Long non-coding RNA PTTG3P promotes hepatocellular carcinoma growth via modulating nuclear-cytoplasm shuttle of ILF2 and stabilizing TOP2A mRNA

doi:10.21203/rs.3.rs-1542711/v1

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Long non-coding RNA PTTG3P promotes hepatocellular carcinoma growth via modulating nuclear-cytoplasm shuttle of ILF2 and stabilizing TOP2A mRNA

https://doi.org/10.21203/rs.3.rs-1542711/v1

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Background: PTTG3P has been characterized as an oncogenic lncRNA in hepatocellular carcinoma (HCC). However, the underlying mechanisms have not been fully understood. This study aimed to explore the functional partner of PTTG3P in modulating HCC malignant phenotypes.

Methods: Hep3B and HepG2 were used as representative HCC models. Cellular and molecular studies were performed using RNA pull-down/mass spectrometry (MS), RNA immunoprecipitation (RIP)-qPCR, Chromatin immunoprecipitation (ChIP)-qPCR, and immunofluorescent (IF) staining.

Results: PTTG3P interacts with both ILF2 and ILF3. The binding between PTTG3P and ILF2 helps maintain the cytoplasmic distribution of ILF2. Together with ILF2, PTTG3P binds to the 3'UTR of TOP2A. PTTG3P depletion impairs the binding and reduces TOP2A mRNA stability. Meanwhile, PTTG3P depletion is associated with nuclear accumulation of ILF2 and increased binding of ILF2 to the promoter region of TOP2A. Dual-luciferase assay confirmed that ILF2 activates the TOP2A promoter and increases its transcription.

Conclusion: this study revealed that when PTTG3P is overexpressed in HCC tumor cells, it mainly acts as a TOP2A mRNA stabilizer, via recruiting the ILF2/ILF3 complex to the 3’UTR of TOP2A mRNA. However, PTTG3P depletion results in nuclear accumulation of ILF2 and subsequent transcriptional activation of TOP2A. This might be utilized by HCC cells to compensate the loss of PTTG3P and associated TOP2A instability. These findings help expand our understanding of the oncogenic mechanisms of PTTG3P in HCC.

PTTG3P

ILF2

ILF3

TOP2A

hepatocellular carcinoma

Long non-coding RNAs (lncRNAs) are transcripts with more than 200 nucleotides that are not translated into proteins. During the past decades, a large number of studies have revealed that lncRNAs exert critical regulatory mechanisms in various cellular activities or diseases ¹. Many lncRNAs are aberrantly expressed in hepatocellular carcinoma (HCC) and regulate the carcinogenic and pathological processes ^2,3. The regulatory mechanisms of lncRNAs are complex and can be at both genetic and epigenetic levels. Firstly, they bind to DNA and remodel chromatin, leading to altered gene expression ^4,5. Secondly, some lncRNAs may encode functional peptides⁶. Thirdly, they can bind to mRNAs and influence their stability and transcription ^7,8. Fourthly, they sponge miRNAs and regulate their stability and targeting ⁹. Fifthly, they interact with proteins, modulating their conformation, localization, or stability ^10,11.

Our previous study found that lncRNA PTTG3P upregulation is a biomarker of unfavorable prognosis of HCC ¹². It stimulates PTTG1 expression and activates PI3K/AKT signaling ¹³ and sponges miR-383 and upregulates CCND1 and PARP2 expression in HCC ¹⁴. PTTG3P also interacts with FOXM1 and activates the transcription of the mitotic checkpoint kinase gene BUB1B, leading to facilitated tumor growth and enhanced chemoresistance in lung adenocarcinoma (LUAD) ¹⁵.

Interleukin Enhancer Binding Factor 2 (ILF2, also known as NF45) can form a complex with ILF3 (NF90). ILF2/ILF3 complex acts as a transcriptional activator via binding to the antigen receptor response element (ARRE)-2 (5’-AAGAAAGGAGGAAAAACTGTTT-3') of the proximal promoter of multiple genes, including IL2 ¹⁶, IL13 ¹⁷ and FOS ¹⁸. This complex recognizes and binds to the core sequences, including CTGTT and GGAAA motif ^17,19. The tumor-promoting effect of ILF2 and ILF3 has been reported in HCC ²⁰. This complex induces the elevation of EGF receptor levels and promotes cell proliferation via binding to pri-miR-7-1 and suppressing mature miR-7-1 generation ^20,21. ILF2/ILF3 binds to the 3' untranslated regions (3'UTRs) of multiple mRNAs and increases their stability, leading to facilitated cell-cycle progression and cell proliferation ²¹.

Here, we confirmed that PTTG3P contributes to the malignant phenotype of HCC, typically via enhancing cell proliferation and invasion. Mechanistically, PTTG3P interacts with ILF2 and ILF3, which bind to the 3'UTR of TOP2A (encoding DNA Topoisomerase II α) mRNA and increase its stability. PTTG3P depletion triggers nuclear translocation of ILF2, which binds to the promoter of the TOP2A gene and enhances its transcription.

Bioinformatic analysis

Data extraction from TCGA-LIHC and survival analysis followed the methods as described previously ¹².

Cell culture and treatment

Human HCC cell lines, Hep3B and HepG2 were obtained from ATCC (Manassas, VA, USA) and grown in Dulbecco's modified Eagle's medium (DMEM, Corning, Manassas, VA, USA) with 10% FBS. All cell lines were maintained at 37°C and 5% CO₂ in an incubator.

Lentiviral PTTG3P (NR_002734.2) overexpression plasmid was generated using the shuttle plasmid pLVX-Puro. Lentiviral PTTG3P, ILF2, and TOP2A were constructed using pLKO.1-puro plasmid. The validated shRNA sequences were provided in Table S1. Empty vectors and scramble (scr.) sequences were used as controls, respectively.

Lentiviruses were produced by co-transfecting recombinant shuttle plasmids with lentivirus packaging plasmids (pSPAX2 and pMD2G, HanBio Technology) in 293T cells. Briefly, supernatants 48 h after transfection were harvested, centrifuged, filtered through a 0.45 μm filter and finally ultracentrifuged. Then the lentiviral plasmids were dissolved in PBS and stored at -80 °C. Hep3B and HepG2 cells were infected with lentiviruses at a multiplicity of infection (MOI) of 15.

RNA fluorescence in situ hybridization (RNA-FISH)

RNA-FISH assay was performed according to the methods described previously ²². CY3-conjugated PTTG3P probes were generated according to protocols from RiboBio (Guangzhou, China). The following probe sequence was used: 5′- -3′. Nuclei were stained with 6-diamidino-2-phenylindole (DAPI). Fluorescent images were captured using a fluorescence microscope (Olympus IX83, Tokyo, Japan).

RNA extraction and quantitative Real-Time PCR (qRT-PCR)

In brief, Total RNA was extracted by using the RNeasy minikit (QIAGEN, Hilden, Germany) and was reverse transcribed into the first strand DNA by using a cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. qRT-PCR was performed with TaqMan gene expression master mix (Applied Biosystems), on an ABI Real-Time 7900HT cycler. RNA levels of queried genes were normalized to the level of GAPDH. Results are representative of three individual experiments in triplicate. The primers used in this study were provided in Supplementary Table 1.

CCK-8 assay of cell proliferation

Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) was used to measure cell proliferation following the manufacturer's instructions. Briefly, 24 h after lentiviral infection, cells were plated in 96-well plates (2×10³ cells/well) in a medium containing 10% FBS for 24, 48 or 72 h. Then 10 μL CCK-8 solution was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader. Results are representative of three individual experiments in triplicate.

Flow-cytometric analysis of cell-cycle distribution and apoptosis

48 h after lentiviral infection, cells were trypsinized and washed. For cell cycle analysis, cells were fixed in 95% ethanol at −4 °C overnight and then were stained using 50 μg/ml Propidium Iodide (PI) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min for apoptosis assessment. Then, cells collected were centrifugated for 5 minutes at 2,000rpm and resuspended in 1× binding buffer containing FITC-labeled Annexin V (25ng/ml) and propidium iodide (50ng/ml) (FITC Annexin V Apoptosis detection kit I, BD Pharmingen, San Jose, CA, USA). Then, the samples were immediately analyzed by flow cytometry with a BD FACSCanto II flow cytometer (BD Biosciences) at a low flow rate. Results were quantified with BD FACSDiva software v8.0 (Franklin Lakes, NJ BD Biosciences). A minimum of 10,000 events were gated per sample.

Cell invasion assay

Briefly, 1×10⁵ cells in serum-free EMEM were added to the upper chamber of the Matrigel-coated insert (Merck Millipore, Darmstadt, Germany), and 500 μL of DMEM containing 10% FBS was added to the lower chamber for culture. The cells were cultured in a cell incubator for 24 h. Then, non-invading cells were removed and invading cells were fixed with 4% paraformaldehyde for 30 minutes and stained using 0.5% crystal violet (Sigma-Aldrich, St Louis, MO, USA) for 20 minutes. The invading cells were counted using an inverted microscope (Olympus, Tokyo, Japan).

Wound healing assay

24 h after lentiviral infection, 5×10⁵ cells were seeded into 6‑well culture plates and cultured to 90% confluence. Then, a sterile 200‑µl micropipette tip was utilized to scratch the cell monolayer. The cells were further cultured in serum‑free medium incubated in a cell incubator for another 48 h. Then, wound healing area was captured using an inverted microscope (Olympus) at a magnification of 200× and quantified using ImageJ software (1.8.0) (National Institutes of Health).

RNA Pull‐Down Assay and Mass Spectrometry

PTTG3P and its antisense RNA were chemically synthesized and were inserted into the sites between KpnI and SacI in pBluescript II SK+. Biotin-labelled PTTG3P was first transcribed in vitro using Biotin RNA Labeling Mix (Roche, Germany) by T3 (for antisense)/T7 (for sense) RNA polymerase Mix (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Then, the RNA products were treated with RNase-free DNase I (Roche) and purified with a RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total proteins extracted from Hep3B cells were then mixed with biotinylated RNA, incubated with Dynabeads Myone Streptavidin T1 beads (Invitrogen, Carlsbad, CA), and washed. The proteins binding to the streptavidin‐coupled dynabeads were resolved by SDS‐PAGE and visualized by silver staining. The specific bands were excised and analyzed by a linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific) coupled with a nanoflow LC system (Dina-2A, KYA Technologies). The proteins with Score Sequest HT > 6 were selected.

Western blot analysis

HCC cells were lysed on ice in RIPA buffer supplemented with protease inhibitors. Protein samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and then were electrotransferred onto polyvinylidene fluoride (Millipore) membranes. After that, the membranes were blocked in 5% non-fat milk powder for 2 h at room temperature and incubated with primary antibodies overnight at 4℃. After incubation with corresponding secondary antibodies conjugated to horseradish peroxidase, protein band signals were detected by BeyoECL Star Kit (Beyotime, Wuhan, China). Signal images were taken using a ChemiDoc imaging system (BIO-RAD, USA).

Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)

qRT-PCR was conducted as described previously. In brief, total RNA was extracted from cell samples using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNAs were reversely transcribed with PrimeScript RT reagent kit (TaKara, Dalian, China). Then, qRT-PCR was conducted using a SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China), with an Applied Biosystems 7500 Sequence Detection System (Applied Biosystem). Primer sequences for qRT-PCR were listed in (Supplementary Table 1). GAPDH expression was used as an internal control.

RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP)

RIP and ChIP assays were conducted using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore) and EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Merck Millipore), respectively, according to the manufacturer's instruction.

In brief, Hep3B cells were lysed using RIPA buffer. For RIP, whole-cell lysate was incubated with the RIP immunoprecipitation buffer containing protein A/G magnetic beads coated with ILF2 or ILF3 antibody. Normal mouse IgG was used as the control. After being incubated for 2 h at 4°C, the precipitated RNA fraction was isolated and subjected to qRT-PCR analysis of PTTG3P expression.

For ChIP, the protein-DNA cross-links were collected and sonicated to an average size of 500~1000 bp. The chromatin was immunoprecipitated with mouse anti-ILF2 (ab154791, Abcam, Cambridge, UK) or mouse IgG overnight at 4 °C. Then, Immunoprecipitated DNA collected was performed by the EZ-Magna ChIP A/G Kit. The primers for ChIP-qPCR were provided in Supplementary Table 1.

RNA-seq

Total RNA was extracted from Hep3B cells 48 h after lentiviral mediated PTTG3P overexpression and subjected to RNA-seq study. RNA-Seq, data generation and normalization were conducted on the Illumina Cluster Station and Illumina Nova-seq 6000 System at Gene denovo Corp. (Guangzhou, China).

IHC staining

IHC staining data are both from the Human Protein Atlas (https://www.proteinatlas.org/) ²³ and from our lab. For IHC of ENO2 expression in HCC tissues, a human HCC tissue array was obtained from Taibosi Bio (Xian, China). IHC staining was automatically conducted using BOND-III Automated IHC Stainer for Immunostaining (Leica Biosystems, Buffalo Grove, IL, USA). Primary anti-ENO2 (55235-1-AP) was purchased from Proteintech (Wuhan, China). Hematoxylin was used for counterstaining.

Immunofluorescent (IF) staining

Hep3B and HepG2 cells were grown on coverslips and infected for PTTG3P depletion. 24 h later, the cells were fixed in 4% paraformaldehyde, permeabilized, and blocked. Then, the coverslips were incubated with mouse anti-ILF2 (ab154791) and rabbit anti-ILF3 (19887-1-AP, Proteintech) at 4℃ overnight. Then the coverslips were washed and incubated with secondary anti-mouse IgG (Alexa Fluor 594, ab150116, Abcam) and anti-rabbit IgG (Alexa Fluor 488, ab150077, Abcam). Nuclear was stained by DAPI. Immunofluorescent staining was visualized using a fluorescence microscope (Olympus IX83).

Dual-luciferase assay

The full-length wild-type (WT) TOP2A promoter sequence and mutant sequences (with mutations of the potential ILF2/ILF3 binding motif) were chemically synthesized and cloned into the sites between KpnI and HindIII in the pGL3 basic vector (Promega, Madison, WI, USA). HCC cells were plated in 24-well plates at a density of 2 × 10⁵ cells per well and were infected with lentivirus carrying shPTTG3P alone or in combination with shILF2. 24h later, cells were transfected with 1 μg of recombinant plasmids and 0.05 μg of pRL-CMV vector, using Lipofectamine 2000 (Invitrogen). 24 h after transfection, cells were lysed for the measurement of firefly luciferase and renilla luciferase activities, using a dual-specific luciferase assay kit (#E1910, Promega).

Animal study

Briefly, five-week-old male BALB/c nude mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd (Sichuan, China). 5*10⁶ Hep3B cells with PTTG3P overexpression, PTTG3P knockdown, or TOP2A knockdown were subcutaneously into the left or right flanks of mice. Tumor growth was recorded twice a week. Four weeks after, all mice were euthanatized. Tumors were excised, pictured and then, fixed and embedded in paraffin. Paraffin sections were applied for immunohistochemistry (IHC) staining of Ki-67 and IF staining of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The experiment protocol was approved by the Animal Care and Use Committee of the University of Electronic Science and Technology of China.

Statistical analysis

Data analysis was performed using GraphPad Prism 8.1.2 (GraphPad Inc., La Jolla, CA, USA). One-way ANOVA with post hoc Tukey's multiple comparisons test was performed for comparison among three or more groups. For two-group comparisons, Welch's unequal variances t-test was conducted. Kaplan-Meier survival curves were generated by median gene expression, with Log-rank test to check differences. Pearson's coefficients were calculated to estimate correlation. p < 0.05 was considered to be statistically significant.

PTTG3P upregulation is associated with enhanced proliferation and cell-cycle progression of HCC

To validate the dysregulation of PTTG3P in HCC, we retrospectively reviewed its expression in 10 databases with PTTG3P expression data in HCCDB, a database of HCC expression atlas ²⁴. PTTG3P upregulation is observed in 8/10 datasets (Fig. 1A). Notably, HCCDB15 refers to TCGA-LIHC. By RNA-FISH assay, we confirmed the cytoplasmic distribution of PTTG3P in Hep3B and HepG2 cells (Fig. 1B). To explore the functional role of PTTG3P, we designed PTTG3P shRNA and overexpression lentivirus (Fig. 1C-D). PTTG3P knockdown slowed HCC cell proliferation (Fig. 1E-F), increased G1 phase accumulation, and decreased the proportion of cells in the S phase (Fig. 1G-I). In comparison, PTTG3P overexpression significantly enhanced cell proliferation (Fig. 1E-F), decreased G1 phase accumulation but increased the ratio of cells in the S phase (Fig. 1G-I).

PTTG3P upregulation reduces apoptosis and enhances invasion and migration of HCC cells

By performing Annexin V/PI staining, we found that PTTG3P depletion led to increased cell apoptosis. In contrast, its overexpression resulted in reduced cell apoptosis (Fig. 2A-C). Matrigel invasion and scratch assays showed that PTTG3P knockdown impaired HCC cell invasion (Fig. 2D-F) and migration (Fig. 2G-I). However, its overexpression significantly enhanced cell invasion (Fig. 2D-F) and migration (Fig. 2G-I).

PTTG3P physically interacts with ILF2 and ILF3 in HCC

To investigate the functional role of PTTG3P in HCC, we tried to identify its interacting proteins. The schematic map showing the construction of recombinant pBluescript II SK + plasmid is presented in Fig. 3A. Then, RNA pull-down assay was conducted using Biotin-labelled sense PTTG3P and antisense PTTG3P followed by silver staining (Fig. 3B). The bands specific to PTTG3P were excised and subjected to mass spectrometry (Fig. 3B, Supplementary Table 2). Results showed that ILF3 (also known as NF90, 90 kD) has the highest Sequest HT score (Fig. 3C). This protein usually forms a regulatory complex with ILF2 (also known as NF45, 45 kD), which is also observed in the MS results (Fig. 3C). Sense PTTG3P specific 45 kD and 90 kD bands were also observed (Fig. 3D). Then, RNA-IP assay was conducted using anti-ILF2 and anti-ILF3, respectively. The presence of ILF2 and ILF3 in the anti-ILF2 and anti-ILF3 immunoprecipitated samples were confirmed by western blotting (Fig. 3E-F). These samples had significantly higher enrichment of PTTG3P compared to the IgG control group (Fig. 3G).

By checking RNA-seq and survival data in TCGA-LIHC and RNA-seq data from GTEx, we confirmed the upregulation of ILF2 and ILF3 in HCC tissues compared to normal liver and tumor-adjacent normal tissues (Fig. 3H). Survival analysis confirmed the association between upregulated ILF2/ILF3 and unfavorable prognosis (Fig. 3I-L).

PTTG3P depletion increases the binding of ILF2 to the promoter of TOP2A

Since we characterized that PTTG3P interacts with ILF2 and ILF3 in HCC, while the ILF2/ILF3 complex as a transcriptional enhancer/factor, we tried to explore their downstream targets in common. Firstly, we overexpressed PTTG3P in Hep3B cells. 48 h later, total RNA was extracted for RNA-seq. By setting adj. p < 0.05 and log 2(Fold Change, FC) ≥ 1 as the cutoff, we identified 215 dysregulated genes (Supplementary Table 3). One recent study explored dysregulated genes in HepG2 cells after ILF2 depletion ²⁵. Using their data, we identified 125 genes downregulated after ILF2 inhibition (adj.p < 0.05 and log 2(FC) ≤-1) (Supplementary Table 4). These two sets shared six genes, including TOP2A, KIF14, BUB1, SERPINA3, STRA6, and KREMEN1 (Fig. 4A). For validation purposes, we further assessed the correlation of these genes with PTTG3P and ILF2 in TCGA-LIHC (Fig. 4B). By setting moderate correlations as the cutoff (| Pearson's r ≥ 0.4,| adj. p < 0.05), we found that TOP2A, KIF14, and BUB1 as potential candidates. Besides, we manually added LOXL2 and ENO2, two overexpressed genes after PTTG3P overexpression (Supplementary Table 3), as potential candidates.

We conducted ChIP-qPCR assay to check whether PTTG3P depletion alters the binding of ILF2 to the promoter of these genes. Results showed that after PTTG3P knockdown, ILF2 presented enhanced binding to the promoter of TOP2A, but showed weakened binding to the promoter of ENO2 (Fig. 4C-D). By checking protein level expression, we observed negative ENO2 expression in both normal liver and HCC tissues (Fig. 4E). In consistence with previous reports, we confirmed negative TOP2A expression in hepatocytes in normal liver (all negative by three primary antibodies in the HPA, HPA006458, HPA026773, and CAB002448) (Fig. 4F, green frame). In comparison, medium to high TOP2A expression was observed in HCC tissues (Fig. 4F, brown frame).

PTTG3P inhibition results in nuclear translocation of ILF2

After PTTG3P depletion, we observed nuclear accumulation of ILF2 by IF staining (Fig. 5A). However, no alteration was observed in the cellular distribution of ILF3 (Fig. 5A). Colocalization of ILF2 and ILF3 was observed in both cytoplasma and nuclear (Fig. 5A). By performing fractional western blotting, we confirmed this distributional alteration (Fig. 5B).

Via scanning the promoter region of TOP2A, we observed two potential ILF2/ILF3 binding sites (Fig. 5C). Then, we generated luciferase reporter plasmids carrying wild-type (pGL3-TOP2A-WT) and mutant TOP2A promoter sequences (pGL3-TOP2A-MT1, pGL3-TOP2A-MT2, and pGL3-TOP2A-MT3) based on a pGL3-basic backbone. ILF2 inhibition significantly reduced the luciferase activity of the reporters carrying at least one wild-type ILF2/ILF3 binding site (Fig. 5D-F), but had no influence on the reporter with two mutant sites (Fig. 5G). In contrast, PTTG3P inhibition significantly increased the luciferase activity of the reporters carrying at least one wild-type ILF2/ILF3 binding site (Fig. 5D-F), but did not influence the luciferase activity of the reporter with two mutant sites (Fig. 5G). These findings suggest that PTTG3P depletion might enhance TOP2A expression via promoting nuclear translocation of ILF2. To test this hypothesis, we checked the mRNA expression of TOP2A expression after PTTG3P knockdown. Interestingly, PTTG3P depletion did not increase but decreased TOP2A mRNA levels (Fig. 5H). This discrepancy implies that some other mechanisms are involved in regulating TOP2A expression/stability.

ILF2/ILF3 complex stabilizes TOP2A mRNA in the cytoplasm

Since the ILF2/ILF3 complex may act as an mRNA stabilizer, we tested whether ILF2/ILF3 binds to the 3'UTR of TOP2A mRNA and regulates its stability. RIP-qPCR confirmed significantly higher enrichment of TOP2A 3'UTR in the samples precipitated by anti-ILF2 than the IgG control (Fig. 6A-B). PTTG3P depletion significantly reduced the enrichment level (Fig. 6A-B). These findings suggest that ILF2 binds to the 3'UTR of TOP2A mRNA, the effect of which was enhanced by PTTG3P. ILF2 or PTTG3P depletion significantly reduced the stability of TOP2A mRNA (Fig. 6C-D), the effect of which was enhanced by simultaneous ILF2 and PTTG3P inhibition (Fig. 6C-D).

PTTG3P or TOP2A inhibition impairs HCC growth in-vivo

By generating Hep3B derived in vivo tumor model in mice, we found that PTTG3P overexpression significantly enhanced tumor growth (Fig. 7A-B). PTTG3P or TOP2A inhibition significantly slowed the growth rate (Fig. 7A-B). HE and TUNEL staining showed that PTTG3P and TOP2A inhibition are associated with enhanced tumor necrosis and apoptosis (Fig. 7C, top and bottom panels). In agree with these data, Ki-67 staining indicated a decreased level of cell proliferation in both PTTG3P KD and TOP2A KD groups (Fig. 7C, middle panel).

Based on these findings, we infer that PTTG3P exerts dual-regulation in TOP2A expression at both transcriptional and post-transcriptional levels. It is usually overexpressed in HCC tumor cells and accumulated in cytoplasm. Under this circumstance, it forms a complex with ILF2/ILF3, which binds to the 3'UTR of TOP2A mRNA and increases its stability. However, PTTG3P depletion can trigger nuclear translocation of ILF2, which binds to the promoter of the TOP2A gene and enhances its transcription (Fig. 8).

PTTG3P has been demonstrated as an oncogenic lncRNA in multiple other cancers, such as gastric cancer ²⁶, colorectal cancer ²⁷, head and neck squamous cell carcinoma ²⁸, and cervical cancer ²⁹. Previous studies found that the regulatory effects of PTT3GP are quite complex. It can act as a miRNA sponge ^14,27 and a functional partner of transcriptional factor ¹⁵. In the current study, we confirmed that PTTG3P overexpression contributed to enhanced proliferation, cell-cycle progression, invasion, and migration of HCC. Then, we found that PTTG3P interacts with both ILF2 and ILF3 in HCC cells. The association between ILF2/ILF3 upregulation and the unfavorable prognosis was also confirmed based on survival data from TCGA-LIHC.

ILF2/ILF3 complex can act as an mRNA stabilizer via binding to the 3'UTR of a cluster of mitotic mRNAs (including NUF2, CDK1, CCNE1, CCNA2, and BIRC5) and reduce Staufen-mediated mRNA decay ^21,25. However, the spectrum of mRNAs controlled by the ILF2/ILF3 complex and other modulators of this complex has not been well characterized. In this study, we revealed that the binding between PTTG3P and ILF2 helps maintain the cytoplasmic distribution of ILF2. Together with ILF2, PTTG3P binds to the 3'UTR of TOP2A. PTTG3P depletion impairs the binding ability and reduces TOP2A mRNA stability.

Similar to our reports, some recent studies also reported the binding of lncRNAs to ILF2/ILF3. LncRNA DANCR interacts with ILF2/ILF3 complex and increases HIF1A mRNA stability in nasopharyngeal carcinoma ³⁰. LINC00470 forms a complex with ILF2/ILF3 and enhances its interaction with CCNE1 mRNA, which further enhances its stability and facilitates cell-cycle progression in HCC ³¹. LncRNA AK085865 specifically interacts with ILF2 and negatively regulates the ILF2/ILF3 complex-mediated miRNA-192 biogenesis in bone marrow-derived macrophages ³². These findings suggest that lncRNAs can act as an important functional modulator of the ILF2/ILF3 complex.

TOP2A is a protein regulating the topological state of DNA during transcription via transiently breaking and rejoining DNA strands. As a critical nuclear enzyme for DNA replication- and cell division‐regulating, TOP2A exerts multiple functions across the cell cycle, and its overexpression is a critical driver of malignant transformation and uncontrolled proliferation of tumor cells ^33,34. Multiple anti-drugs targeting TOP2A, such as mitoxantrone, mAMSA, etoposide, doxorubicin, epirubicin, and idarubicin, have been developed ³⁵. TOP2A overexpression is associated with early age onset, shorter survival, and chemoresistance to doxorubicin of HCC ^36,37. Depletion of TOP2A significantly suppresses the proliferation of HCC cells ³⁸, suggesting that TOP2A is an important therapeutic target in HCC.

Since PTTG3P is usually aberrantly upregulated in HCC, its accumulation in the cytoplasm make it mainly act as a TOP2A stabilizer. Therefore, when it is knocked down, we observed downregulated TOP2A mRNA levels. After PTTG3P depletion, we observed nuclear accumulation of ILF2 and increased binding of ILF2 to the promoter region of TOP2A. Dual-luciferase assay confirmed that ILF2 could activate the TOP2A promoter, suggesting that the ILF2/ILF3 complex acts as a transcriptional enhancer of TOP2A. The translation of ILF2 to the nuclear part might be utilized by HCC cells to compensate the loss of PTTG3P and associated TOP2A instability. These mechanisms help explain why PTTG3P depletion does not lead to drastically downregulated TOP2A mRNA levels.

In conclusion, this study identified PTTG3P as a novel functional partner of the ILF2/ILF3 complex, which enhances the binding of the ILF2/ILF3 complex to the 3'UTR of TOP2A mRNA and increases its stability in HCC. When it is overexpressed in HCC tumor cells, it mainly acts as a TOP2A mRNA stabilizer. PTTG3P depletion results in nuclear accumulation of ILF2, which binds to the promoter of TOP2A and enhances its transcription. These findings help expand our understanding of the oncogenic mechanisms of PTTG3P in HCC.

Acknowledgment

The authors have no conflict of interest.

Authors’ contributions

TL,MZ,HSB conceived the idea; HYZ,JHL performed the analysis; HYJ,LL interpreted the results and made tables. All authors read and approved the final manuscript

Funding

none

Availability of data and materials

the datasets used during the study are available from the corresponding authors on reasonable request

Ethics approval and consent to participate

all animal procedures and care were conducted by institutional guidelines and in compliance with national and international laws and policies

Consent for publication

not applicable

Competing interests

the authors declare that they have no competing interests

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SupplementaryTable1PrimersandshRNA.xlsx
Supplementary Table 1. ShRNA and primers used in this study
SupplementaryTable2RNApulldownproteinlist.xlsx
Supplementary Table 2. MS results of proteins from RNA pull-down assay
SupplementaryTable3.xlsx
Supplementary Table 3. Genes upregulated in Hep3B cells after PTTG3P overexpression
SupplementaryTable4.xlsx
Supplementary Table 4. Genes downregulated in HepG2 cells after ILF2 depletion

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Long non-coding RNA PTTG3P promotes hepatocellular carcinoma growth via modulating nuclear-cytoplasm shuttle of ILF2 and stabilizing TOP2A mRNA

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