Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p

Abstract Long non‐coding RNAs (lncRNAs) could regulate growth and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to investigate the mechanism of lncRNA F11‐AS1 in hepatitis B virus (HBV)–related HCC. The relation of lncRNA F11‐AS1 expression in HBV‐related HCC tissues to prognosis was analysed in silico. Stably HBV‐expressing HepG2.2.15 cells were established to explore the regulation of lncRNA F11‐AS1 by HBx protein, as well as to study the effects of overexpressed lncRNA F11‐AS1 on proliferation, migration, invasion and apoptosis in vitro. Subsequently, the underlying interactions and roles of lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis in HBV‐related HCC were investigated. Additionally, the influence of lncRNA F11‐AS1 and miR‐211‐5p on tumour growth and metastasis capacity of HepG2.2.15 cells were studied on tumour‐bearing nude mice. Poor expression of lncRNA F11‐AS1 was correlated with poor prognosis in patients with HBV‐related HCC, and its down‐regulation was caused by the HBx protein. lncRNA F11‐AS1 was proved to up‐regulate the NR1I3 expression by binding to miR‐211‐5p. Overexpression of lncRNA F11‐AS1 reduced the proliferation, migration and invasion, yet induced apoptosis of HepG2.2.15 cells in vitro, which could be abolished by overexpression of miR‐211‐5p. Additionally, either lncRNA F11‐AS1 overexpression or miR‐211‐5p inhibition attenuated the tumour growth and metastasis capacity of HepG2.2.15 cells in vivo. Collectively, lncRNA F11‐AS1 acted as a modulator of miR‐211‐5p to positively regulate the expression of NR1I3, and the lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis participated in HBV‐related HCC progression via interference with the cellular physiology of HCC.


| INTRODUC TI ON
The leading cause of hepatocellular carcinoma (HCC) is infection caused by hepatotropic viruses such as hepatitis B virus (HBV), HCV and HDV, among which HBV infection serves as the main trigger accounting for approximately 54% of all liver cancer cases. 1 The mechanisms that contribute to HBV-induced HCC include deregulated control of cellular proliferation and apoptosis, which may be partially attributed to abnormal alterations in certain gene expression such as hepatitis B virus X (HBx) protein. 2 Currently, various therapeutic options are applied for HCC, such as chemotherapeutics and miRNA-based therapies combining surgery. 3 Additionally, liver transplantation and resection are considered as the best approaches to treat HCC and to deliver long-term survival for the early-diagnosed HCC patients. However, these methods are not suitable for advanced HCC patients, owing to disappointing 5-year survival rates. 4 Hence, it is trivial to attain a better and deeper understanding of the pathophysiology of HCC to effectively treat this liver cancer.
Recently, dysregulation of long non-coding RNAs (lncRNAs) has been highlighted to share association with tumorigenesis and metastasis of HCC. 5 Particularly, lncRNA F11-antisense 1 (lncRNA F11-AS1) has been uncovered to be implicated in the progression of some carcinomas. For instance, down-regulated levels of lncRNA F11-AS1 have been documented in pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that its misregulation might exert critical roles in PDAC. 6 In addition, lncRNA F11-AS1 is also poorly expressed in ovarian cancer; moreover, differential expression of lncRNA F11-AS1 in cell lines that exhibited platinum-based drug resistance or non-resistance implied that lncRNA F11-AS1 could be regarded as a prognostic marker to predict the incidence of platinum-based chemoresistance. 7 All these previous findings and results indicate the prognostic and therapeutic values of lncRNA F11-AS1 in multiple cancers; however, the exact role of lncRNA F11-AS1 in HCC has not been investigated yet.
More recently, emerging evidence has demonstrated that ln-cRNAs can function as a competing endogenous RNA (ceRNA) for specific miRNAs and regulate their function and downstream targets of miRNA to influence the development of HCC. 8,9 For example, lncRNA F11-AS1 is reported to bind to miR-3146 to affect the HCC progression. 10 microRNA-211 (miR-211) is closely related to the progression and development of human cancers, and lncRNA SNHG15 promotes the occurrence and progression of lung cancer by enhancing the proliferative and migration capacity of lung cancer cells through binding to miR-211-3p. 11 In oral squamous cell carcinoma, miR-211-3p directly suppresses the transcription factor 12, which regulates the activities of cancer-related suppressors in tumour cells. 12 In silico analysis and luciferase experiment in the current study confirmed that lncRNA F11-AS1 could potentially bind to miR-211-5p and nuclear receptor subfamily 1, group I, member 3 (NR1I3), also known as CAR, was a target gene of miR-211-5p. The functionality of NR1I3, an important regulator of drug metabolism and cancer development, is mediated via modulation of transcription of target genes. 13 Therefore, we proposed a hypothesis that the interactions among lncRNA F11-AS1/ miR-211-5p/NR1I3 axis might be involved in tumorigenesis of HBV-related HCC, and the subsequent experiments in the current study were performed to study the effects of this axis on the physiological functions of stably HBV-expressing HepG2.2.15 cells.

| Ethics statement
The current study was approved by the Ethics Committee of the Affiliated Hospital of Youjiang Medical College for Nationalities.   Table 1. The tissue samples were frozen in liquid nitrogen and stored in a −80°C refrigerator for subsequent use. cells were incubated in minimum essential medium (MEM)-α medium (M8042) containing 10% FBS, and additionally supplemented with 100 U/mL penicillin/streptomycin, 200 μg/mL G418 (1013102700) and 2 mmo/L l-glutamine. Then, the cells were maintained under a condition of 5% CO 2 at 37°C. 14 The cell culture medium and reagents used in this part were all purchased from Gibco BRL/Invitrogen. Expression of lncRNA F11-AS1 in each cell line was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the cell line exhibiting the lowest lncRNA F11-AS1 expression was selected for subsequent experimentation. The experiments were repeated 3 times to obtain the mean value.

| Cell transfection
Cell transfection was conducted according to the instructions of Lipofectamine 2000 transfection kit (11668019; Invitrogen). A total of 1 × 10 6 cells were treated with 50 μg plasmids or lncRNA F11-AS1 overexpression plasmid (oe-lncF11-AS1), miR-211-5p mimic, miR-211-5p inhibitor, oe-NR1I3 and their corresponding negative controls (oe-NC and miR-NC), mixed well and later incubated for 6 h at 37°C. Afterwards, the culture medium was renewed with complete medium, following incubation for another 24-48 hours. RNA content extraction from cells was performed in order to assess the transfection efficiency. All the plasmids used in this study were constructed by Shanghai Sangon Biotechnology Co., Ltd.

| Flow cytometry
After transfection for 48 hours, the cells were treated with ethylenediaminetetraacetic acid (EDTA)-free 0.25% trypsin (YB15050057; Shanghai Yubo Biotechnology Co., Ltd.) and collected in a flow tube.
The cells were then centrifuged with the supernatant discarded.
Subsequently, cell apoptosis assay was performed with Annexin

| RNA isolation and quantitation
Total RNA content extraction from tissues or cells after 36-hours transfection was performed according to the manufacturer's protocols provided by the TRIzol reagent (10296010; Invitrogen). All the primers (Table 2) were synthesized by BGI Biotech Co., Ltd.
(Beijing, China). For RT-qPCR of miRNAs, 100 ng of total RNA was reverse-transcribed and subjected to TaqMan TM MicroRNA assay (4366596; Applied Biosystems, Inc). Subsequently, the expression of miR-211-5p was determined by qPCR with a TaqMan TM MicroRNA assay kit (4427975; Applied Biosystems, Inc) and standardized using U6. For RT-qPCR of mRNAs, cDNA synthesis was performed with the total RNA using EasyScript First-Strand cDNA Synthesis SuperMix (AE301-02; Beijing TransGen Biotech Co., Ltd.) and standardized using housekeeping coding gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Subsequently, the quantitative analysis of mRNA expression was conducted according to the instructions of SYBR ® Premix Ex TaqTM II kit (RR820A; TaKaRa) on an ABI7500 real-time PCR instrument (Applied Biosystems).
Finally, the fold changes were calculated by means of relative quantification (2 −∆∆Ct ). The experiments were repeated 3 times to obtain the mean value.
Abbreviations: AFP, alpha-fetoprotein; TNM, tumour node metastasis; lncRNA F11-AS1, long non-coding RNA F11-antisense 1. and the other portion was incubated with antibodies for coprecipitation. The magnetic bead-antibody complex was resuspended in 900 μL RIP immunoprecipitation buffer after washing. The samples were placed on the magnetic seat to obtain a magnetic bead-antibody complex. The antibodies used for RIP were 5 μg Ago2 antibody (39854; Active Motif) and 5 μg mouse anti-IgG (ab200699; Abcam).
The extracted RNA was purified using the conventional TRIzol method followed by RT-qPCR assay.

| Fluorescence in situ hybridization (FISH)
The subcellular localization of lncRNA F11-AS1 and its colocalization with miR-211-5p were detected using FISH kits (BIS-P0001; BersinBio, Guangzhou  using a 10-μL sterile pipette. After being washed two times with PBS, the cells were further cultured in MEM-α complete medium containing 10% FBS. Afterwards, images of each well at 0 and 24 hours postscratching were pictured under the inverted microscope, with three replicates set for each group. The width of each scratch was determined using the ImageJ software.

| Transwell assay
The invasion ability of HepG2.

| Western blot analysis
Western blot analysis was performed according to the previous literature. 16 Total protein in tissues and cells was extracted using RIPA cell lysis buffer (R0010; Solarbio) containing PMFS, which was then separated through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the protein was transferred onto the polyvinylidene fluoride membrane (PVDF, FFP36; Beyotime Biotechnology Co., Ltd.) and blocked with 5% bovine serum albumin (BSA) at 37°C for 2 hours. Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl-2 (SAB4500005, 1:1000) from Sigma-Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C. The next day, the membrane was incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (ab6721, 1:20 000) or goat antimouse IgG (ab6789, 1:10 000) from Abcam for 1 h at room temperature. Finally, the protein on the membrane was visualized using an image developing kit (P0020; Beyotime Biotechnology Co., Ltd.), and photographed using a Bio-Rad gel imaging analysis system (Bio-Rad). The protein expression was analysed using the ImageJ software (National Institutes of Health).GAPDH was used as an internal control. The experiment was repeated three times.

| Xenograft tumour in nude mice
Specific-pathogen-free athymic female BALB/C nude mice (aged

| Immunocytochemistry and immunohistochemistry
The coverslips of HepG2. HBV + or HBV − HCC tissue samples were fixed in 4% paraformaldehyde, paraffin-embedded and prepared into 5-μm serial sections.
Subsequently, the paraffin-embedded sections were deparaffinized in xylene, rehydrated with distilled water and then subjected to antigen retrieval with 0.01 mol/L citrate buffer at 95°C for 30 minutes.
The sections were then incubated with the primary antibody at 4°C overnight. The following observatory and calculation procedures were performed as previously mentioned.

| Nuclear/cytoplasmic fractionation
When cell density reached 1 × 10 6 cells/mL, the cells were lysed with 200 μL lysis buffer. After centrifugation, the supernatant (cytoplasmic fractions) and the precipitation (nuclear fractions) were added with Buffer SK and anhydrous ethanol, respectively. After centrifugation, the cytoplasmic fractions and nuclear fractions were harvested with elution.

| Northern blot analysis
Total RNA content was extracted from each cell line using the TRIzol

| Statistical analysis
Each experiment was repeated at least three times, and the statistics were analysed using the SPSS 21.0 software (IBM Corp.). All data were subjected to analysis of normal distribution and variance homogeneity, and measurement data were expressed by mean ± standard deviation.
If data were consistent with normal distribution and even variance, Consequently, a higher survival rate was determined in HBV + HCC patients with a higher lncRNA F11-AS1 expression than those with lower lncRNA F11-AS1 expression ( * P < .05) ( Figure 1C). Subsequently, lncRNA F11-AS1 was found to be primarily localized in the cytoplasm of HepG2.2.15 cells as demonstrated by FISH assay. After the cytoplasmic RNA and nuclear RNA were isolated, the expression of U1 and GAPDH in nucleus and cytoplasm was detected by qPCR. As U1 was known to be mainly expressed in the nucleus, whereas GAPDH was expressed in the cytoplasm, the cytoplasmic and nuclear lncRNA F11-AS1 were normalized to GAPDH and U1, respectively. It was observed that the expression of lncRNA F11-AS1 in the cytoplasm was higher than that in the nucleus ( * P < .05) ( Figure 1D). Collectively, the results indicated that poorly expressed lncRNA F11-AS1 might serve as an indicator of poor prognosis in patients with HBV-related HCC. Comparison of data between matched HCC and normal tissues was analysed by paired t test and that between the two groups was analysed by unpaired t test, and that among multiple groups was examined by one-way analysis of variance followed by Tukey's post hoc test

| lncRNA F11-AS1 up-regulation suppresses proliferation, migration and invasion yet induces apoptosis of HBV + HCC cells
To
Based on the prediction results of the RNA22 database (https :// cm.jeffe rson.edu/rna22/ Inter activ e/), lncRNA F11-AS1 could potentially bind to miR-211-5p in HCC ( Figure 4A). Interestingly, miR-211-5p has also been reported to be differentially expressed in numerous malignancies, as well as to be involved in the regulation of cellular processes such as proliferation, migration and apoptosis. [20][21][22][23][24] Therefore, the focus of the experiment was shifted to the exploration of the relationship between lncRNA F11-AS1 and miR-211-5p.
The expression patterns of miR-211-5p in HBV ± HCC tissues and corresponding adjacent tissues were determined by RT-qPCR, which revealed that there were no significant differences in miR-211-5p expression between the HBV − HCC tissues and corresponding adjacent tissues ( * P > .05). However, a relatively higher expression of miR-211-5p was observed in HBV + HCC tissues compared with that in corresponding adjacent tissues ( * P < .05). Additionally, miR-211-5p was found to be highly expressed in HBV + HCC tissues in comparison with the HBV − HCC tissues ( * P < .05) ( Figure 4B). Then, miR-   Together, these results supported the conclusion that miR-211-5p could abolish the anti-cancer effect of lncRNA F11-AS1 on HCC cells.

| lncRNA F11-AS1 up-regulates NR1I3 expression by competitively binding to miR-211-5p
Considering the eliminating effects exerted by miR-211-5p on tumour suppressive effects of lncRNA F11-AS1, the focus of the study shifted onto exploration of the possible mechanism of miR-211-5p.
Subsequently, a binding site between miR-211-5p and NR1I3 was found according to bioinformatics analysis ( Figure 6A). In addition, NR1I3 has also been documented to exert its regulatory role in breast cancer and colorectal cancer, especially in hepatocarcinogens. 25 Then, NR1I3 expression in HBV + /HBV − HCC tissues and corresponding adjacent tissues was examined by RT-qPCR and Western blot analysis. As depicted in Figure 6B, no significant differences were observed regarding NR1I3 expressions in HBV − HCC tissues and corresponding adjacent tissues ( * P > .05); however, HBV + HCC tissues exhibited relatively lower NR1I3 expression compared with corresponding adjacent tissues ( * P < .05). Furthermore, compared with HBV − HCC tissues, HBV + HCC tissues displayed significantly low levels of NR1I3 expression ( * P < .05). As shown in Figure 6C, A lower NR1I3 expression was observed in HBV + HepG2.2.15 cells in comparison with HBV − HepG2 cells by RT-qPCR and Western blot analysis ( * P < .05). Following transfection with pHBV1.3 into HepG2 and Huh7 cell lines, a significant down-regulation of NR1I3 was determined in both the cell lines ( * P < .05) ( Figure 6D). Subsequent dual-luciferase reporter gene assay and RNA pull-down experiments verified that miR-211-5p could specifically bind to NR1I3 ( * P < .05) ( Figure 6E,F).
Immunocytochemistry revealed that these results were similar to that observed in HepG2.2.15 cells ( Figure 6I). At the same time, the relationship between miR-211-5p and NR1I3 was detected by Northern blot analysis, and consequently, the binding of miR-211-5p to NR1I3 was substantiated ( Figure 6J). We analysed the correlations between the expression of lncRNA F11-AS1, miR-211-5p and NR1I3 in HCC tissues using Pearson's correlation coefficient. The results revealed that the expression of lncRNA F11-AS1 was negatively correlated with that of miR-211-5p, and the same negative correlation was also observed between miR-211-5p and NR1I3. On the contrary, the expression of lncRNA F11-AS1 and NR1I3 was positively correlated ( Figure 6K).
These results suggested that NR1I3 was a target gene of miR- Compared with mice receiving an injection of oe-NC plasmid-delivered cells, the tumour volume and tumour weight and the number of metastatic nodules were significantly decreased in mice receiving injections of either miR-211-5p-antagomir-transfected cells or oe-lncF11-AS1 or NC-agomir-cotransfected cells ( * P < .05). There were no significant differences observed in relation to these parameters between mice administrated with miR-211-5p-antagomir-transfected cells and those injected with oe-lncF11-AS1 and NC-agomircotransduced cells ( * P > .05). However, the tumour volume, tumour weight and the number of metastatic nodules were found to be reduced by overexpression of lncF11-AS1, while they were remarkably increased by overexpression of miR-211-5p ( * P < .05) ( Figure 8A-D).
All these results led to the conclusion that overexpression of lncRNA F11-AS1 inhibited the expression of miR-211-5p to suppress tumour growth and metastasis in vivo.

| D ISCUSS I ON
The lack of reliable early-stage diagnostic modalities has resulted in extremely poor prognoses for HCC patients, and therefore, it is trivial to discover more specific and reliable biomarkers for the early detection of HCC to improve prognostic outcomes. 28 Interestingly, a previous review highlighted the use of lncRNAs as potential markers for cancer diagnosis and prognosis. 29 In the current study, we aimed to investigate the underlying mechanism of lncRNA F11-AS1 regulating NR1I3 by binding to miR-211-5p, thus serving as a tumour suppressor in HBV-related HCC progression.
Initially, we uncovered that lncRNA F11-AS1 was poorly ex- have also been demonstrated to be capable of regulating miRNAs at a post-transcription level through competitively binding to shared miRNAs. 37 For example, lncRNA KCNQ1OT1 stimulates the proliferation and cisplatin resistance of tongue cancer cells by acting as a ceRNA for miR-211-5p. 24 lncRNA tumour suppressor candidate 7 also acts as a miR-211 sponge to inhibit colorectal cancer cell proliferation by down-regulating CDK6. 38 Principally, our results indicated that lncRNA F11-AS1 competes with miR-211-5p to exert its function in HCC. Later, we also observed that miR-211-5p inhibition brought about the same inhibitory effects on HCC cells as lncRNA F11-AS1 overexpression. Some reports have illustrated that the HBx protein-induced miRNA expression also shares association with the carcinogenic network of HCC, [39][40][41] but whether the aberrantly up-regulated miR-211-5p in HCC is directly induced by HBx protein still needed to be investigated.
Additionally, our findings also verified that NR1I3 was tar- stresses. 42 Previously, decreased expression of NR1I3 due to promoter hypermethylation has been strongly correlated with a decline in cytochrome P450 (CYP) 2C19 expression in HBV-related HCC tissues, which might play a role in the tumorigenic processes of HCC. 43 Remarkably, we found that overexpression of lncRNA F11-AS1 could up-regulate the expression of NR1I3 by competitively sponging miR-211-5p, and the lncRNA F11-AS1/miR-211-5p/ NR1I3 axis might participate in HBV-related HCC. Previous studies have also shown that the regulatory axis containing lncRNA and miRNA is widely present in multiple diseases. For example, F I G U R E 9 lncRNA F11-AS1 upregulates the expression of NR1I3 via miR-211-5p to hinder the progression of HBV-related HCC. In HBV-related HCC, HBV-encoded HBx protein inhibited the expression of lncRNA F11-AS1. lncRNA F11-AS1 could up-regulate NR1I3 via binding to miR-211-5p, whereas downregulation of lncRNA F11-AS1 caused by HBx protein weakened its ability to bind to miR-211-5p, which could target to decrease NR1I3 expression. As a result, the proliferation, migration and invasion of HBV-related HCC cells were enhanced, yet cell apoptosis was attenuated. Importantly, overexpression of lncRNA F11-AS1 could enhance the NR1I3 expression by acting as a ceRNA of miR-211-5p, ultimately impeding the progression of HBV + HCC a former study implied that lncRNA MEG3 confers a protective role against congenital intestinal atresia injury via regulation of the miR-221-5p/glial cell-derived neurotrophic factor (GDNF) axis, which suppresses hypoxia-induced intestinal ganglion cell apoptosis. 44 Moreover, the miR-211-5p-mediated inhibition of GDNF was impaired by another lncRNA myocardial infarction-associated transcript, which attenuates neuron apoptosis to alleviate hypoxic-ischaemic injury. 45 And both lncRNA F11-AS1 overexpression and miR-211 suppression were revealed to increase the expression of Bax protein, while reducing that of Bcl-2 and PCNA.
The regulation of lncRNA F11-AS1/miR-211-5p/NR1I3 axis in HBVrelated HCC cell line and in nude mice was preliminarily investigated in the current study. However, the clinical efficacy and future potential use of lncRNA F11-AS1/miR-211-5p/NR1I3 axis in treatment of HBV-related HCC warrant further studies to improve the overall outcomes of HCC patients.

ACK N OWLED G EM ENTS
We would like to acknowledge the helpful comments on this paper received from our reviewers.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated/analysed during the current study are available.