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LncRNA PVT1 activated by TGF-β1/Smad3 facilitates proliferation and metastasis of hepatocellular carcinoma via upregulating Smad6 and NRG1

Journal of Translational Medicine volume 23, Article number: 500 (2025) Cite this article

Abstract

Background

Hepatocellular carcinoma (HCC) significantly affects the patient’s physical and mental health. Long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) has been associated with the progression of HCC. However, the current effectiveness of HCC treatment is considered insufficient, and the scope of its therapeutic targets is highly limited. The purpose of this investigation is to investigate the pathogenic mechanism of PVT1 in HCC and assess its potential for gene therapy in HCC.

Methods

This study assessed cycle phases and proliferative capacity of HCC cells through flow cytometry, CCK-8 assay, EdU, and colony formation assays. Chromatin Immunoprecipitation (ChIP) and Dual-Luciferase Reporter Assays were conducted to investigate the interactions among the promoter and PVT1, PVT1 and its target miRNAs, as well as miRNAs and their target genes. BALB/c nude mice were employed to establish models for studying the proliferation and metastasis of HCC in vivo.

Results

The data revealed that TGF-β1 upregulates PVT1, while Smad3 functions as a transcription factor to modulate PVT1. PVT1, in turn, upregulates Smad6 and NRG1 (Neuregulin 1). Moreover, PVT1 combines with miR-186-5p and miR-143-3p, while miR-186-5p inhibits Smad6 and miR-143-3p inhibits NRG1. Further, in vivo and in vitro analyses revealed that PVT1 stimulates the expression of Smad6, thereby promoting the proliferation of HCC. In addition, PVT1 also promotes the spread of HCC by upregulating NRG1.

Conclusion

This study validated that PVT1 activated by TGF-β1/Smad3 facilitates HCC progression and metastasis by upregulating the miR-186-5p/Smad6 and miR-143-3p/NRG1 axes, indicating its potential as a biological target for treating HCC.

Introduction

Hepatocellular carcinoma (HCC) has high morbidity and mortality rates and is the most prevalent form of liver cancer. It is ranked 6th among all cancers and is the 3rd highest cause of cancer-related death globally, posing a significant threat to human physical and mental health [1, 2]. Alcoholic liver disease (ALD), hepatitis B (HBV), and chronic hepatitis C (CHC) are considered the primary cause of liver cancer [3]; however, the pathogenic mechanism of HCC remains unclear. lncRNA PVT1, does not encode proteins, is > 200 nt long, and has been documented to accelerate the development of numerous types of tumors [4,5,6,7]. Furthermore, several studies have proved that PVT1 not only enhances the proliferation of HCC but also boosts its metastatic ability, significantly impacting the overall survival rates of HCC patients. However, the exact mechanism remains elusive [8,9,10]. It has been recognized that TGF-β1 can inhibit the growth of HCC but enhance their dissemination, which is crucial for the advancement of HCC [11,12,13]. However, TGF-β1 also plays a significant role in various normal physiological processes; therefore, its inhibition has marked safety risks, and patients cannot receive this treatment for long. Therefore, identifying molecules that can counteract the suppressive effect of TGF-β1 on HCC proliferation and enhance its metastatic capabilities is significant. This study identified a novel downstream mechanism of TGF-β1 in HCC and the comprehensive promoting mechanism of PVT1 in HCC, thereby providing new evidence and potential targets for gene therapy in HCC.

Materials and methods

Cell culture and transfections

HepG2, Hep3B, Huh7, PLC/PRF/5 and HCCLM3 cells were acquired from Pricella Biology (Wuhan, China), MHCC97H cells were acquired from Servicebio (Wuhan, China), and THLE-3 cells were procured from Chinese Academy of Sciences (Shanghai, China). The cells were grown in a medium augmented with 10% fetal bovine Serum (Pricella, Wuhan) and 1% penicillin-streptomycin (Gibico, USA). Genechem (Shanghai, China) synthesized the lentiviruses, while plasmids, miRNA inhibitors, and mimics were developed by Tsingke Biotechnology (Beijing, China). Wuhan JTS Scientific constructed siRNAs. The transfection reagents were acquired from Servicebio (Wuhan, China). The cells were transfected at the confluency of about 50%. Supplementary Table 1 enlists the sequences of small interferences RNAs and shRNAs.

Quantitative real-time PCR analysis (qRT-PCR)

The total RNA was extracted from the processed cells using Trizol. RNA purity and concentration were assessed using the NANO2000. The acquired RNA was then reverse transcribed to synthesize cDNA via TRUEscript RT MasterMix (keep Biotechnologies Co., Ltd), which was then used for qRT-PCR analysis using SYBR Mix (Servicebio) and BIO-RAD equipment. The specific primers, microRNA mimics and inhibitors were synthesized by Tsingke Biotechnology Co., Ltd and the sequences are included in Supplementary Tables 2 and 3.

Western blotting (WB)

The proteins were extracted with RIPA buffer (Biyuntian, Shanghai), quantified with the BCA kit (Biyuntian), electrophoresed through 10% SDS-PAGE gel, transferred to the PVDF membrane, which was then blocked for 2 h with 5% skim milk, and then treated with the corresponding antibody. The membrane was then visualized using a Tanon multi-function imaging machine. Supplementary Table 4 enlists the antibodies utilized in this research.

Cell cycle analysis

Briefly, the cells (5 × 105) were collected, fixed with 75% ethanol, then stained with the working solution as described in the cell cycle analysis kit (Servicebio). The cell cycle was then analyzed via flow cytometry.

Cell proliferation assays

The cell counting kit 8 (CCK-8) assay was carried out to determine cell proliferation ability. Briefly, 3 × 103 cells were plated in a 96-well plate with 200µL medium and incubated. Then, 10µL of CCK-8 reagent (biosharp, Hebei, China) was added to each well and after 2 h, the absorbance was measured at 450 nm.

For the EdU assay, the log growth phase HCC cells were grown in 24-well plates, labeled by the EdU kit (Biyuntian), and then observed and photographed with a microscope.

For colony formation assay, 2 × 103 cells were cultured in a 6-well plate for 2 weeks. The cells were then fixed, stained, and counted for colony formation.

Wound healing assays

To assess the cell metastasis ability, a wound healing assay was carried out. Briefly, the cells were cultured in 6 well-plate and upon near confluency, wounded with a pipette tip. The width of the scratch was then monitored and photographed under an inverted microscope.

Transwell experiment

Briefly, 600µL of complete medium was added in the bottom chamber of a 24-well transwell plate (LABSELECT, Beijing China 6.5 mm diameter/well-plate). In the upper chamber 2 × 104 cells were suspended in a serum-free medium for 48 h. Then, the upper layer of the chamber was wiped clean, and the cells that had migrated through the chamber were fixed, stained with Crystal Violet Ammonium Oxalate Solution (Solarbio, Beijing, China), and photographed.

Chromatin immunoprecipitation

For this analysis, about 1 × 107 cells were collected, cross-linked with 1% formaldehyde + 0.125 M glycine, and lysed with 720µL buffer. The supernatant was preserved as the input group and split into IgG and IP groups. To verify that the DNA is purified, qRT-PCR was performed using the DNA purification kit (CWbiotech, Beijing, China). PCR products were then analyzed using 2% agarose gel electrophoresis.

Dual-luciferase reporter assay

To prove that Smad3 interacts with PVT1, HCCLM3 cells were transfected with pGL3-PVT1-WT, the negative control plasmid pGL3-PVT1-MUT, and pRL-TK renilla. After 48 h, Firefly&Renilla Luciferase Reporter Assay Kit (meilunbiotech, Dalian, China) was employed to measure the activity of firefly and Renilla luciferases.

Furthermore, pmirGLO plasmids containing the PVT1 3’UTR, NRG1 3’UTR, Smad6 3’UTR, and miR-186-5p and miR-143-3p binding series were synthesized. When the HCCLM3 cell density reached approximately 60%, the miRNAs and pmirGLO plasmids were co-transfected. After 48 h, cell fluorescence intensity was detected using the kit.

Animal experiments

The SPF standard female BALB/c nude mice aged 4–6 weeks were obtained from Bent Laboratory Animal in Wuhan, China, and housed at Wuhan University’s Animal Experimental Center. To establish a subcutaneous tumor model, HCCLM3 cells were transfected with shPVT1-NC, shPVT1, shPVT1 + Smad6-Vector, and shPVT1 + Smad6. Transfected cells were then injected into the axilla of mice (1 × 106 cells/mouse). The treatment mice were analyzed at least twice weekly. Tumor dimensions [length (L) and width (W)] were measured using a vernier caliper, and tumor volume was assessed using the following formula V = 0.5 × L × W2. The mice were euthanized after 4 weeks via cervical dislocation and subcutaneous tumors were excised, photographed, weighed, and subjected to hematoxylin and eosin (H&E) staining and immunohistochemical analysis. Furthermore, for establishing a lung metastasis model, transfected HCCLM3 cells (1 × 105) were injected into the tail veins of mice. After 5 weeks, the mice were euthanized, and their lungs were removed for H&E staining.

Immunohistochemistry

The paraffin-embedded Sect. (4 mm thick) were placed on glass slides, dehydrated through a series of graded alcohols and xylene, and then stained using the UltraSensitiveTM S-P Kit (Maixin, Fuzhou, China) as per the manufacturer’s guidelines.

Statistical analyses

All statistical analyses were carried out using GraphPad Prism Software version 9.0 (GraphPad, USA), with results expressed as mean ± standard deviation of at least three independent replicates. Intergroup differences were assessed using the Student’s t-test, while one-way ANOVA was employed to evaluate differences among multiple groups. The threshold for statistical significance was set at p < 0.05, and p-values were designated as follows: *p < 0.05, **p < 0.01 ***p < 0.001, and ****p < 0.0001.

Results

PVT1 was highly expressed in HCC and up-regulated by TGF-β1

According to the GEPIA database, PVT1 expression was significantly upregulated in liver cancer tissues compared to normal tissues (Fig. 1A). The Kaplan-Meier Plotter database further demonstrated that patients with high PVT1 expression had significantly shorter overall survival (OS) compared to those with low PVT1 expression (Fig. 1B). It has been shown that the level of PVT1 expression is significantly elevated in liver cancer patients, and increased PVT1 expression has unfavorable clinical outcomes [8,9,10]. In this study, PVT1 expression in various cell lines, including hepatocyte THLE-3, hepatoma HepG2, Hep3B, Huh7, PLC/PRF/5 (low metastatic), and MHCC97H, HCCLM3 (high metastatic) was evaluated (Fig. 1C). This study selected 4 types of HCC cells (Huh7, PLC/PRF/5, MHCC97H, and HCCLM3) for subsequent analyses.

Fig. 1
figure 1

PVT1 was highly expressed in HCC and up-regulated by TGF-β1. (A) The GEPIA database indicated the PVT1 expression in liver cancer and normal tissue. (B) The Kaplan-Meier Plotter platform assessed the overall survival (OS) in HCC patients. (C) qRT-PCR was carried out to assess the expression of PVT1 in hepatocytes and hepatoma cells. (D-G) qRT-PCR was carried out to assess the expression of PVT1 in Huh7, PLC/PRF/5, MHCC97H, and HCCLM3 cells after treatment with TGF-β1 (10 ng/mL) for 1 and 2 days. (H-K) qRT-PCR was carried out to assess the PVT1 expression after the treatment of SB431542 (5 µM). The data are expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Previous studies have indicated that PVT1 expression in HCC cells rises following TGF-β1 treatment (GSE54797); however, the underlying mechanism has not been reported. To explore whether TGF-β1 can upregulate PVT1, the Huh7, PLC/PRF/5, MHCC97H, and HCCLM3 cells were exposed 10 ng/mL of TGF-β1. Following an incubation period of 24 to 48 h, qRT-PCR analysis was performed to quantify PVT1 expression in the cells. The results showed significant upregulation of PVT1 in all four HCC cell lines (Fig. 1D-G). Moreover, treatment with SB431542 (5 µM), a TGF-β1 inhibitor, reduced PVT1 expression in these four cell types (Fig. 1H-K). These data proved that TGF-β1 is the upstream regulator of PVT1.

Smad3 binds to PVT1 and positively regulates its expression as a transcription factor

In the canonical TGF-β/Smad signaling pathway, TGF-β1 can activate and induce Smad3 accumulation in the nucleus, where it acts as a transcription factor to regulate downstream target genes [14, 15]. Since TGF-β1 can regulate the expression of PVT1; therefore, whether TGF-β1 affects PVT1 via the canonical Smad pathway was also assessed. Small interference RNA (siRNA) was transfected into MHCC97H, HCCLM3, Huh7, and PLC/PRF/5 cells to temporarily silence Smad3 expression. The knockdown efficiency was validated via qRT-PCR and Western blotting (WB) analyses, which indicated a significant decrease in Smad3 expression in all four cell lines (Fig. 2A-D). The data revealed that PVT1 expression markedly reduced with Smad3 knockdown (Fig. 2E-H). These results showed that smad3 can regulate PVT1.

Fig. 2
figure 2

Smad3 binds to PVT1 and positively regulates its expression as a transcription factor. (A-D) qRT-PCR and WB analyses were performed to assess the knockdown efficiency of Smad3 in MHCC97H, HCCLM3, Huh7, and PLC/PRF/5 cells. (E-H) qRT-PCR data confirmed that PVT1 expression was decreased in four HCC cell types after Smad3 knockdown. (I, J) The ChiP experiment results confirmed the combination of Smad3 and PVT1. (K) The binding and mutation sites of Smad3 and PVT1. (L) The result of dual-luciferase reporter assay in HCCLM3 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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To detect whether Smad3 functions as a transcription factor to regulate PVT1 expression, the chip experiment and dual-luciferase reporter gene assay were performed. The results of chip experiment demonstrated that Smad3 can bind with PVT1 (Fig. 2I, J). Then, the JASPAR bioinformatics platform was employed to predict the binding site of Smad3 and the PVT1 promoter region (Fig. 2K). Furthermore, wild-type PVT1 plasmid (PVT1-WT) and mutant PVT1 plasmid (PVT1-MUT) were constructed and transfected into HCCLM3 cells, along with the addition of TGF-β1. After 48 h, the dual-luciferase reporter assay indicated a marked increase in fluorescence intensity of PVT1-WT after TGF-β1 treatment, whereas PVT1-MUT showed no significant change (Fig. 2L). These outcomes indicated that Smad3 acts as a transcription factor, stimulated by TGF-β1 to enhance the expression of PVT1.

PVT1 promotes the proliferation, migration, and invasion of HCC cells

The lentivirus was transfected into the HCCLM3 and MHCC97H cells to knock down PVT1 (shPVT1), as well as into Huh7 and PLC/PRF/5 cells to overexpress PVT1. The qRT-PCR data confirmed the successful establishment of knockdown and overexpression models (Fig. 3A, H; Supplementary Fig. 1A, H). The flow cytometry analysis revealed that PVT1 knockdown in HCCLM3 and MHCC97H cells halted the cell cycle at the G0/G1 phase, thereby significantly reducing the percentage of cells in the S-phase (Fig. 3B; Supplementary Fig. 1B). Whereas PVT1 overexpression in Huh7 and PLC/PRF/5 cells had the opposite effects (Fig. 3I; Supplementary Fig. 1I). Moreover, the CCK-8, EdU staining, and colony formation assays revealed that PVT1 knockdown substantially decreased the proliferation of the two HCC cells (Fig. 3C-E; Supplementary Fig. 1C-E), while the proliferation potential of HCC cell was enhanced as PVT1 overexpressing (Fig. 3J-L; Supplementary Fig. 1J-L). Similarly, the transwell experiments and wound healing tests confirmed that PVT1 knockdown significantly impaired the migratory and invasive capabilities of HCCLM3 and MHCC97H cells (Fig. 3F, G; Supplementary Fig. 1F, G), while PVT1 overexpression particularly increased the metastasis potential of Huh7 and PLC/PRF/5 cells (Fig. 3M, N; Supplementary Fig. 1M, N). Overall, this evidence suggests that PVT1 markedly increases the growth and aggressive behavior of HCC cells.

Fig. 3
figure 3

PVT1 promotes the proliferation, migration, and invasion of HCC cells. (A, H) qRT-PCR was performed to assess the knockdown efficiency of PVT1 in HCCLM3 cells and the overexpression efficiency in huh7 cells. (B, I) Flow cytometry was conducted to assess the cell cycles of HCCLM3 cells (PVT1 knockdown) and huh7 cells (PVT1 overexpression). (C-E, J-L) The CCK-8, EdU, and colony formation analyses were performed to measure the proliferation of HCCLM3 and huh7 cells after PVT1 knockdown and overexpression. (F, G, M, N) Wound healing and transwell tests were conducted to evaluate the migration and invasion ability of HCCLM3 and huh7 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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The downstream target of PVT1

Fang et al. studied PVT1 and revealed that the most significantly affected genes in PVT1 overexpressing HCC cells were those related to the cell cycle. Furthermore, KEGG pathway analysis revealed that certain molecules in the TGF-β pathway were also influenced [8]; however, the specific mechanisms remain elusive. This study comprehensively analyzed the RNA sequencing results of Fang et al. and found that PVT1 overexpression in HCC cells had upregulated Smad6 and downregulated P21 expressions significantly (GSE57786). Smad6 is a crucial member of the TGF-β superfamily and can specifically inhibit the bone morphogenetic protein (BMP) pathway [16,17,18], which can substantially increase P21 expression, as P21 is a crucial gene that inhibits cell cycle progression [19, 20]. However, no previous study has documented the relationship between PVT1 and Smad6. Furthermore, TGF-β1 itself can directly upregulate p21 [21,22,23]. Therefore, it was predicted that PVT1 may antagonize the growth inhibitory effect of TGF-β1 through the Smad6/BMP/P21 pathway, ultimately promoting cancer progression. To verify the reliability of the sequencing results, RNA and proteins were extracted from HCCLM3 and MHHCC97H cells with knocked-down PVT1 expression, as well as from Huh7 and PLC/PRF/5 cells with overexpressed PVT1. The qRT-PCR and WB analyses revealed a significant downregulation of Smad6 expression and upregulation of P21 expression when PVT1 was knocked down (Fig. 4A; Supplementary Fig. 2A). Whereas PVT1 overexpression cells indicated an opposite trend (Fig. 4B; Supplementary Fig. 2B).

Fig. 4
figure 4

The downstream target of PVT1. (A, B) qRT-PCR and WB analyses were performed to assess the Smad6 and P21 expression in HCCLM3 (PVT1 knockdown) and huh7 (PVT1 overexpression) cells. (C, D) qRT-PCR and WB analyses were performed to assess NRG1 expression in HCCLM3 (PVT1 knockdown) and huh7 (PVT1 overexpression) cells. (E) Bioinformatics predicted miRNAs binding with PVT1 and Smad6. (F) The miRNAs binding to PVT1 and NRG1 were identified via a biometrics database. (G) miR-186-5p mimics transfection efficiency was assessed using qRT-PCR. (H) qRT-PCR and WB analyses were performed to assess Smad6 expression after miR-186-5p mimics transfection. (I) miR-186-5p inhibitor transfection efficiency was detected by qRT-PCR. (J) qRT-PCR and WB analyzed analyses were performed to assess Smad6 expression after miR-186-5p inhibitor transfection. (K) The miR-143-3p mimics’ transfection was verified by qRT-PCR. (L) NRG1 expression was examined by WB and qRT-PCR after miR-143-3p mimics transfection (M, N) The transfection efficiency of the miR-143-3p inhibitor was detected by qRT-PCR, and the expression of NRG1 was analyzed via WB and qRT-PCR. (O-P) The results of luciferase reporter assay of PVT1/miR-186-5p/Smad6 interaction. (Q-R) The results of reporter gene assays of PVT1/miR-143-3p/NRG1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Epithelial-mesenchymal transformation (EMT) is the core event in cancer invasion and metastasis [24,25,26]. By comparing the differential gene sets between EMT key molecules and those from PVT1 overexpressing HCC cells, it was found that PVT1 can significantly upregulate NRG1 expression [8]. NRG1 is an important upstream ligand in the epidermal growth factor receptor (EGFR) family, which can foster the migration and invasion of HCC by activating its receptor ERBB2/ERBB3 and up-regulating the expression of downstream RAS/MAPK in HCC [27]. Therefore, it was speculated that PVT1 may promote the metastasis of HCC by up-regulating NRG1. To validate this hypothesis, qRT-PCR and WB analyses were performed, and the data indicated that PVT1 knocked down downregulated NRG1 (Fig. 4C. Supplementary Fig. 2C), whereas PVT1 overexpression upregulated NRG1 (Fig. 4D; Supplementary Fig. 2D).

This study also assessed how PVT1 interacts with Smad6 and NRG1. LncRNA commonly modulates gene expression post-transcriptionally via the competitive endogenous RNA (ceRNA) network. It functions as a ceRNA sponge, sequestering microRNAs (miRNAs) and consequently reducing their suppressive influence on target mRNAs [28, 29]. Therefore, the miRDB bioinformatics platform and TargetScan website were employed to predict the potential miRNAs that may interact with PVT1, Smad6, and NRG1. The data indicated two miRNAs common to PVT1 and Smad6 (Fig. 4E) and three miRNAs common to PVT1 and NRG1 (Fig. 4F). Previous studies have indicated that PVT1 can interact with miR-186-5p [30, 31] and miR-186-5p to regulate the TGF-β1 pathway as well as bind to and inhibit Smad6 [32]. Furthermore, miR-186-5p can also inhibit HCC [33]. Thus, here, whether PVT1 can competitively bind with miR-186-5p and stabilize Smad6 mRNA through a molecular sponge mechanism was also assessed. Briefly, the miR-186-5p mimics were transfected into Huh7 cells and subjected to a qRT-PCR experiment to prove their high transfection efficiency (Fig. 4G). Furthermore, Smad6 expression was detected by qRT-PCR and WB analyses, which revealed a significant decrease (Fig. 4H). Moreover, treatment with miR-186-5p inhibitors increased the Smad6 expression levels (Fig. 4I, J). In addition, the binding sites of PVT1 3’UTR and miR-186-5p were identified via the miRDB database. The TargetScan website was utilized to predict the binding sites of miR-186-5p and Smad6 3’UTR. Additionally, the PVT1 wild-type reporter plasmid (PVT1 3’UTR-WT) and the Smad6 3’UTR wild-type reporter plasmid (Smad6 3’UTR-WT) as well as the PVT1 mutant plasmid (PVT1 3’UTR-MUT) and the Smad6 3’UTR mutant reporter plasmid (Smad6 3’UTR-MUT) were also constructed. These plasmids were utilized for dual-luciferase reporter assays, which indicated significantly reduced fluorescence intensity of both PVT1 3’UTR-WT and Smad6 3’UTR-WT after transfection with miR-186-5p mimics. However, no significant difference was noted in the fluorescence intensity of PVT1 3’UTR-MUT and Smad6 3’UTR-MUT post-transfection (Fig. 4O, P). The above data proved that PVT1 binds competitively with miR-186-5p/Smad6.

Among the microRNAs (miRNAs) capable of binding PVT1 and NRG1, miR-143-3p interacts with NRG1 and suppresses its expression [34]. Furthermore, a recent review systematically assessed the inhibitory effect of miR-143-3p on HCC and revealed that various lncRNAs competitively bind with miR-143-3p to regulate downstream target genes, ultimately resulting in the promotion of HCC metastasis [35]. However, no study suggests that miR-6088 or miR-4770 bind to either PVT1 or NRG1. Therefore, it was hypothesized that PVT1 might sponge miR-143-3p to control the expression of NRG1. The miR-143-3p mimics and miR-143-3p inhibitor were transfected into Huh7 and HCCLM3 cells. Then, their transfection efficiency was verified (Fig. 4K, M). The results of qRT-PCR and WB experiments showed that miR-143-3p mimics inhibited NRG1, while miR-143-3p inhibitor upregulated NRG1(Fig. 4L, N). For further verification, NRG1 3’UTR wild-type reporter plasmid (NRG1 3’UTR-WT) and mutant plasmid (NRG1 3’UTR-MUT) were constructed for reporter assay. The data revealed that miR-143-3p mimics transfection markedly decreased the fluorescence intensity of both PVT1 3’UTR-WT and NRG1 3’UTR-WT plasmids. However, there was no specific alteration in the fluorescence intensity of PVT1 3’UTR-MUT and NRG1 3’UTR-MUT post-transfection with miR-143-3p mimics. (Fig. 4Q, R). Overall, it was inferred that PVT1 functions as a molecular sponge for miR-143-3p, competitively binding it and diminishing its inhibitory effect on NRG1, thereby upregulating NRG1 expression.

Smad6 deletion reversed the proliferative effect of PVT1 on HCC

To further confirm that PVT1 impacts HCC proliferation by modulating the expression of downstream Smad6, rescue experiments were performed using PVT1 knockdown and Smad6 overexpression models. These experiments were categorized into four groups: shPVT1-NC, shPVT1, shPVT1 + Smad6-Vector, and shPVT1 + Smad6. After constructing the four stably transfected cells, the expression of Smad6 and P21 was analyzed via qRT-PCR and WB analyses. The results indicated that shPVT1 increased P21 expression, which was reversed by Smad6 overexpression (Supplementary Fig. 3A-C). Furthermore, these cell lines were also subjected to cell cycle, CCK-8, EdU tests, and colony formation experiments, which revealed that Smad6 overexpression could inhibit the shPVT1-induced G0/G1 cell cycle arrest and decreased HCC cell proliferation in vitro (Fig. 5A, B, E, G). However, altering the expression of Smad6 does not affect the PVT1-induced migration and invasion in HCC (Supplementary Fig. 5E-H). Moreover, rescue analysis was carried out in vivo by injecting the above cell lines into the armpits of immunocompromised nude mice. After 4 weeks, the subcutaneous tumors were weighed and measured. Overall, it was observed that Smad6 overexpression could reverse the PVT1 knockdown-induced inhibition of subcutaneous tumor progression (Fig. 5I-k). Furthermore, the subcutaneous tumors of the mice were subjected to H&E staining and immunohistochemical analysis, which showed that Smad6 overexpression could reverse the decreased Ki67 ratio and rescue the increased cleaved-caspase3 ratio observed after PVT1 knockdown (Fig. 5L-N).

Fig. 5
figure 5

Smad6 deletion reversed the proliferative effect of PVT1 on HCC. (A, B, E, G) Flow cytometry, CCK-8, EdU, and colony formation analyses were carried out to assess the proliferation potential of HCC cells when co-transfected with shPVT1 and Smad6. (C, D, F, H) The growth ability of PVT1 overexpression and shSmad6 in HCC cells was evaluated by Flow cytometry, CCK-8, EdU, and colony formation assays. (I) Representative images of mice’s axillary subcutaneous tumors after four weeks of HCC cell injection. (J, K) The tumor’s weight and volume were recorded and analyzed. (L) H&E staining of the tumor. (M, N) Immunohistochemical staining with Ki67 and cleaved-caspase3 markers was employed to assess the tumor’s proliferative potential. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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For another rescue experiment, the cells were categorized into the following four groups: PVT1-Vector, PVT1, PVT1 + shSmad6-NC, and PVT1 + shSmad6. These cells were used to assess the expression of Smad6 and P21 via qRT-PCR and WB analyses. The data showed that shSmad6 could restore P21 levels to normal (Supplementary Fig. 3D-F). Moreover, the flow cytometry cycle, CCK-8, EdU and colony formation assays indicated that shSmad6 could counteract the enhanced PVT1 overexpression-induced HCC cell proliferation (Fig. 5C, D, F, H). Overall, these in vitro and in vivo analyses revealed that Smad6 overexpression can rescue the inhibition of HCC proliferation caused by PVT1 knockdown, whereas Smad6 knockout can reverse the increased HCC proliferation induced by PVT1 overexpression.

Silencing of NRG1 rescued PVT1-induced migration and invasion activities of HCC

To demonstrate that PVT1 modulates the metastatic potential of HCC by altering NRG1 expression, rescue experiments were carried out in four groups: shPVT1-NC, shPVT1, shPVT1 + NRG1-Vector and shPVT1 + NRG1. qRT-PCR and WB analyses were performed to assess NRG1 expression levels (Fig. 6A, B). Furthermore, migration assay, invasion assay, and wound healing analysis were performed to evaluate metastatic potential. The data revealed that NRG1 overexpression reversed the reduced migration and invasion capabilities of cells caused by shPVT1 (Fig. 6E, G), however, NRG1 did not affect the proliferation of PVT1-induced HCC (Supplementary Fig. 5A-D). Moreover, rescue experiments on the shPVT1 and NRG1 overexpression were conducted in vivo by injecting stably transfected HCCLM3 cells into the tail veins of nude mice to establish a lung metastasis model of HCC. After 5 weeks, the mice’s lungs were dissected and analyzed via H&E staining. The lung nodules were also counted. The outcomes demonstrated that NRG1 overexpression reversed the PVT1 knockdown-induced reduction in the lung metastatic ability (Fig. 6I). The in vivo and in vitro analyses verified that NRG1 overexpression mitigated the PVT1 knockdown-induced reduction in HCC metastatic potential.

Fig. 6
figure 6

Silencing of NRG1 rescued PVT1-promoted migration and invasion activities of HCC. (A) qRT-PCR test and WB analyses showed the level of NRG1 overexpression. (B) The qRT-PCR and WB techniques were employed to measure NRG1’s expression in shPVT1 and the overexpression of NRG1 in HCC cells. (C) The silencing efficiency of NRG1 was measured by qRT-PCR and WB assays. (D) qRT-PCR and WB analyses were performed to assess the expression of NRG1 in PVT1 overexpression and shNRG1 conditions. (E, G) Wound healing and transwell tests analyzed the metastatic potential of HCC cells with shPVT1 and NRG1 overexpression. (F, H) The ability of HCC cells to migrate and invade after PVT1 overexpression and NRG1 knockdown was estimated by transwell and wound healing assays. (I) Mouse lungs were subjected to H&E staining, and the lung nodules were enumerated. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Furthermore, another rescue experiment was conducted for the following groups: PVT1-Vector, PVT1, PVT1 + shNRG1-NC, and PVT1 + shNRG1. qRT-PCR and WB evaluations were carried out to assess the changes in NRG1 expression (Fig. 6C, D). The transwell and wound healing experiments were carried out to elucidate the migration and invasion capabilities of these four groups of Huh7 cells. The results verified that NRG1 knockdown rescued the advanced metastasis of HCC cells caused by PVT1 overexpression (Fig. 6F, H).

PVT1 up-regulates Smad6 by competitive binding miR-186-5p to facilitate HCC proliferation

To verify that PVT1 regulates HCC cell proliferation by interacting competitively with miR-186-5p to modulate Smad6 expression, two sets of rescue experiments involving PVT1 and miR-186-5p were carried out. One set was divided into four groups: shPVT1-NC, shPVT1, shPVT1 + inhibitor NC, and shPVT1 + miR-186-5p inhibitor. qRT-PCR and WB experiments revealed that the miR-186-5p inhibitor reversed the PVT1 knockdown-induced suppression of Smad6 and elevation of P21 at both the mRNA and protein levels (Supplementary Fig. 4A, C). Furthermore, flow cell cycle, CCK-8, EdU, and colony formation trials also validated that the miR-186-5p inhibitor can rescue the proliferation inhibition induced by shPVT1 (Fig. 7A, B, E, G). The other set of rescue experiments included PVT1-Vector, PVT1, PVT1 + mimics NC, and PVT1 + miR-186-5p mimics. qRT-PCR and WB revealed that miR-186-5p mimics rescued the PVT1 overexpression-induced Smad6 upregulation and P21 downregulation (Supplementary Fig. 4B, D). Moreover, flow cell cycle, CCK-8, EdU and colony formation assays also validated that miR-186-5p mimics could reverse the PVT1 overexpression-induced HCC cell proliferation (Fig. 7C, D, F, H). Whereas the presence of miR-186-5p does not alter the PVT1-enhanced migration and invasion activities (Supplementary Fig. 6E, H). Overall, these results imply that PVT1 promotes HCC proliferation via the miR-186-5p/Smad6 axis.

Fig. 7
figure 7

PVT1 up-regulates Smad6 by competitive binding miR-186-5p to facilitate HCC proliferation. (A, B, E, G) The proliferation of HCC cells after PVT1 knockdown and transfection with miR-186-5p inhibitor was investigated using flow cytometry, CCK-8, EdU, and colony formation assays. (C, D, F, H) HCC cells overexpressing PVT1 and harboring transfected miR-186-5p mimics were evaluated for their growth potential via flow cytometry, CCK-8, EdU, and colony formation assays. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Full size image

PVT1 up-regulates NRG1 by competitively binding with miR-143-3p to promote the migration and invasion of HCC

To further substantiate that PVT1 increases NRG1 expression to promote the metastatic potential of HCC cells by competitively interacting with miR-143-3p, rescue experiments were performed on miR-143-3p and PVT1. miR-143-3P was transfected into the already stable transfected cells and then assayed for NRG1 expression via qRT-PCR and WB analyses. It was found that in the rescue experiments of shPVT1-NC, shPVT1, shPVT1 + inhibitor NC, and shPVT1 + miR-143-3p inhibitor, the miR-143-3p inhibitor reversed the PVT1 knockdown-induced reduction in NRG1 expression (Fig. 8A). Furthermore, among PVT1-Vector, PVT1, PVT1 + mimics NC, and PVT1 + miR-143-3p mimics, the miR-143-3p mimics significantly reversed the PVT1 overexpression-induced increase in NRG1 expression (Fig. 8B). Moreover, the metastatic potential of these rescue experiments was assessed by transwell and wound healing analyses, which revealed that the miR-143-3p inhibitor could counteract the suppression of metastasis resulting from PVT1 knockdown (Fig. 8C, E). Whereas the miR-143-3p mimics rescued the PVT1 overexpression-induced increase in metastasis (Fig. 8D, F). However, the expression of miR-143-3p does not affect the proliferative effect of PVT1 (Supplementary Fig. 6A-D). Overall, these data suggested that PVT1 competitively binds with miR-143-3p to up-regulate NRG1 and facilitate the spread of HCC cells Fig. 9.

Fig. 8
figure 8

PVT1 up-regulates NRG1 by competitively binding with miR-143-3p to promote the migration and invasion of HCC. (A) qRT-PCR and WB assays were performed to analyze NRG1 expression in HCC cells after co-transfection with shPVT1 and a miR-143-3p inhibitor. (B) qRT-PCR and WB analyses were performed to assess NRG1 expression levels in HCC cells that overexpressed PVT1 and had been transfected with miR-143-3p mimics. (C, E) Wound healing and transwell experiments were conducted to evaluate the migratory and invasive potentials of HCC cells transfected with shPVT1 and miR-143-3p inhibitor (D, F) After the miR-143-3p mimics and PVT1 were transfected into HCC cells, wound healing and transwell assays performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Full size image
Fig. 9
figure 9

The mechanism of PVT1 regulating proliferation and metastasis in HCC

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Discussion

In recent years, despite substantial progress in the diagnosis and treatment of HCC, the incidence and mortality rates of HCC patients remain substantially high due to the limited understanding of its underlying mechanisms of occurrence and development. Previous literature has indicated that PVT1 holds a crucial significance in the development and progression of HCC [8,9,10]; however, its underlying molecular mechanism remains undetermined. TGF-β1 has also been observed to have an essential impact on HCC, indicating both tumorigenic and tumor-suppressive effects [36, 37]. In the classical TGF-β/Smad signaling pathway, TGF-β binds to type I and II receptors, thereby inducing phosphorylation and activation of the type I receptor. This activation recruits and activates downstream Smad proteins, which accumulate in the nucleus and function as transcription factors. Moreover, TGF-β can also activate a non-classical pathway independent of the Smad protein to regulate gene expression [14, 15]. Our previous study indicated that the TGF-β1/Smad classic signaling pathway promotes the progression of liver fibrosis by upregulating the lncRNA UCA1, which subsequently activates the Smad3 molecule through a positive feedback mechanism [38]. Although it has been shown that PVT1 is upregulated in HCC cells after TGF-β1 (GSE54797) treatment, the underlying mechanism remains elusive. This is the first study to verify that PVT1 is regulated by the TGF-β1 canonical signaling pathway.

Smad6, an inhibitory SMAD, transduces signals from the TGF-β superfamily, competitively binds to type I receptors or Smad4, and inhibits Smad1/5/8 phosphorylation or nuclear translocation [39,40,41,42], primarily inhibiting BMP signal transduction [16, 17]. Furthermore, BMP signaling has been observed to markedly activate P21 expression, which can bind to cyclin-dependent kinases (CDKs) to suppress their kinase activity, thereby causing cells to arrest at the G1/S and G2/M transition phases [19, 20]. Here, it was validated that PVT1 can upregulate Smad6 and downregulate P21, and these results are consistent with the RNA sequencing findings of Fang et al. [8]. PVT1, a lncRNA, is commonly reported to regulate downstream molecules through the competitive endogenous RNA (ceRNA) mechanism [5, 43,44,45]. The literature suggests that PVT1 can interact with miR-186-5p [30, 31], which can suppress Smad6 to regulate colorectal cancer progression [32]. However, whether PVT1 can regulate Smad6 by competitively binding miR-186-5p has not been previously studied. This study performed various experiments, including dual-luciferase reporter, qRT-PCR, and WB analyses, to indicate that PVT1 stabilizes Smad6 mRNA by competitively binding miR-186-5p. Furthermore, this is the first investigation to indicate that PVT1 enhances the proliferation of HCC in vivo and in vitro by upregulating Smad6.

NRG1, a key subunit involved in EMT, is upregulated upon PVT1 overexpression [8]. NRG1 is a crucial upstream ligand within the family of EGFRs and has been documented to promote the spread of diverse tumor types, including gastric, esophageal, breast cancers, etc [46,47,48]. In HCC, NRG1 has been reported to promote migration and invasion by activating its receptors, ERBB2/ERBB3, and upregulating the expression of downstream RAS/MAPK signaling pathways [27]. This is the first study to demonstrated that PVT1 promotes the metastasis of HCC by the miR-143-3p/NRG1 pathway.

PVT1 has great significance in the development and progression of HCC, whereas TGF-β1 can inhibit HCC proliferation and paradoxically promote its metastasis. Pure antagonism of the TGF-β signal, however, poses significant safety concerns. This study evaluated the underlying mechanisms by which PVT1 promotes HCC proliferation and metastasis as well as validated that PVT1 acts as a downstream effector in the classical TGF-β1/Smad3 pathway and is a potential target in HCC metastasis and proliferation, thus providing a potential target for future TGF-β signaling pathway-based therapies and gene therapy against HCC.

Conclusions

This study revealed that PVT1 is activated by the TGF-β1/Smad3 pathway and promotes HCC proliferation by the miR-186-5p/Smad6 axis while enhancing migration and invasion through the miR-143-3p/NRG1 pathway, indicating that PVT1 may act as potential therapeutic targets for HCC.

Abbreviations

HCC:

Hepatocellular carcinoma

lncRNA:

Long non-coding RNA

PVT1:

Plasmacytoma variant translocation 1

NRG1:

Neuregulin 1

ceRNA:

Competing endogenous RNA

miRNAs:

MicroRNAs

qRT-PCR:

Quantitative real-time PCR

WB:

Western blotting

H&E:

Hematoxylin and eosin

ChIP:

Chromatin Immunoprecipitation

CCK-8:

Cell counting kit 8

EMT:

Epithelial-mesenchymal transformation

ALD:

Alcoholic liver disease

HBV:

Hepatitis B

CHC:

Chronic hepatitis C

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Acknowledgements

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Funding

This study was supported by the National Natural Science Foundation of China (81903001) and the talent introduction project of Zhongnan Hospital of Wuhan University (20190101).

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  1. Shuaihui Wu, Qian Cheng and Yang Shi have contribute equally to this work.

Authors and Affiliations

  1. Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China

    Shuaihui Wu, Qian Cheng, Yang Shi, Kunlei Wang, Zhinan Chen, Xinyin Li, Ping Jiang, Zhixiang Cheng, Zhiyong Yang & Bo Liao

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Contributions

SW, ZY, and BL designed the research and experiments. SW, QC, and YS performed the experiments. ZC and KW evaluated and explained the results. ZC, PJ, and XL wrote the paper. ZC, ZY, and BL reviewed the writing. All of the authors read and approved the final version of the manuscript.

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Correspondence to Zhixiang Cheng, Zhiyong Yang or Bo Liao.

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Wu, S., Cheng, Q., Shi, Y. et al. LncRNA PVT1 activated by TGF-β1/Smad3 facilitates proliferation and metastasis of hepatocellular carcinoma via upregulating Smad6 and NRG1. J Transl Med 23, 500 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06229-4

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Journal of Translational Medicine

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