Abstract

Long non-coding RNAs (lncRNAs) play important roles in a variety of tumours; however, their biological function and clinical significance in hepatocellular carcinoma (HCC) are still unclear. In the present study, the clinical significance, biological function and regulatory mechanisms of lncRNA DCST1-AS1 in HCC were investigated. Differential lncRNAs in HCC were identified based on The Cancer Genome Atlas (TCGA) database. The biological function and mechanism of DCST1-AS1 were studied in vitro and in vivo. LncRNA DCST1-AS1 was highly expressed in HCC tissues, and the high expression of DCST1-AS1 was significantly correlated with larger tumours and shorter survival time. Moreover, DCST1-AS1 knockout significantly inhibited proliferation, promoted apoptosis and cycle arrest of HCC cells, and inhibited tumour growth in vivo. According to functional analysis, DCST1-AS1 competitively bound miR-1254, thus blocking the silencing effect of miR-1254 on the target gene Fas apoptosis inhibitor 2 (FAIM2). A novel lncRNA DCST1-AS1 that functions as an oncogene in HCC was discovered. DCST1-AS1 up-regulates the expression of FAIM2 by up-regulating the expression of miR-1254, ultimately promoting the proliferation of HCC cells. This research provides new therapeutic targets for HCC.

Introduction

Hepatocellular carcinoma (HCC) is a type of malignant tumour with the fifth highest incidence rate and third highest mortality rate in the world [1] and is a common malignant tumour in patients’ digestive systems in China [2]. Almost 50% of new HCC cases and cases that lead to death occur in China. Due to the hidden onset, rapid progression, proneness to early metastasis, high mortality rate and poor overall prognosis, the 5-year survival rate of HCC patients remains low, seriously threatening human health [3]. Currently, there is a lack of an effective early diagnosis index, and recurrence and metastasis after operation are still main reasons for the poor prognosis of HCC. The key molecular mechanisms affecting this process remain unclear. Therefore, searching for molecular targets in the occurrence and development of HCC and determining how these may affect prognosis would be of great value in improving the early diagnosis of HCC and the long-term survival rate of patients.

With the rapid development of high-throughput sequencing techniques and the continuous decline in cost in recent years, researchers at home and abroad have made great achievements in tumour regulation at the transcriptional and post-transcriptional levels using high-throughput techniques, such as gene chips, whole genome sequencing and tumour metabolomics [4]. Long non-coding RNAs (lncRNAs) are a type of RNA more than 200 nts in length without protein-coding function and were originally considered to be ‘transcriptional noise’ [5]. In recent years, studies have shown that 95% of miRNA and lncRNA arising from non-coding regions of the genome play important regulatory roles in the occurrence and progression of tumours [6]. There is a complex functional regulatory relationship among non-coding RNAs and between non-coding RNA and protein-coding RNA [6]. Increasingly, more studies have revealed that lncRNA has important physiological functions and plays important regulatory roles in epigenetic modification, transcriptional regulation, post-transcriptional regulation and protein modification [7,8]. Previous studies mainly focused on the role and mechanism of protein-coding genes in HCC. The function of lncRNA in HCC is far less well understood compared with that of protein-coding genes. In the present study, the Cancer Genome Atlas (TCGA) data were screened to identify differentially expressed lncRNAs in HCC tissues and para-carcinoma tissues, and more samples were collected for quantificative real-time PCR (qRT-PCR). DCST1-AS1 was selected as the object of study. A series of bioinformatics and molecular biological methods were adopted to investigate the regulatory correlation and mechanisms of the DCST1-AS1 lncRNA, miRNA and target genes in the occurrence and development of HCC to further clarify the molecular mechanism of lncRNA DCST1-AS1 in regulating the occurrence and development of HCC and provide new intervention targets for HCC treatment and a theoretical basis for the optimisation of treatment strategies.

Materials and methods

Ethical statement

Signed informed consents were received from all patients and the present study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University. All experimental protocols were approved by the Animal Care and Use Committee of Soochow University and followed the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Tissue specimens from patients

A total of 60 pairs of HCC and para-carcinoma tissue specimens were collected from the Specimen Bank of the Third Affiliated Hospital of Soochow University from 2009 to 2013. They were pathologically confirmed as HCC and para-carcinoma normal tissues. After sampling, one part of tissue was immediately transferred to liquid nitrogen, and stored in a low-temperature refrigerator at −80°C to be used for qRT-PCR after RNA extraction. In addition, tissues of patients receiving radical resection of HCC in the hospital from 2009 to 2014 were collected to study lncRNA DCST1-AS1 and the prognosis of HCC patients. All subjects signed informed consents. The study protocol was performed in accordance with the guidelines outlined in the Declaration of Helsinki. The present study was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University.

Cell lines and cell cultures

The cell lines (LO2, HepG2, SMMC-7721, Bel-7404 and SK-hep-1) were purchased from the Cell Bank of the Chinese Academy of Science. Cells were cultured in DMEM or 1640 medium under 5% CO2 at 37°C. The fluid was replaced once every 2–3 days, followed by passage when 90% of the cells had fused. Cells in the logarithmic growth phase were used for experiments.

Cell transfection

At 1 day before transfection, cells were applied on to a six-well plate so that the cell density would reach 30–50% for transfection. The DCST1-AS1 plasmid, DCST1-AS1 siRNA, Fas apoptosis inhibitor 2 (FAIM2) plasmid, FAIM2 siRNA and negative controls were all purchased from Sigma–Aldrich (U.S.A.), and miR-1254 mimics, miR-1254 inhibitors and negative controls were purchased from RiboBio (Guangzhou, China). Transfection was performed according to instructions of the Lipofectamine 2000 transfection reagent, and cells were collected after 48 h for subsequent experiments.

qRT-PCR

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, CA, U.S.A.). RNA purity and integrity were determined using a UV spectrophotometer and agarose gel electrophoresis. RNA was stored at −80°C. qRT-PCR amplification detection was performed using SYBR Green fluorescence dye. GAPDH was used as an internal reference for mRNA, and U6 was used as an internal reference for miRNA. Each experiment was repeated three times. Primer sequences in qRT-PCR are shown in Supplementary Figure S1.

Western blot analysis

Cells in each group were collected, and protein was extracted using routine methods, followed by protein quantification. An appropriate amount of sample separated by SDS/PAGE, and the protein was transferred on to a nitrocellulose membrane and sealed in 5% sealing solution for 4 h at 4°C. Then, LATS2 was added to detect the antibodies and corresponding secondary antibodies. Electrochemiluminescence detection was performed according to instructions of the ECL kit.

Nuclear-cytoplasmic fractionation

Nuclear and cytoplasmic fractions were separated following the instructions of the Protein and RNA Isolation System. U6 was used as an internal reference for the nucleus, and GAPDH was used as an internal reference for the cytoplasm.

Luciferase reporter

A prediction database was used to predict the binding region of miR-1254 and DCST1-AS1, and a wild-type DCST1-AS1 fluorescence plasmid containing the binding site and a DCST1-AS1 fluorescence plasmid with a binding site mutation were constructed. The control DCST1-AS1 WT and DCST1-AS1 MUT fluorescence plasmids were co-transfected with the miR-1254 mimic, and the control was simulated in HepG2 cells. Forty-eight hours after transfection, luciferase activity was measured using a dual luciferase reporter gene assay system according to the instructions. In addition, a database was used to predict the binding sites of the miR-1254 and FAIM2 3′-UTR to construct a wild-type FAIM2 3′-UTR fluorescence plasmid containing the binding site and a FAIM2 3′-UTR fluorescence plasmid with a mutated binding site.

RNA immunoprecipitation

The extent of RNA enrichment was examined by qRT-PCR using a Magna RIP RNA binding protein immunoprecipitation kit (Millipore, Billerica, MA, U.S.A.) according to the instructions using AGO2 or a control IgG as an antibody.

Cell counting kit-8 assay

According to the experimental requirements, the cells were pre-incubated at 37°C and at 5% CO2 for 4 h, and the cells were adhered for 0, 24, 48 and 72 h. After the sample, cell counting kit-8 (CCK-8) solution was added to each well, the plates were incubated in the dark at 37°C and 5% CO2 for 2 h, and the absorbance (A) was measured at 450 nm using a microplate reader. The cell growth curve on the ordinate was plotted and the differences between the groups were compared.

Colony formation assay

Cells were seeded in six-well plates and incubated for 10–14 days. The clones were appropriately sized, washed twice with PBS, fixed in paraformaldehyde for 30 min, and Crystal Violet-stained at room temperature for 30 min. The number of clones greater than 50 units was calculated. Each group had three complex holes.

Flow cytometry analysis

Apoptosis analysis was performed by collecting transfected cells, washing them three times with PBS, resuspending the cells by adding 500 µl of binding buffer, 5 µl Annexin V-FITC, and 5 µl of PI and incubating them in the dark at room temperature for 15 min, which was followed by flow cytometry. For cell cycle assays, the cells were washed twice with pre-chilled PBS, collected, added to 70% pre-cooled ethanol solution, and fixed at 4°C overnight. Cells were analysed by flow cytometry after staining with PI. The percentages of cells in the G0/G1, S, and G2/M phases were counted and compared.

Animal experiments

The immune-deficient mice used in the experiment were all purchased from the Experimental Animal Center of Soochow University, and all of them were subjected to humanitarian treatment throughout the experiment. Stable HepG2 cell lines with 5 × 106 control or DCSTl-ASl interference were inoculated on the back of the mice. A subcutaneous tumour model was established to observe the effect of DCST1-AS1 on tumour growth. After implantation, the tumour was measured every 5 days, the size and diameter of the tumour were recorded, and tumour size (long diameter × short diameter 2 × 1/2) was calculated. All subcutaneous tumour-bearing mice were killed at the fifth week by cervical dislocation. The tumour tissue was removed and imaged. Protein and RNA were extracted and fixed, and pathological examinations such as HE staining were performed.

Statistical analysis

Measurement data are expressed as the mean ± S.D. GraphPad Prism 5 statistics were used for statistical analysis and plotting. One-way ANOVA was used for the comparison between groups, and Student’s t test was adopted for the comparison of means. The survival curve was plotted using the Kaplan–Meier method, and significant differences were analysed via log-rank test. Differences with P<0.05 were taken as statistically significant.

Results

LncRNA DCST1-AS1 is up-regulated in HCC and associated with poor prognosis

To search for lncRNAs associated with HCC, sequencing data from 50 pairs of carcinoma and para-carcinoma HCC in TCGA database were analysed (http://ibl.mdanderson.org/tanric/_design/basic/index.html). Based on the fold-length difference (fold change > 4.0, t test P<0.05) between HCC and para-carcinoma tissues, the expression of lncRNAs was mostly up-regulated (1412 up-regulated and 68 down-regulated) in HCC tissue (Figure 1A). However, the levels of most lncRNAs in carcinoma and para-carcinoma tissues were low; therefore, only lncRNAs with an average FPKM greater than 2 in carcinoma or para-carcinoma tissues were selected for further study. There was a total of 50 lncRNAs (44 up-regulated and 6 down-regulated) that met the requirements of the study (Figure 1B). Then, the survival status of 50 lncRNAs in 180 HCC patients in the TCGA database was analysed, and only 9 lncRNAs (ENSG00000225210, ENSG00000266976.1, ENSG00000249395, ENSG00000233461, ENSG00000228288, ENSG00000222041, ENSG00000270346, ENSG00000232093 and ENSG00000257698) were found to be related to HCC patient prognosis (Figure 1C). These 9 lncRNAs in 20 HCC carcinomas and para-carcinoma tissues were verified via qRT-PCR. ENSG00000232093 (lncRNA DCST1-AS1) was highly expressed in HCC and showed the largest significant difference (Figure 1D). Therefore, we chose DCST1-AS1 as the research target. In addition, DCST1-AS1 expression in the normal liver cell line LO2 and HCC cell lines (HepG2, Bel-7404, SK-Hep-1, Smmc-7721) was determined. The expression of DCST1-AS1 was significantly higher in HCC cell lines than in control cells (P<0.05, Figure 1E).

LncRNA DCST1-AS1 is up-regulated in HCC and associated with poor prognosis

Figure 1
LncRNA DCST1-AS1 is up-regulated in HCC and associated with poor prognosis

(A) Volcano plots were constructed based on TCGA (fold-change > 4.0 and P<0.01). The red points represent differentially up-regulated genes, and green points represent down-regulated genes. (B) Analysis of differentially expressed genes in HCC based on TCGA (fold change > 4.0, P<0.05 in t test, an average FPKM greater than 2 in carcinoma or para-carcinoma tissues). (C) LncRNAs found to be related to the prognosis of HCC patients based on TCGA. (D) Validation of lncRNAs in HCC via qRT-PCR. ENSG00000232093 (lncRNA DCST1-AS1) is the most significantly up-regulated lncRNA in HCC. (E) qRT-PCR analysis of DCST1-AS1 expression in a normal liver cell line (LO2) and HCC cells. Kaplan–Meier curve showing the overall survival (OS) of HCC patients stratified by lncRNA DCST1-AS1 expression based on our dataset (F) and a TCGA dataset (G). *P<0.05, ***P<0.001.

Figure 1
LncRNA DCST1-AS1 is up-regulated in HCC and associated with poor prognosis

(A) Volcano plots were constructed based on TCGA (fold-change > 4.0 and P<0.01). The red points represent differentially up-regulated genes, and green points represent down-regulated genes. (B) Analysis of differentially expressed genes in HCC based on TCGA (fold change > 4.0, P<0.05 in t test, an average FPKM greater than 2 in carcinoma or para-carcinoma tissues). (C) LncRNAs found to be related to the prognosis of HCC patients based on TCGA. (D) Validation of lncRNAs in HCC via qRT-PCR. ENSG00000232093 (lncRNA DCST1-AS1) is the most significantly up-regulated lncRNA in HCC. (E) qRT-PCR analysis of DCST1-AS1 expression in a normal liver cell line (LO2) and HCC cells. Kaplan–Meier curve showing the overall survival (OS) of HCC patients stratified by lncRNA DCST1-AS1 expression based on our dataset (F) and a TCGA dataset (G). *P<0.05, ***P<0.001.

To test the clinical role of DCST1-AS1 in HCC, the correlation of DCST1-AS1 expression with clinicopathological parameters was evaluated. The expression of DCST1-AS1 in HCC was determined by qRT-PCR. The 90 HCC patients were divided into high- and low-expression groups based on median expression levels. As shown in Table 1, high expression of DCST1-AS1 was closely related to tumour size in HCC patients but was not correlated with other clinicopathological factors. Subsequently, the relationship between DCST1-AS1 expression and overall survival (OS) in HCC patients was examined using survival curves. The OS rate of HCC patients with high expression of DCST1-AS1 was significantly lower than the survival rate of the low-expression group (Figure 1F). The TCGA database also confirmed this result (Figure 1G).

Table 1
Relationship between lncRNA DCST1-AS1 expression and clinicopathologic features of HCC patients
Clinicopathological features Patients (n=90) LncRNA DCST1-AS1 χ2 P-value 
  Low High   
Gender      
  Men 78 39 39 1.00 
  Women 12   
Age (years)      
  ≤50 31 14 17 0.44 0.506 
  >50 59 31 28   
Tumour size      
  <5 cm 36 26 10 11.85 0.001 
  ≥5 cm 54 19 35   
AJCC stage      
  I–II 43 27 16 3.78 0.052 
  III–IV 43 18 25   
  Unknown   
Histological grade      
  Well 0.78 0.676 
  Moderate 50 25 25   
  Poor 34 16 18   
Liver cirrhosis      
  Yes 33 14 19 1.20 0.274 
  No 57 31 26   
Clinicopathological features Patients (n=90) LncRNA DCST1-AS1 χ2 P-value 
  Low High   
Gender      
  Men 78 39 39 1.00 
  Women 12   
Age (years)      
  ≤50 31 14 17 0.44 0.506 
  >50 59 31 28   
Tumour size      
  <5 cm 36 26 10 11.85 0.001 
  ≥5 cm 54 19 35   
AJCC stage      
  I–II 43 27 16 3.78 0.052 
  III–IV 43 18 25   
  Unknown   
Histological grade      
  Well 0.78 0.676 
  Moderate 50 25 25   
  Poor 34 16 18   
Liver cirrhosis      
  Yes 33 14 19 1.20 0.274 
  No 57 31 26   

AJCC, American Joint Committee on Cancer

LncRNA DCST1-AS1 promotes proliferation of HCC cells

Because DCST1-AS1 is overexpressed in HepG2 and Bel-7404, these two cell lines were chosen for further study. To investigate the function of lncRNA DCST1-AS1 in HCC cells, DCST1-AS1 was knocked out or overexpressed by transfection with siRNA or overexpression plasmids (pcDNA3.1-DCST1-AS1) in HepG2 and Bel-7404 cells (Figure 2A,B). CCK-8 proliferation experiments showed that knockdown of DCST1-AS1 significantly inhibited the proliferation of HepG2 and Bel-7404 cells (Figure 2C), whereas overexpression of DCST1-AS1 significantly promoted the proliferation of both cell types (Figure 2D). Similarly, colony formation assays showed that DCST1-AS1 knockdown significantly inhibited colony formation and survival of HepG2 and Bel-7404 cells (Figure 2E), whereas DCST1-AS1 overexpression significantly increased colony survival of HepG2 and Bel-7404 cells (Figure 2F). Therefore, we concluded that DCST1-AS1 promotes HCC proliferation.

LncRNA DCST1-AS1 promotes proliferation of HCC cells

Figure 2
LncRNA DCST1-AS1 promotes proliferation of HCC cells

(A) qRT-PCR analysis of DCST1-AS1 expression in control, DCST1-AS1 siRNA 1-, and DCST1-AS1 siRNA 2-treated HCC cells. (B) qRT-PCR analysis of DCST1-AS1 expression in control and overexpression plasmid (pcDNA3.1-DCST1-AS1)-treated HCC cells. (C) With down-regulation of DCST1-AS1 expression, CCK-8 assays revealed that the proliferation of HepG2 and Bel-7404 was significantly less. (D) With up-regulation of DCST1-AS1 expression, CCK-8 assays revealed that the proliferation of HepG2 and Bel-7404 was significantly enhanced. (E) With down-regulation of DCST1-AS1 expression, colony formation assays revealed that the proliferation of HepG2 and Bel-7404 was significantly less. (F) With up-regulation of DCST1-AS1 expression, colony formation assays revealed that the proliferation of HepG2 and Bel-7404 was significantly greater. **P<0.01, ***P<0.001.

Figure 2
LncRNA DCST1-AS1 promotes proliferation of HCC cells

(A) qRT-PCR analysis of DCST1-AS1 expression in control, DCST1-AS1 siRNA 1-, and DCST1-AS1 siRNA 2-treated HCC cells. (B) qRT-PCR analysis of DCST1-AS1 expression in control and overexpression plasmid (pcDNA3.1-DCST1-AS1)-treated HCC cells. (C) With down-regulation of DCST1-AS1 expression, CCK-8 assays revealed that the proliferation of HepG2 and Bel-7404 was significantly less. (D) With up-regulation of DCST1-AS1 expression, CCK-8 assays revealed that the proliferation of HepG2 and Bel-7404 was significantly enhanced. (E) With down-regulation of DCST1-AS1 expression, colony formation assays revealed that the proliferation of HepG2 and Bel-7404 was significantly less. (F) With up-regulation of DCST1-AS1 expression, colony formation assays revealed that the proliferation of HepG2 and Bel-7404 was significantly greater. **P<0.01, ***P<0.001.

Apoptosis and cell cycle arrest are two important processes that affect the proliferation of tumour cells. Therefore, flow cytometry and TUNEL staining were performed to verify whether DCST1-AS1 affects these two factors. DCST1-AS1 knockdown significantly promoted apoptosis in HepG2 and Bel-7404 cells compared with control treatment. Additionally, siRNA-transfected HepG2 and Bel-7404 cells showed more cell cycle arrest than the control group (Figure 3A). The number of cells in the G1/G0 phase was significantly higher in cells with DCST1-AS1 knocked out, and the number of cells in G2/S phase was lower (Figure 3B). In addition, down-regulated DCST1-AS1 in HepG2 and Bel-7404 cells decreased the expression levels of Cyclin D1, Cyclin D3 and CDK4 proteins and increased the expression levels of cleaved caspase 3, cleaved PARP and p21 proteins, but no changes in the protein levels of Cyclin A, Cyclin B1, Cyclin E, P27 or pHH3 were observed (Figure 3C). These results demonstrate that knocking out DCST1-AS1 to inhibit HCC cell proliferation is primarily achieved by promoting apoptosis and cell cycle arrest at G1/S.

LncRNA DCST1-AS1 affects apoptosis and cell cycle arrest of HCC cell

Figure 3
LncRNA DCST1-AS1 affects apoptosis and cell cycle arrest of HCC cell

(A) The apoptotic rates of cells and (B) cell cycle as analysed by flow cytometry. Representative images and quantificative data are shown. Each column represents the means of three separate experiments. (C) Western blot analysis of apoptosis-related and cell cycle-related proteins after control siRNA, DCST1-AS1 siRNA 1, and DCST1-AS1 siRNA 2 transfection in HepG2 and Bel-7404. GAPGH protein was used as an internal control. *P<0.05, **P<0.01.

Figure 3
LncRNA DCST1-AS1 affects apoptosis and cell cycle arrest of HCC cell

(A) The apoptotic rates of cells and (B) cell cycle as analysed by flow cytometry. Representative images and quantificative data are shown. Each column represents the means of three separate experiments. (C) Western blot analysis of apoptosis-related and cell cycle-related proteins after control siRNA, DCST1-AS1 siRNA 1, and DCST1-AS1 siRNA 2 transfection in HepG2 and Bel-7404. GAPGH protein was used as an internal control. *P<0.05, **P<0.01.

LncRNA DCST1-AS1 functions as a competing endogenous RNA through regulating FAIM2 expression by binding to miR-1254

To investigate the molecular mechanism by which DCST1-AS1 promotes HCC proliferation, nuclear-cytoplasmic separation assays were performed to explore the nuclear and cytoplasmic localisation of DCST1-AS1 in HCC cells. DCST1-AS1 was present in both the nucleus and cytoplasm of HCC cells, but more was found in the cytoplasm and less in the nucleus (Figure 4A), suggesting that DCST1-AS1 may be involved in post-transcriptional regulation of target genes. Therefore, we speculated that DCST1-AS1 may function by competitive adsorption of miRNAs. To determine which miRNAs DCST1-AS1 can bind, an online bioinformatics database (RegRNA 2.0) (http://regrna2.mbc.nctu.edu.tw/detection.html) was used. miR-197, miR-1291, miR-1304, miR-1254, miR-3135, miR-4732, miR-4766 and miR-5193 have corresponding binding sites. Then, an immunofluorescence reporter assay was used to verify whether these candidate miRNAs could bind to DCST1-AS1, and we found that miR-1254 was the most effective inhibitor of these candidate miRNAs (Figure 4B). Subsequently, the expression of miR-1254 in HCC and para-carcinoma tissues was examined, and we found that the expression of miR-1254 in HCC was significantly lower than in adjacent tissues (Figure 4C). Then, a mutated form of DCST1-AS1 was constructed in which the miR-1254 binding sites were mutated (Figure 4D). The miR-1254 mimic resulted in less luciferase activity only in DCST1-AS1-wt but not in DCST1-AS1-mut (Figure 4E). In addition, RNA immunoprecipitation (RIP) assays showed that the expression of DCST1-AS1 and miR-1254 was higher in the anti-Ago2 group than that in the anti-normal IgG group (Figure 4F). The expression level of miR-1254 in HepG2 and Bel-7404 cells after knockout or overexpression of DCST1-AS1 was evaluated. DCST1-AS1 knockdown correlated with significantly higher miR-1254 expression levels, whereas DCST1-AS1 overexpression resulted in significantly lower miR-1254 expression levels (Figure 4G). In addition, the expression of DCST1-AS1 was negatively correlated with the expression of miR-1254 in 60 HCC tissues as evaluated by qRT-PCR (Figure 4H). In addition, miR-1254 had no significant correlation with HCC patients’ survival in TCGA datasets (Supplementary Figure S1A).

Regulation relationship between lncRNA DCST1-AS1 and miR-1254

Figure 4
Regulation relationship between lncRNA DCST1-AS1 and miR-1254

(A) qRT-PCR analysis of DCST1-AS1 nuclear and cytoplasmic levels in HepG2 and Bel-7404 cells. U6 was used as a nuclear marker, and GAPDH was used as a cytosolic marker. (B) Relative luciferase activity from HepG2 and Bel-7404 cells co-transfected with DCST1-AS1 reporter plasmid and candidate miRNAs. (C) Expression of miR-1254 was significantly down-regulated in HCC tissues compared with adjacent normal tissues. (D) Construction of the psiCheck2 reporter plasmid, containing wild-type or mutant DCST1-AS1. (E) MiR-1254 mimic resulted in lower luciferase activity in DCST1-AS1-WT but not in DCST1-AS1-MUT. (F) RIP experiments were performed in HepG2, and co-precipitated miR-1254 was subjected to qRT-PCR for DCST1-AS1. (G) qRT-PCR analysis of miR-1254 expression in HepG2 and Bel-7404 transfected with control, DCST1-AS1 siRNA or DCST1-AS1 overexpression plasmid. (H) Association analysis of the relationship between DCST1-AS1 and miR-1254 expression levels in 60 paired HCC tissues. *P<0.05, **P<0.01, ***P<0.001.

Figure 4
Regulation relationship between lncRNA DCST1-AS1 and miR-1254

(A) qRT-PCR analysis of DCST1-AS1 nuclear and cytoplasmic levels in HepG2 and Bel-7404 cells. U6 was used as a nuclear marker, and GAPDH was used as a cytosolic marker. (B) Relative luciferase activity from HepG2 and Bel-7404 cells co-transfected with DCST1-AS1 reporter plasmid and candidate miRNAs. (C) Expression of miR-1254 was significantly down-regulated in HCC tissues compared with adjacent normal tissues. (D) Construction of the psiCheck2 reporter plasmid, containing wild-type or mutant DCST1-AS1. (E) MiR-1254 mimic resulted in lower luciferase activity in DCST1-AS1-WT but not in DCST1-AS1-MUT. (F) RIP experiments were performed in HepG2, and co-precipitated miR-1254 was subjected to qRT-PCR for DCST1-AS1. (G) qRT-PCR analysis of miR-1254 expression in HepG2 and Bel-7404 transfected with control, DCST1-AS1 siRNA or DCST1-AS1 overexpression plasmid. (H) Association analysis of the relationship between DCST1-AS1 and miR-1254 expression levels in 60 paired HCC tissues. *P<0.05, **P<0.01, ***P<0.001.

Next, to identify the target genes of miR-1254, TargetScan (http://www.targetscan.org) and miRDB (http://mirdb.org/) databases were searched. Each of the databases predicted the first 100 target genes, and both predicted that the target genes with binding sites were MGAT5B, KCTD5, RASL12, and FAIM2. Subsequently, miR-1254 mimics or miR-1254 inhibitors were transfected in the HepG2 and Bel-7404 cell lines to observe the expression of candidate target genes. Of the candidate target genes, only FAIM2 mRNA and protein were altered in the transfected cell lines (Figure 5A,B). FAIM2-wt-3′-UTR and FAIM2-mut-3′-UTR was constructed and co-transfected with the fluorescent reporter plasmid miR-1254 into HepG2 (Figure 5C). The immunoluciferase assay results showed that FAIM2-wt-3′-UTR fluorescence plasmid resulted in significantly less fluorescence intensity than the FAIM2-mut-3′-UTR plasmid (Figure 5D). These results indicate that miR-1254 can directly regulate the expression of FAIM2, and the binding site is the site that was mutated. In addition, FAIM2 was highly expressed in HCC tissues (Figure 5E), but there was no significant negative correlation between FAIM2 and miR-1254 in HCC tissues. We also analysed the relationship between DCST1-AS1 and FAIM2. The expression of FAIM2 was positively correlated with the expression of DCST1-AS1 in 60 HCC tissues (Figure 5F). In addition, FAIM2 had no significant correlation with HCC patients’ survival in TCGA datasets (Supplementary Figure S1B).

MiR-1254 can directly regulate the expression of FAIM2

Figure 5
MiR-1254 can directly regulate the expression of FAIM2

FAIM2 mRNA and protein expression in HepG2 and Bel7404 cells by qRT-PCR (A) and Western blot (B) after transfection of miR-1254 mimics, miR-1254 inhibitor or control miRNA. (C) The construction of the psiCheck2 reporter plasmid containing wild-type or mutant FAIM2 3′UTR. (D) The MiR-1254 mimic resulted in lower luciferase activity in FAIM2 3′UTR–WT, but not in FAIM2 3′UTR-MUT. (E) The expression of FAIM2 mRNA was significantly higher in HCC tissues compared with adjacent normal tissues. (F) Association analysis of the relationship between DCST1-AS1 and FAIM2 expression levels in 60 paired HCC tissues. **P<0.01, ***P<0.001.

Figure 5
MiR-1254 can directly regulate the expression of FAIM2

FAIM2 mRNA and protein expression in HepG2 and Bel7404 cells by qRT-PCR (A) and Western blot (B) after transfection of miR-1254 mimics, miR-1254 inhibitor or control miRNA. (C) The construction of the psiCheck2 reporter plasmid containing wild-type or mutant FAIM2 3′UTR. (D) The MiR-1254 mimic resulted in lower luciferase activity in FAIM2 3′UTR–WT, but not in FAIM2 3′UTR-MUT. (E) The expression of FAIM2 mRNA was significantly higher in HCC tissues compared with adjacent normal tissues. (F) Association analysis of the relationship between DCST1-AS1 and FAIM2 expression levels in 60 paired HCC tissues. **P<0.01, ***P<0.001.

Rescue assays were performed twice to verify whether miR-1254 and FAIM2 participate in DCST1-AS1-mediated cell proliferation, apoptosis and cell cycle arrest. First, HepG2 cells were divided into four groups: control siRNA + miR-NC, DCST1-AS1 si1 + miR-NC, miR-1254 inhibitor + control siRNA and DCST1-AS1 si1 + miR-1254 inhibitor. The expression of DCST1-AS1, miR-1254 and FAIM2 in four groups are shown in Figure 6A. Down-regulation of miR-1254 alone was correlated with significantly enhanced cell proliferation and inhibition of apoptosis and cell cycle arrest. When miR-1254 was down-regulated in DCST1-AS1 down-regulated cells, the changes in cell function induced by DCST1-AS1 were completely reversed (Figure 6B,C). Second, HepG2 cells were divided into the following four groups: control siRNA + miR-NC, FAIM2si + miR-NC, miR-1254 inhibitor + control siRNA, and FAIM2si + miR-1254 inhibitor. The expression of DCST1-AS1, miR-1254 and FAIM2 in four groups are shown in Figure 7A. Down-regulation of FAIM2 alone significantly reduced cell proliferation and promoted apoptosis and cell cycle arrest. Down-regulation of miR-1254 and down-regulation of FAIM2 in cells completely reversed the changes in cell function induced by down-regulation of miR-1254 (Figure 7B,C). Finally, HepG2 cells were divided into the following four groups: control siRNA + control, DCST1-AS1 si1 + control, control siRNA + FAIM2 and DCST1-AS1 si1 + FAIM2. The expression of DCST1-AS1, miR-1254 and FAIM2 in four groups are shown in Figure 8A. Overexpression of FAIM2 and down-regulation of DCST1-AS1 in cells completely reversed the changes in cell function induced by down-regulation of DCST1-AS1 (Figure 8B,C). In conclusion, we determined that DCST1-AS1 up-regulates the expression of FAIM2 by up-regulating the expression of miR-1254 and ultimately promotes the proliferation of HCC cells.

LncRNA DCST1-AS1 regulates cell proliferation through miR-1254 in HCC

Figure 6
LncRNA DCST1-AS1 regulates cell proliferation through miR-1254 in HCC

(A) The expression of DCST1-AS1, miR-1254 and FAIM2 in HepG2 cells after co-transfection with DCST1-AS1 siRNA1, miR-1254 inhibitor or control siRNA. Growth curves (B) and apoptotic rates (C) in four groups were determined by CCK-8 and flow cytometry assay. *P<0.05, **P<0.01, ***P<0.001.

Figure 6
LncRNA DCST1-AS1 regulates cell proliferation through miR-1254 in HCC

(A) The expression of DCST1-AS1, miR-1254 and FAIM2 in HepG2 cells after co-transfection with DCST1-AS1 siRNA1, miR-1254 inhibitor or control siRNA. Growth curves (B) and apoptotic rates (C) in four groups were determined by CCK-8 and flow cytometry assay. *P<0.05, **P<0.01, ***P<0.001.

MiR-1254 regulates cell proliferation through FAIM2 in HCC

Figure 7
MiR-1254 regulates cell proliferation through FAIM2 in HCC

(A) The expression of DCST1-AS1, miR-1254 and FAIM2 in HepG2 cells after co-transfection with FAIM2 siRNA, miR-1254 inhibitor or control siRNA. Growth curves (B) and apoptotic rates (C) in four groups were determined by CCK-8 and flow cytometry assay. *P<0.05, **P<0.01, ***P<0.001.

Figure 7
MiR-1254 regulates cell proliferation through FAIM2 in HCC

(A) The expression of DCST1-AS1, miR-1254 and FAIM2 in HepG2 cells after co-transfection with FAIM2 siRNA, miR-1254 inhibitor or control siRNA. Growth curves (B) and apoptotic rates (C) in four groups were determined by CCK-8 and flow cytometry assay. *P<0.05, **P<0.01, ***P<0.001.

LncRNA DCST1-AS1 regulates cell proliferation through FAIM2 in HCC

Figure 8
LncRNA DCST1-AS1 regulates cell proliferation through FAIM2 in HCC

(A) The expression of DCST1-AS1, miR-1254 and FAIM2 in HepG2 cells after co-transfection with DCST1-AS1 siRNA, FAIM2 or control. Growth curves (B) and apoptotic rates (C) in four groups were determined by CCK-8 and flow cytometry assay. *P<0.05, **P<0.01, ***P<0.001.

Figure 8
LncRNA DCST1-AS1 regulates cell proliferation through FAIM2 in HCC

(A) The expression of DCST1-AS1, miR-1254 and FAIM2 in HepG2 cells after co-transfection with DCST1-AS1 siRNA, FAIM2 or control. Growth curves (B) and apoptotic rates (C) in four groups were determined by CCK-8 and flow cytometry assay. *P<0.05, **P<0.01, ***P<0.001.

Knockdown of lncRNA DCST1-AS1 can inhibit tumour growth in vivo

To verify whether DCST1-AS1 can influence tumour suppression in model animals, stably transfected DCST1-AS1 shRNA or control HepG2 cells were implanted into the back of nude mice. Tumour volume in the DCST1-AS1 knockout group were significantly lower than in the negative control group (Figure 9A). In addition, the expression of DCST1-AS1, miR-1254 and FAIM2 in mouse tissues of the DCST1-AS1 shRNA and control groups was analysed. In DCST1-AS1 shRNA mouse tissues, miR-1254 was highly expressed and FAIM2 showed low expression (Figure 9B,C). In conclusion, we determined that DCST1-AS1 might provide a new target for HCC treatment.

Knockdown of lncRNA DCST1-AS1 can inhibit tumour growth in vivo

Figure 9
Knockdown of lncRNA DCST1-AS1 can inhibit tumour growth in vivo

(A) The stable DCST1-AS1 knockdown HepG2 cells were used for the in vivo assays. The tumours from two groups of nude mice were shown and tumour growth curves were measured and shown after the injection of HepG2 cells. The tumour volume was calculated every 3 days. (B) The expression of DCST1-AS1, miR-1254 and FAIM2 in mouse tissues of the DCST1-AS1 shRNA and control groups was analysed by qRT-PCR. (C) HE staining of mouse tissues. MiR-1254 expression was detected via ISH. FAIM2 protein expression was detected via IHC. **P<0.01.

Figure 9
Knockdown of lncRNA DCST1-AS1 can inhibit tumour growth in vivo

(A) The stable DCST1-AS1 knockdown HepG2 cells were used for the in vivo assays. The tumours from two groups of nude mice were shown and tumour growth curves were measured and shown after the injection of HepG2 cells. The tumour volume was calculated every 3 days. (B) The expression of DCST1-AS1, miR-1254 and FAIM2 in mouse tissues of the DCST1-AS1 shRNA and control groups was analysed by qRT-PCR. (C) HE staining of mouse tissues. MiR-1254 expression was detected via ISH. FAIM2 protein expression was detected via IHC. **P<0.01.

Discussion

Many studies have shown that lncRNAs are involved in the occurrence of multiple tumours and are correlated with tumour cell proliferation, apoptosis, invasion and metastasis, and drug resistance [6,9–16]. LncRNA expression levels are often related to phenotypic effects: usually high expression of lncRNA exerts a carcinogenic effect, and low expression of lncRNA plays a role in tumour suppression [17]. In this study, a novel HCC-associated lncRNA, DCST1-AS1, was discovered, and found to be significantly up-regulated in HCC tissues and cell lines. High expression of DCST1-AS1 was closely related to tumour size. In addition, the OS rate of HCC patients with high DCST1-AS1 expression was significantly lower than the survival rate of those with low DCST1-AS1 expression. Subsequent in vitro and in vivo experiments showed that knockdown of DCST1-AS1 inhibits HCC cell proliferation and tumour growth and induces apoptosis and cell cycle arrest, whereas overexpression of DCST1-AS1 promotes cell proliferation. These findings suggest that DCST1-AS1 plays a role as an oncogene in HCC and may be considered a potential prognostic indicator of HCC.

With the discovery and functional characterisation of a large number of lncRNAs, the mechanism by which lncRNAs and their target molecules carry out their biological function has become a research hotspot. LncRNAs mainly play a regulatory role at the molecular level, including chromatin remodelling, transcriptional activation, mRNA stability regulation, mRNA and post-translational transcriptional regulation, and multiple other modes of action to form complex regulatory pathways for target genes [18,19]. The subcellular localisation of LncRNA is closely related to its function. LncRNAs in the nucleus mainly function through chromatin remodelling and directly activate or suppress the transcription of target genes through in situ or remote regulation [20]. In the cytoplasm, lncRNAs can bind to target mRNAs to affect their translation or mediate their stability. In addition, lncRNAs may act in the cytoplasm as competing endogenous RNAs (ceRNAs) [20]. CeRNAs are endogenous competitive RNAs. CeRNAs can compete with miRNAs and adsorb miRNAs like sponges to block miRNA silencing of target mRNAs [21,22]. In this study, DCST1-AS1 was mainly distributed in the cytoplasm of HCC, and we speculate that it functions as a ceRNA. Subsequent bioinformatics, immunoluciferase assays, and RIP assays confirmed our hypothesis that DCST1-AS1 acts as a ceRNA in the cytoplasm by adsorbing miR-1254. Many studies have confirmed the role of miR-1254 as a tumour suppressor gene in various tumours [23–25]. In non-small cell lung cancer (NSCLC), miR-1254 induces apoptosis and cell cycle arrest in human NSCLC cells by inhibiting the expression of HO-1, thereby inhibiting the growth of NSCLC cells. In mouse xenograft studies, miR-1254 was demonstrated to inhibit NSCLC tumour growth in vivo [23]. In thyroid cancer, miR-1254 inhibits the stimulation of thyroid cancer cell growth and invasion by lncRNA n340790 [24]. miR-1254 is down-regulated in colorectal cancer CRC tissues and cells. Up-regulation of miR-1254 significantly inhibited the proliferation and migration of colon cancer cell lines SW1116 and HCT116 [25]. In our study, miR-1254 was significantly down-regulated in HCC, and inhibition of miR-1254 expression significantly enhanced the proliferation of HCC cells. In addition, remedial experiments confirmed that miR-1254 completely reversed the changes in cell function caused by DCST1-AS1 down-regulation.

In the ceRNA network, the lncRNA DCST1-AS1 can compete with miR-1254 to block the silencing effect of miR-1254 on the target gene mRNA, which indicates that the target gene of miR-1254 plays an important role in HCC. A potential target gene of miR-1254, FAIM2, was predicted using a network database. Subsequent immunofluorescence studies confirmed that FAIM2 is a direct target of miR-1254. FAIM2 is highly expressed in breast cancer tissues and cells, and overexpression of FAIM2 inhibits breast cancer cell apoptosis [26]. In addition, activation of the Akt/LEF-1 pathway in breast cancer up-regulates FAIM2 and inhibits apoptosis [27], and down-regulation of FAIM2 promotes chemosensitivity of the breast cancer cell line MDA-MB-231 to cisplatin [28]. FAIM2 is highly expressed in small cell lung cancer (SCLC), and knockdown of FAIM2 expression increases Fas-induced apoptosis in SCLC cells [29]. It has also been found that enhanced FAIM2 expression promotes proliferation, migration and invasion and inhibits apoptosis of lung cancer cells [30,31]. Therefore, FAIM2 plays a role as oncogene in breast cancer and lung cancer. In this study, FAIM2 was highly expressed in HCC, and inhibition of FAIM2 expression significantly inhibited the proliferation of HCC cells. In addition, remedial experiments confirmed that down-regulation of FAIM2 completely reversed the down-regulation of cell function induced by miR-1254.

In conclusion, a novel lncRNA DCST1-AS1 that functions as an oncogene in HCC was discovered. DCST1-AS1 up-regulates the expression of FAIM2 by up-regulating the expression of miR-1254, ultimately promoting the proliferation of HCC cells. This research provides new therapeutic targets for HCC.

Clinical perspectives

  • The molecular mechanisms of hepatocarcinogenesis are not fully understood, and the biomarkers capable of predicting clinical outcomes and serving as potential therapeutic targets are limited.

  • LncRNA DCST1-AS1 was found to be a new potential biomarker for HCC. DCST1-AS1 is up-regulated in HCC and associated with poor prognosis. DCST1-AS1 promotes proliferation of HCC cells in vitro and in vivo. DCST1-AS1 functions as a ceRNA through regulating FAIM2 by binding to miR-1254.

  • Our work led to the identification of a novel functional pathway controlled by DCST1-AS1/miR-1254/FAIM2, and DCST1-AS1 may represent a novel potential therapeutic target for the treatment of HCC.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Author contribution

J.C, Y.D. and Y.A. conceived and designed the study and helped to draft the manuscript. D.W., Y.Z. and Y.Y. performed the data collection. Y.Y. performed the statistical analysis. D.W, Y.Z. and Y.Y. helped acquire or generate the data. All authors read and critically revised the manuscript for intellectual content and approved the final manuscript.

Funding

This work was supported by the Changzhou High-Level Medical Talents Training Project [grant number 2016CZBJ044]; the National Natural Science Foundation of China [grant number 81502002]; and the Natural Science Foundation of Jiangsu Province [grant number BK20150254].

Abbreviations

     
  • CCK-8

    cell counting kit-8

  •  
  • ceRNA

    competing endogenous RNA

  •  
  • FAIM2

    Fas apoptosis inhibitor 2

  •  
  • HCC

    hepatocellular carcinoma

  •  
  • lncRNA

    long non-coding RNA

  •  
  • NC

    normal control

  •  
  • NSCLC

    non-small cell lung cancer

  •  
  • OS

    overall survival

  •  
  • qRT-PCR

    quantificative real-time PCR

  •  
  • RIP

    RNA immunoprecipitation

  •  
  • SCLC

    small cell lung cancer

  •  
  • TCGA

    The Cancer Genome Atlas

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Supplementary data