression. Importantly, miR-642a inhibitor could rescue the sh-NR2F1-AS1 effect on HCC cell invasion and migration.DEK is a potential biomarker and oncogene, which is reported in many cancers [25–27]. DEK was found to involve in the development of pancreatic cancer as a target of miR-200a [28]. Also, miR-1292 inhibited gastric cancer cell growth, migration, and invasion via targeting DEK [29]. However, whether DEK served as the target of miR-642a was not reported until now. In this study, we found that DEK served as the direct target of miR-642a following the luciferase and RIP assays. Prior research has shown that abnormal expression of DEK exists in various tumors. For instance, DEK was significantly elevated in pancreatic cancer and cervical cancer [30, 31]. Soo Yeon Lee et al. discovered that DEK was highly expressed in HCC, and its high expression was associated with poor prognosis of HCC patients [27, 32], which is consistent with our study that DEK was increased significantly in HCC tissues. Our study also found that NR2F1-AS1 aggravated the expression of DEK in HCC cells by decreasing the expression of miR-642a.There are some limitations to this study that we only investigated the biological functions and mechanism of NR2F1-AS1 in cell migration and invasion in vitro. The functional role of NR2F1-AS1 in vivo animal experiments is required to explore in the future studies.Taken together, our results demonstrated that NR2F1-AS1 was highly expressed in HCC, and silence of NR2F1-AS1 suppressed cell invasion and migration in HCC cells via miR-642a/DEK axis. The NR2F1-AS1/miR-642a/DEK axis might provide new insights for designing promising therapeutic strategies for HCC.



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