Published online May 21, 2026. doi: 10.3748/wjg.v32.i19.115332
Revised: December 28, 2025
Accepted: February 14, 2026
Published online: May 21, 2026
Processing time: 214 Days and 19.4 Hours
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Dysregulation of the epigenetic modifier PRMT5 contributes to the proliferation and metastasis of cancers, and its inhibition has displayed pro
To elucidate the molecular mechanisms by which PRMT5 promotes HCC pro
The correlation between PRMT5 expression and patient prognosis was analyzed using an online database (GEPIA2). Subsequently, the functional impact of PRMT5 was evaluated in vitro through cell viability, clonogenic formation, and apoptosis assays in HCC cell lines. Mechanistically, chromatin immunoprecipitation, co-immunoprecipitation, and promoter activity assays were used to investigate the binding PRMT5 and histone modification (H4R3me2) on the DDIT3 promoter, and its interaction with STAT3. The anti-tumor efficacy of HLCL-61 was evaluated in subcutaneous xenograft mouse models using both cell line-derived xenografts and patient-derived xenografts.
Our study found that high PRMT5 expression was correlated with a worse prognosis of HCC, and inhibition of PRMT5 expression significantly decreased the viability of HCC cells by inducing apoptosis. In the mechanistic study, we discovered that PRMT5 could bind to the DDIT3 promoter and increase the H4R3me2 level on it to repress DDIT3 transcription. Meanwhile, PRMT5 interacted with the coiled-coil domain of STAT3 and recruited it to the DDIT3 promoter to conjointly inhibit promoter activity. In addition, we evaluated that the PRMT5 inhibitor HLCL-61 and found that it exhibited excellent inhibitory efficacy on HCC cells and tissue derived tumors.
PRMT5 served as an adaptor of STAT3, displaying a dual inhibitory role in DDIT3 transcription to promote apoptosis resistance, and its inhibitor HLCL-61 represents a potential alternative therapeutic approach to treat HCC.
Core Tip: High PRMT5 expression was correlated with a worse prognosis of hepatocellular carcinoma (HCC), and inhibition of PRMT5 expression significantly decreased the viability of HCC cells by inducing apoptosis. Mechanistically, PRMT5 dually suppressed the promoter activity of the apoptosis-inducing factor DDIT3 by increasing H4R3me2 modification and recruiting STAT3 to its promoter. The PRMT5 inhibitor HLCL-61 exerted excellent inhibitory efficacy on HCC cells and tissue derived tumors. In general, our study demonstrated that PRMT5 served as an adaptor for STAT3, displaying a dual inhibitory role on DDIT3 transcription to promote apoptosis resistance, and provided that its inhibitor HLCL-61 was an alternative therapeutic approach of HCC.
- Citation: Jiang H, Yan JH, Tang WJ, Shen B, Mo S, Wang Y, Hu DH, Dong ZX, Zhang SB. PRMT5 imposed a dual repression on DDIT3 transcription to promote the malignancy of hepatocellular carcinoma. World J Gastroenterol 2026; 32(19): 115332
- URL: https://www.wjgnet.com/1007-9327/full/v32/i19/115332.htm
- DOI: https://dx.doi.org/10.3748/wjg.v32.i19.115332
Hepatocellular carcinoma (HCC) is the fifth most common malignant cancer and the third leading cause of cancer-related death worldwide due to its strong heterogeneity and complex genetic background[1]. Despite the recent clinical application of atezolizumab and bevacizumab for unresectable HCC, the median overall survival remains limited to approximately 19.2 months[2,3]. These findings indicate that the prognosis is still poor, largely because of late diagnosis and the limited efficacy of existing treatments. Thus, deeply comprehending pathological mechanism and exploring novel approaches to effectively predict and treat HCC are of paramount importance.
Epigenetic modifiers are frequently disordered and positively associated with tumor progression by modulating oncogenic signaling pathways[4,5]. PRMT5 is a type II protein arginine methyltransferase that symmetrically dimethylate arginine residues in substrate proteins, and regulates a diverse range of biological processes, including translation, RNA processing, signal transduction, cell cycle and DNA damage response[6,7]. It is frequently dysregulated and involved in cancer progression, high expression of PRMT5 is positively associated with the malignant capacity and poor prognosis of HCC[8,9]. Therefore, PRMT5 is a promising therapeutic target, and its inhibitors (such as S-adenosyl-L-methionine analogues, CMP5 and its derivatives) exerts excellent efficacy in several kinds of cancers, including non-small cell lung cancer and lymphoma[10]. However, the deeply malevolent mechanism and therapeutic potential of targeting PRMT5 in HCC remains not well known.
DDIT3, also known as growth arrest- and DNA damage-inducible gene 153, is a transcription factor induced by DNA damage, hypoxia, amino acid starvation and endoplasmic reticulum stress[11,12]. Its expression accumulation triggers cell cycle G1/S phase arrest and cell apoptosis by enhancing the biological function of BCL2 interacting mediator of cell death and inhibiting the antiapoptotic function of BCL2[13]. In HCC, DDIT3 serves as an apoptosis-related tumor suppressor gene, and inhibits HCC progression by inducing cell apoptosis[14]. Uncommonly, the repression of DDIT3 messenger RNA (mRNA) expression is directly regulated by STAT3, which displays an oncogenic role in HCC de
In this study, we found that PRMT5 suppressed the expression of the apoptosis-related gene DDIT3 and promoted the malignant progression of HCC, leading to poor prognosis of HCC patients. Mechanistically, PRMT5 bound to the DDIT3 promoter and increased the level of H4R3me2 modification, thereby inhibiting DDIT3 promoter activity. Meanwhile, PRMT5 interacted with the coiled-coil domain of STAT3, recruited it to the DDIT3 promoter, and promoted STAT3-mediated transcriptional repression of DDIT3. Additionally, the PRMT5 inhibitor HLCL-61 exerted preeminent inhibition efficacy of HCC in intro and in vivo. Collectively, our study revealed that PRMT5 served as a new adaptor of STAT3, displayed a dual inhibitory effect on DDIT3 transcription to confer apoptosis resistance and promoted HCC malignancy progression. These findings further clarify that HLCL-61 represents a promising therapeutic agent for HCC treatment.
HEK293T, HepG2, Hep3B, and Huh7 cells were purchased from the Cell Bank of the type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). portal vein tumor thrombus (PVTT) cells were obtained from Professor Xie D’s lab, the detailed procedures for establishing the PVTT cell line and relevant clinical information of the patient who donated the tumor tissue have been described previously[16,17]. All cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% of fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C with humidity with 5% carbon dioxide. After obtaining written informed consent, all HCC and paired adjacent tissues were collected from the Second Xiangya Hospital of Central South University.
PRMT5 and DDIT3 coding sequences were respectively inserted into the pHAGE-full-EF1a-MCS-IZsGreen and pCDH-CMV-MCS-EF1-GreenPuro-CD513B lentiviral vector. Short hairpin RNAs (shRNAs) targeting PRMT5 and DDIT3 were designed by Qiagen (Valencia, CA, United States) and inserted into the pLKO.1-TRC vector. The procedures for lentivirus packaging and stale cell line construction were performed following the methods described in previous study[18]. The sequences of the cloned genes and shRNAs were listed in Tables 1 and 2.
| Primer | Sequence |
| PRMT5 qPCR | F: 5’-CGATCAGACCTACTGCTGTCA-3’ |
| R: 5’-CTCGGAGTTCCTGCGAATCT-3’ | |
| DDIT3 qPCR | F: 5’-CTCGCTCTCCAGATTCCAGTC-3’ |
| R: 5’-CTTCATGCGTTGCTTCCCA-3’ | |
| DDIT3 promoter | F: 5’-CCCTCGAGTCATTTTTAAAGAATGAGTTAAGGG-3’ |
| R: 5’-GATATCTGGGGAATGACCACTCTGTTTCC-3’ | |
| PRMT5 clone | F: 5’-CGGGATCCATGGCGGCGATGGCGGTC-3’ |
| R: 5’-CGACGCGTCGAGGCCAATGGTATATGAGCG-3’ | |
| DDIT3 clone | F: 5’-GGAATTCATGGAGCTTGTTCCAGCCACT-3’ |
| R: 5’-CGGGATCCTCATGCTTGGTGCAGATTCACCAT-3’ |
| Primer | Sequence |
| shPRMT5-1 | F: 5’-CCGGAGTACCAGCAGGCCATCTATACTCGAGTATAGATGGCCTGCTGGTACTTTTTTG-3’ |
| R: 5’-AATTCAAAAAAGTACCAGCAGGCCATCTATACTCGAGTATAGATGGCCTGCTGGTACT-3’ | |
| shPRMT5-2 | F: 5’-CCGGGCCCAGTTTGAGATGCCTTATCTCGAGATAAGGCATCTCAAACTGGGCTTTTTG-3’ |
| R: 5’-AATTCAAAAAGCCCAGTTTGAGATGCCTTATCTCGAGATAAGGCATCTCAAACTGGGC-3’ | |
| shDDIT3-1 | F: 5’-CCGGCTGCACCAAGCATGAACAATTCTCGAGAATTGTTCATGCTTGGTGCAGTTTTTG-3’ |
| R: 5’-AATTCAAAAACTGCACCAAGCATGAACAATTCTCGAGAATTGTTCA TGCTTGGTGCAG-3’ | |
| shDDIT3-2 | F: 5’-CCGGTGAACGGCTCAAGCAGGAAATCTCGAGATTTCCTGCTTGAGCCGTTCATTTTTG-3’ |
| R: 5’-AATTCAAAAATGAACGGCTCAAGCAGGAAATCTCGAGATTTCCTGCTTGAGCCGTTCA-3’ |
Total RNA was extracted from the HCC cells and samples with TRIzol reagent (Cat. No. 15596026, Invitrogen, United States). RNA reverse transcription and quantitative polymerase chain reaction were performed as described in Fan et al’s article[18]. Primers sequences were shown in Table 1.
Of 2 × 103 cells were seeded into 96 and 6-wells plates. The crystal violet assay and methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (Cat. No. C0009S, Beyotime, China) were performed as described in our previous article[19].
Gene expression data from GSE168745 were quantile-normalized using the normalize between arrays function in the limma package. Differential gene expression analysis between normal and PRMT5 knockdown JHH-7 cells were conducted using DESeq2 version 1.40.2, with thresholds set as |log2 fold change| > 1.0 and adjusted P value < 0.05 (Benjamini-Hochberg correction). Heatmaps visualizing the expression patterns of differentially expressed genes were generated on the basis of scaled expression values.
Cells were lysed and proteins were quantified by Bradford reagent (Cat. No. P0006, Beyotime, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Antibodies against PRMT5 (Cat. No. 79998S, 1:1000, Cell Signaling Technology, United States), STAT3 (Cat. No. 12640S, 1:1000, Cell Signaling Technology, United States), phospho-STAT3 (Y705) (Cat. No. 9145S, 1:1000, Cell Signaling Technology, United States) and DDIT3 (Cat. No. 5554S, 1:1000, Cell Signaling Technology, United States), GAPDH (Cat. No. G9545, 1:10000, Sigma, United States), Flag (Cat. No. F1804, 1:4000, Sigma, United States), and HA (Cat. No. sc-7392, 1:1000, Santa Cruz Biotechnology, United States) were used. Horseradish peroxidase-conjugated secondary antibodies (Cat. No. AP307P, 1:2000, Sigma, United States) were used for detection.
Tris-buffered saline (potential of hydrogen = 7.4) was used to lysis the cells, and the cell lysates were incubated with primary antibodies against HA, PRMT5, STAT3 or phospho-STAT3 at 4 °C overnight. Then, protein A or G beads was added and incubated for 4-6 hours. The beads were centrifuged at 500 g for 2 minutes at 4 °C and washed with Tris-buffered saline for three times. Proteins bound to the beads were eluted with 2 × loading buffer, and the proteins were examined by western blotting.
Stably transfected HCC cell lines and their corresponding control cells were co-transfected with 50 ng of reporter plasmid and 20 ng of Renilla luciferase plasmid and cultured for 24 hours. The experiments were performed in triplicate. Then, cells were lysed by passive lysis buffer (Promega, Madison, WI, United States), and reporter activities were investigated by the dual-luciferase reporter assay.
Cells were washed with phosphate-buffered saline (PBS) buffer three times, fixed with methanol, blocked with 5% bovine serum albumin for 1 hour at 37 °C. The cells were incubated with primary antibodies against PRMT5 and STAT3 (1:100, Cell Signaling Technology) overnight at 4 °C. After three washes with PBS, the cells were incubated with secondary antibodies (Alexa Fluor 488- and 555-conjugated donkey anti-rabbit immunoglobulin G, 1:1000) for 1 hour at room temperature and washed three times with PBS. The nuclei were stained with 4’,6-diamidino-2-phenylindole dye. The fluorescence signals were monitored using an inverted confocal laser scanning microscope (Carl Zeiss, Thornwood, NY, United States).
The procedures for chromatin immunoprecipitation (ChIP) and ChIP-Re-ChIP were performed following the methods described in previous study[20]. Five paired-primers were employed to assess the enrichment of PRMT5 at the DDIT3 promoter and the primer sequences were provided in Table 3.
| Primer | Sequence |
| ChIP primer 1 | F: 5’-TTGCCAAACATTGCATCATC-3’ |
| R: 5’-AGCGCCTGGCTGTAATCTT-3’ | |
| ChIP primer 2 | F: 5’-CAAACAGAATCGGGTCCACT-3’ |
| R: 5’-GGCAGAAGAATCGCTTGAAC-3’ | |
| ChIP primer 3 | F: 5’-ACCACATGCCTCTGTTTTC-3’ |
| R: 5’-TGTGACTCCCTGTGTGAGGA-3’ | |
| ChIP primer 4 | F: 5’-GTAGAGACGGGGTTTCACCA-3’ |
| R: 5’-TAGTCGGTCGTGAGCCTCTT-3’ | |
| ChIP primer 5 | F: 5’-CCCTTCCAAAGCCACAGTCT-3’ |
| R: 5’-TTGTGACTGGAGTGGTGTGG-3’ |
DMEM/Matrigel mixed suspensions (contained 1 × 106 cells) or patients derived HCC tissues (5 mm × 5 mm) were subcutaneously injected into nude mice. When the diameter of the tumors reached 5 mm, the mice were randomly divided into two groups. Beginning on day 7 post-inoculation, and the mice in the treatment group were orally administered HLCL-61 (S8209; Selleck, United States) by gavage at a dose of 50 mg/kg. Mice in the control group received an equal volume of vehicle solution via the same route. Treatments were administered once every two days for a total of 21 days. Tumor volumes were monitored every 2 days. Until tumors growth to appropriative size, the mice were sacrificed and tumors were excised for further analysis.
Survival curves were plotted by the Kaplan-Meier method and analyzed by the log-rank test. Statistical analyses were performed by GraphPad Prism 5 and SPSS 17.0 software. The results are representative of at least three independent experiments performed in triplicate and are expressed as the means ± SDs. The data were analyzed using Student’s t test.
High PRMT5 expression was observed in HCC in previous study[6]. Thus, we investigated the correlation between PRMT5 expression and prognosis of HCC, and found that upregulated PRMT5 expression was positively associated with worse overall survival and disease-free survival of HCC patients from GEPIA2 online tool. Figure 1 revealed that high expression of PRMT5 predicted poor prognosis of HCC patients and PRMT5 might played vital role in HCC (Figure 1A and B). Therefore, we overexpressed and knocked down of PRMT5 in HCC cells, and observed that overexpression of PRMT5 conferred the malignant growth of HCC cells (Figure 1C and E), while knockdown of PRMT5 notably decreased the growth of HCC cells (Figure 1D and F). Those results indicated that PRMT5 promoted the malignant growth of HCC cells, which suggested that inhibition of PRMT5 might be a potential strategy for suppressing HCC progression. In order to demonstrate above hypothesis, we suppressed PRMT5 using its inhibitor HLCL-61 in HCC cells, and the results showed that HLCL-61 obviously repressed the growth of HCC cells in a dose-dependent manner (Figure 1G and H). Collectively, our results demonstrated that PRMT5 inhibition could significantly suppress the viability of HCC cells, supporting that PRMT5 as a potential therapeutic target for treating HCC.
Given that PRMT5 inhibition potently suppressed HCC cell growth, the effects of PRMT5 on the apoptosis and necrosis of HCC cells was examined (Figure 2). We found that knockdown of PRMT5 remarkably increased the distribution of apoptotic HCC cells (Figure 2A and B). Meanwhile, the consistent results were observed in HCC cells treated with HLCL-61 (Figure 2C and D). These results determined that inhibition of PRMT5 could induced apoptosis in HCC cells.
In order to explore how PRMT5 played the role of inducing apoptosis, we analyzed the Gene Expression Omnibus data (GSE168745) and found that the mRNA level of the apoptosis-related factor DDIT3 was notably upregulated in PRMT5 knockdown JHH-7 cells (Figure 2E). Besides, overexpression of PRMT5 effectively decreased the expression of DDIT3 mRNA and protein (Figure 2F and J). Conversely, knockdown of PRMT5 notably increased the DDIT3 mRNA and protein levels (Figure 2G and J). Additionally, increases in DDIT3 mRNA and protein expression were detected in HCC cells treated with HLCL-61, and these increases were dependent on the HLCL-61 concentration (Figure 2H, I and K). To further confirm the correlation between PRMT5 and DDIT3 expression, we examined the expression of PRMT5 and DDIT3 in HCC tissues. The results indicated that compared with normal tissues, HCC tissues with high expression of PRMT5 presented lower levels of DDIT3 expression (Figure 2L). Correlation analysis revealed that there was a negative correlation between PRMT5 and DDIT3 expression in HCC tissues (Figure 2M). Based on above results, these results demonstrated that PRMT5 suppressed the DDIT3 expression in HCC.
These obtained results implied that PRMT5 might suppress the transcription of DDIT3. Therefore, we performed a DDIT3 promoter activity assay. Overexpression of PRMT5 obviously attenuated the promoter activity of DDIT3 in HCC cells, and knockdown of RPMT5 notably elevated the DDIT3 promoter activity (Figure 3A and B). To investigate whether PRMT5 directly regulated DDIT3 transcription, we designed five pairs of ChIP primers according to the DDIT3 promoter sequence (Figure 3C). An obvious enrichment of PRMT5 was detected at the -313 bp to -4 bp region of the DDIT3 promoter in HCC cells (Figure 3D). Previous studies have shown that PRMT5 always inhibited the transcription of its target genes by inducing symmetrical dimethylation of histone H4R3[21]. In order to verified that PRMT5 inhibited DDIT3 transcription by regulating the level of H4R3me2, we examined the H4R3me2 modification at the DDIT3 promoter in stably transfected HCC cells. The level of H4R3me2 at the -313 bp to -4 bp region of the DDIT3 promoter was upre
It has been reported that PRMT5 can form a complex with transcription factors, and that STAT3 inhibited DDIT3 transcription. To explore the potential regulatory mechanism, we analyzed transcription factors that bind to the DDIT3 promoter region using the JASPAR online database. The results indicated that STAT3 might bind to the -213 bp to -203 bp region of the DDIT3 promoter (Figure 4A). Therefore, we speculated that PRMT5 and STAT3 might form a transcriptional regulatory complex to repress DDIT3 transcription. To test our hypothesis, we performed a promoter activity assay and observed that STAT3 elevated the inhibition of DDIT3 promoter activity when co-transfected with PRMT5 (Figure 4B). Both exogenous and endogenous co-immunoprecipitation assays demonstrated that PRMT5 interacted with STAT3 (Figure 4C and D). Besides, the results of immunofluorescence staining further confirmed the interaction between the colocalization of PRMT5 and STAT3 in the nucleus, which corroborated their interaction (Figure 4E). Because STAT3 localized in the nucleus was phosphorylated, we detected that PRMT5 interacted with phosphorylated STAT3 by immune-coprecipitation assay, which explained the colocalization of PRMT5 and STAT3 (Figure 4F).
Various truncated mutants of STAT3 were constructed (Figure 4G), and truncation assay demonstrated that the DNA-binding domain (DBD) of STAT3 was indispensable for the PRMT5-STAT3 interaction (Figure 4H). Moreover, ChIP assays demonstrated that STAT3 also is enriched in the -313 bp to -4 bp region of the DDIT3 promoter (Figure 4I), and ChIP-re-ChIP assays detected that the interaction between PRMT5 and STAT3 occurred on the DDIT3 promoter (Figure 4J). Additionally, overexpression of PRMT5 promoted STAT3 binding on the DDIT3 promoter, while knockdown of PRMT5 reduced the enrichment (Figure 4K and L), which indicated that STAT3 binding to the DDIT3 promoter was dependent on PRMT5 expression. Altogether, our results corroborated that PRMT5 interacted with STAT3 through the DBD domain and recruited STAT3 to the DDIT3 promoter to conjointly repress its transcriptional activity.
To verify that PRMT5 resisted apoptosis through repressing the transcription of DDIT3 in HCC cells (Figure 5). We forced to express DDIT3 in PRMT5 overexpressing HCC cells and attenuated DDIT3 expression in PRMT5-knockdown HCC cells (Figure 5A and B). Results showed that overexpression of DDIT3 significantly suppressed cell growth and colony formation in PRMT5 overexpressing HCC cells (Figure 5C and E). Reversely, downregulation of DDIT3 promoted growth and colony formation in PRMT5 knockdown HCC cells (Figure 5D and F). Apoptotic staining indicated that the attenuation of DDIT3 antagonized the cell apoptosis induced by knockdown PRMT5 in HCC cells (Figure 5G and H). Taken together, our results showed that DDIT3 restored the phenotype mediated by PRMT5 and confirmed that PRMT5 promoted the malignant growth of HCC cells by inhibiting DDIT3-induced apoptosis.
Our results hinted that HLCL-61 was a potential way of HCC treatment. Therefore, we constructed a tumor xenograft mouse model using highly (Huh7) and less (HepG2) malignant HCC cells to investigate the inhibitory effect of HLCL-61 in vivo. Compared with the control group, HLCL-61 treatment (50 mg/kg) significantly inhibited tumor volume and weight (Figure 6A-F). These results suggested that HLCL-61 had an excellent suppression on tumors with different malignancy. To further evaluate the inhibitory effects in clinical HCC models, we constructed patient-derived xenografts (PDX) mouse models. Consistent results revealed that HLCL-61 significantly reduced the volume and weight of tumors, and exerted a stronger inhibitory effect on tumorigenesis in PDX models (Figure 6G-I). Additionally, immunohistochemical staining showed that HLCL-61 notably decreased the expression of Ki-67 (a biomarker reflecting malignant proliferation of cancer) in tumor tissues, and increased the DDIT3 expression when compared with control group (Figure 6J and K). In general, our results testified that HLCL-61 effectively suppressed the tumorigenesis of HCC, and provided that HLCL-61 was an alternative therapeutic way of HCC (Figure 6).
Aberrantly high PRMT5 expression is widely observed in various cancers and its overexpression enhanced malignant potential and chemoresistance in tumor cells[22,23], those findings indicate that PRMT5 serves as a malignant marker in cancers. Studies have shown that PRMT5 promotes proliferation and metastasis in HCC, but the deeply pathological mechanism by which PRMT5 contributes to HCC progression is still vague. Our study indicated that high PRMT5 expression confer the rapid growth of HCC cells through resistance to apoptosis. Therefore, our findings reveal an oncogenic function of PRMT5 in antagonizing apoptosis in HCC.
PRMT5 inhibitors are currently being evaluated in clinical trials for advanced solid tumors. Its inhibitor HLCL-61 exerted excellent suppression acute myeloid leukaemia[24], however, the therapeutic efficacy of HLCL-61 in HCC remains unknown. In this study, we found that knockdown of PRMT5 using shRNA and pharmacological inhibition with HLCL-61 notably repressed the viability of HCC cells. Those results were further confirmed by in vivo experiments in which HLCL-61 effectively suppressed tumorigenesis in cell line-derived xenografts and PDX models. Our study demonstrated that PRMT5 was a promising target for HCC therapy, and provided that HLCL-61 was a potential therapeutic strategy of HCC.
PRMT5 commonly catalyses the symmetric dimethylarginine of arginine residues on histone 3 and H4. Among these modifications, H4R3me2 is a well-documented epigenetic marker that mediates the transcriptional repression of target genes, including DNMT3A, RBL1, and Gas1[25]. Based on our results, we determined the suppressive role of PRMT5 in the regulation of DDIT3 expression. To supposed the hypothesis that PRMT5 suppressed DDIT3 transcription, we corroborated the transcriptional repression of DDIT3 by PRMT5 using promoter activity and ChIP assays. Since H4R3 is the main catalytic site of PRMT5 on histones, H4R3me2 modification is widely recognized as an indicator of gene transcriptional silencing[26]. We validated that PRMT5 remarkably elevated the H4R3me2 levels on the DDIT3 promoter, thereby repressing its transcription.
PRMT5 can form a complex with transcription factors to co-regulate gene expression. The PRMT5/β-catenin/JDP2 complex plays a vital role in gene regulation, thereby re-establishing glutathione homeostasis in ovarian cancer[27]. Similarly, PRMT5 interacts with the oncoprotein BCL6 and is required for lymphoma cell survival. Whether PRMT5 forms complexes with other transcription factors for gene regulation. Online database analysis and previous studies have indicated that the transcription factor STAT3 may bind to the DDIT3 promoter[28]. Consistent with this prediction, our experiments confirmed that STAT3 binds to the DDIT3 promoter. Besides, PRMT5 interacted and colocalized with STAT3, and STAT3 enhanced the PRMT5-mediated suppression of DDIT3 transcriptional activity. Additionally, the binding of STAT3 to the DDIT3 promoter was depended on PRMT5 expression, which suggested that PRMT5 could recruit STAT3 to the DDIT3 promoter. Therefore, we determined that PRMT5 functions as an adaptor for STAT3, and explored the underlying molecular mechanism by which the PRMT5/STAT3 complex exerted a dual inhibitory effect on DDIT3 transcription, ultimately promoting the malignant growth of HCC. Moreover, our results also hint that PRMT5 is potentially a vital therapeutic target for STAT3-activated HCC.
STAT3 is a multifunctional transcription factor involved in oncogenic transformation, cell cycle progression, anti-apoptosis, displaying transcriptional activator role on oncogenes (such as c-Myc, c-Jun, Bcl-2 and VEGF) and transcriptional repressor role on tumor suppressor genes (such as P53, SHP-1 and DDIT3)[29,30]. In this study, we discovered that PRMT5 could recruit STAT3 to the DDIT3 promoter and co-suppress its transcription, indicating that the inhibitory effect of STAT3 on DDIT3 expression was dependent on PRMT5. Previous studies have reported that STAT3 cooperates with DNMT1 to silence the transcription of SHP-1. Based on these discoveries, we inferred that the transcriptional repression of STAT3 on its target genes might require the assistance of epigenetic modifiers.
In summary, we disclosed the underlying mechanism by which PRMT5 played dual roles in the inhibition of DDIT3 transcription in HCC: (1) PRMT5 directly binds to the DDIT3 promoter and elevated H4R3me2 modification levels, thereby suppressing its transcriptional activity; and (2) PRMT5 recruited STAT3 to the DDIT3 promoter, which further enhanced the repression of DDIT3 transcription (Figure 7). In addition, we evaluated the anti-tumor efficacy of HLCL-61 in HCC and demonstrated that HLCL-61 was an alternative strategy of HCC therapy.
This work was supported in part by the High Performance Computing Center of Central South University, Center for Computational Biology and Bioinformatics, Furong Laboratory, Bioinformatics Center, Xiangya Hospital, Central South University.
| 1. | Filho AM, Laversanne M, Ferlay J, Colombet M, Piñeros M, Znaor A, Parkin DM, Soerjomataram I, Bray F. The GLOBOCAN 2022 cancer estimates: Data sources, methods, and a snapshot of the cancer burden worldwide. Int J Cancer. 2025;156:1336-1346. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 615] [Cited by in RCA: 471] [Article Influence: 471.0] [Reference Citation Analysis (2)] |
| 2. | Shen YC, Liu TH, Nicholas A, Soyama A, Yuan CT, Chen TC, Eguchi S, Yoshizumi T, Itoh S, Nakamura N, Kosaka H, Kaibori M, Ishii T, Hatano E, Ogawa C, Naganuma A, Kakizaki S, Cheng CH, Lin PT, Su YY, Chuang CH, Lu LC, Wu CJ, Wang HW, Rau KM, Hsu CH, Lin SM, Huang YH, Hernandez S, Finn RS, Kudo M, Cheng AL. Clinical Outcomes and Histologic Findings of Patients With Hepatocellular Carcinoma With Durable Partial Response or Durable Stable Disease After Receiving Atezolizumab Plus Bevacizumab. J Clin Oncol. 2024;42:4060-4070. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 22] [Cited by in RCA: 21] [Article Influence: 10.5] [Reference Citation Analysis (0)] |
| 3. | Cappuyns S, Piqué-Gili M, Esteban-Fabró R, Philips G, Balaseviciute U, Pinyol R, Gris-Oliver A, Vandecaveye V, Abril-Fornaguera J, Montironi C, Bassaganyas L, Peix J, Zeitlhoefler M, Mesropian A, Huguet-Pradell J, Haber PK, Figueiredo I, Ioannou G, Gonzalez-Kozlova E, D'Alessio A, Mohr R, Meyer T, Lachenmayer A, Marquardt JU, Reeves HL, Edeline J, Finkelmeier F, Trojan J, Galle PR, Foerster F, Mínguez B, Montal R, Gnjatic S, Pinato DJ, Heikenwalder M, Verslype C, Van Cutsem E, Lambrechts D, Villanueva A, Dekervel J, Llovet JM. Single-cell RNA sequencing-derived signatures define response patterns to atezolizumab + bevacizumab in advanced hepatocellular carcinoma. J Hepatol. 2025;82:1036-1049. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 34] [Cited by in RCA: 24] [Article Influence: 24.0] [Reference Citation Analysis (0)] |
| 4. | Chaudhri A, Lizee G, Hwu P, Rai K. Chromatin Remodelers Are Regulators of the Tumor Immune Microenvironment. Cancer Res. 2024;84:965-976. [RCA] [PubMed] [DOI] [Full Text] [Cited by in RCA: 15] [Reference Citation Analysis (0)] |
| 5. | Yang J, Xu J, Wang W, Zhang B, Yu X, Shi S. Epigenetic regulation in the tumor microenvironment: molecular mechanisms and therapeutic targets. Signal Transduct Target Ther. 2023;8:210. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 141] [Cited by in RCA: 276] [Article Influence: 92.0] [Reference Citation Analysis (0)] |
| 6. | Elurbide J, Colyn L, Latasa MU, Uriarte I, Mariani S, Lopez-Pascual A, Valbuena E, Castello-Uribe B, Arnes-Benito R, Adan-Villaescusa E, Martinez-Perez LA, Azkargorta M, Elortza F, Wu H, Krawczyk M, Schneider KM, Sangro B, Aldrighetti L, Ratti F, Casadei Gardini A, Marin JJG, Amat I, Urman JM, Arechederra M, Martinez-Chantar ML, Trautwein C, Huch M, Cubero FJ, Berasain C, G Fernandez-Barrena M, Avila MA. Identification of PRMT5 as a therapeutic target in cholangiocarcinoma. Gut. 2024;74:116-127. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 4] [Cited by in RCA: 18] [Article Influence: 9.0] [Reference Citation Analysis (1)] |
| 7. | Sloan SL, Brown F, Long M, Weigel C, Koirala S, Chung JH, Pray B, Villagomez L, Hinterschied C, Sircar A, Helmig-Mason J, Prouty A, Brooks E, Youssef Y, Hanel W, Parekh S, Chan WK, Chen Z, Lapalombella R, Sehgal L, Vaddi K, Scherle P, Chen-Kiang S, Di Liberto M, Elemento O, Meydan C, Foox J, Butler D, Mason CE, Baiocchi RA, Alinari L. PRMT5 supports multiple oncogenic pathways in mantle cell lymphoma. Blood. 2023;142:887-902. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 6] [Cited by in RCA: 26] [Article Influence: 8.7] [Reference Citation Analysis (0)] |
| 8. | Yan W, Liu X, Qiu X, Zhang X, Chen J, Xiao K, Wu P, Peng C, Hu X, Wang Z, Qin J, Sun L, Chen L, Wu D, Huang S, Yin L, Li Z. PRMT5-mediated FUBP1 methylation accelerates prostate cancer progression. J Clin Invest. 2024;134:e175023. [RCA] [PubMed] [DOI] [Full Text] [Cited by in RCA: 12] [Reference Citation Analysis (0)] |
| 9. | Liu A, Yu C, Qiu C, Wu Q, Huang C, Li X, She X, Wan K, Liu L, Li M, Wang Z, Chen Y, Hu F, Song D, Li K, Zhao C, Deng H, Sun X, Xu F, Lai S, Luo X, Hu J, Wang G. PRMT5 methylating SMAD4 activates TGF-β signaling and promotes colorectal cancer metastasis. Oncogene. 2023;42:1572-1584. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 3] [Cited by in RCA: 61] [Article Influence: 20.3] [Reference Citation Analysis (0)] |
| 10. | Belmontes B, Slemmons KK, Su C, Liu S, Policheni AN, Moriguchi J, Tan H, Xie F, Aiello DA, Yang Y, Lazaro R, Aeffner F, Rees MG, Ronan MM, Roth JA, Vestergaard M, Cowland S, Andersson J, Sarvary I, Chen Q, Sharma P, Lopez P, Tamayo N, Pettus LH, Ghimire-Rijal S, Mukund S, Allen JR, DeVoss J, Coxon A, Rodon J, Ghiringhelli F, Penel N, Prenen H, Glad S, Chuang CH, Keyvanjah K, Townsley DM, Butler JR, Bourbeau MP, Caenepeel S, Hughes PE. AMG 193, a Clinical Stage MTA-Cooperative PRMT5 Inhibitor, Drives Antitumor Activity Preclinically and in Patients with MTAP-Deleted Cancers. Cancer Discov. 2025;15:139-161. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 9] [Cited by in RCA: 56] [Article Influence: 56.0] [Reference Citation Analysis (0)] |
| 11. | Li M, Thorne RF, Shi R, Zhang XD, Li J, Li J, Zhang Q, Wu M, Liu L. DDIT3 Directs a Dual Mechanism to Balance Glycolysis and Oxidative Phosphorylation during Glutamine Deprivation. Adv Sci (Weinh). 2021;8:e2003732. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 15] [Cited by in RCA: 63] [Article Influence: 12.6] [Reference Citation Analysis (0)] |
| 12. | Ni R, Cao T, Ji X, Peng A, Zhang Z, Fan GC, Stathopulos P, Chakrabarti S, Su Z, Peng T. DNA damage-inducible transcript 3 positively regulates RIPK1-mediated necroptosis. Cell Death Differ. 2025;32:306-319. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 8] [Cited by in RCA: 13] [Article Influence: 13.0] [Reference Citation Analysis (0)] |
| 13. | Abbonante V, Malara A, Chrisam M, Metti S, Soprano P, Semplicini C, Bello L, Bozzi V, Battiston M, Pecci A, Pegoraro E, De Marco L, Braghetta P, Bonaldo P, Balduini A. Lack of COL6/collagen VI causes megakaryocyte dysfunction by impairing autophagy and inducing apoptosis. Autophagy. 2023;19:984-999. [RCA] [PubMed] [DOI] [Full Text] [Cited by in RCA: 30] [Reference Citation Analysis (0)] |
| 14. | Xiao F, Li H, Feng Z, Huang L, Kong L, Li M, Wang D, Liu F, Zhu Z, Wei Y, Zhang W. Intermedin facilitates hepatocellular carcinoma cell survival and invasion via ERK1/2-EGR1/DDIT3 signaling cascade. Sci Rep. 2021;11:488. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 14] [Cited by in RCA: 11] [Article Influence: 2.2] [Reference Citation Analysis (0)] |
| 15. | Canino C, Luo Y, Marcato P, Blandino G, Pass HI, Cioce M. A STAT3-NFkB/DDIT3/CEBPβ axis modulates ALDH1A3 expression in chemoresistant cell subpopulations. Oncotarget. 2015;6:12637-12653. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 68] [Cited by in RCA: 70] [Article Influence: 6.4] [Reference Citation Analysis (0)] |
| 16. | Wang T, Hu HS, Feng YX, Shi J, Li N, Guo WX, Xue J, Xie D, Liu SR, Wu MC, Cheng SQ. Characterisation of a novel cell line (CSQT-2) with high metastatic activity derived from portal vein tumour thrombus of hepatocellular carcinoma. Br J Cancer. 2010;102:1618-1626. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 43] [Cited by in RCA: 54] [Article Influence: 3.4] [Reference Citation Analysis (2)] |
| 17. | Cai Z, Qian ZY, Jiang H, Ma N, Li Z, Liu LY, Ren XX, Shang YR, Wang JJ, Li JJ, Liu DP, Zhang XP, Feng D, Ni QZ, Feng YY, Li N, Zhou XY, Wang X, Bao Y, Zhang XL, Deng YZ, Xie D. hPCL3s Promotes Hepatocellular Carcinoma Metastasis by Activating β-Catenin Signaling. Cancer Res. 2018;78:2536-2549. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 26] [Cited by in RCA: 27] [Article Influence: 3.4] [Reference Citation Analysis (0)] |
| 18. | Fan L, Tian C, Yang W, Liu X, Dhungana Y, Yang W, Tan H, Glazer ES, Yu J, Peng J, Ma L, Ni M, Zhu L. HKDC1 promotes liver cancer stemness under hypoxia through stabilizing β-catenin. Hepatology. 2025;81:1685-1699. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 6] [Cited by in RCA: 17] [Article Influence: 17.0] [Reference Citation Analysis (0)] |
| 19. | Jiang H, Ma N, Shang Y, Zhou W, Chen T, Guan D, Li J, Wang J, Zhang E, Feng Y, Yin F, Yuan Y, Fang Y, Qiu L, Xie D, Wei D. Triosephosphate isomerase 1 suppresses growth, migration and invasion of hepatocellular carcinoma cells. Biochem Biophys Res Commun. 2017;482:1048-1053. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 30] [Cited by in RCA: 53] [Article Influence: 5.3] [Reference Citation Analysis (0)] |
| 20. | Jiang H, Cao HJ, Ma N, Bao WD, Wang JJ, Chen TW, Zhang EB, Yuan YM, Ni QZ, Zhang FK, Ding XF, Zheng QW, Wang YK, Zhu M, Wang X, Feng J, Zhang XL, Cheng SQ, Ma DJ, Qiu L, Li JJ, Xie D. Chromatin remodeling factor ARID2 suppresses hepatocellular carcinoma metastasis via DNMT1-Snail axis. Proc Natl Acad Sci U S A. 2020;117:4770-4780. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 35] [Cited by in RCA: 89] [Article Influence: 14.8] [Reference Citation Analysis (0)] |
| 21. | Borbora SM, Rajmani RS, Balaji KN. PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection. PLoS Pathog. 2022;18:e1010095. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in RCA: 17] [Reference Citation Analysis (0)] |
| 22. | Zhu Y, Xia T, Chen DQ, Xiong X, Shi L, Zuo Y, Xiao H, Liu L. Promising role of protein arginine methyltransferases in overcoming anti-cancer drug resistance. Drug Resist Updat. 2024;72:101016. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 50] [Cited by in RCA: 52] [Article Influence: 26.0] [Reference Citation Analysis (0)] |
| 23. | Lin CC, Chang TC, Wang Y, Guo L, Gao Y, Bikorimana E, Lemoff A, Fang YV, Zhang H, Zhang Y, Ye D, Soria-Bretones I, Servetto A, Lee KM, Luo X, Otto JJ, Akamatsu H, Napolitano F, Mani R, Cescon DW, Xu L, Xie Y, Mendell JT, Hanker AB, Arteaga CL. PRMT5 is an actionable therapeutic target in CDK4/6 inhibitor-resistant ER+/RB-deficient breast cancer. Nat Commun. 2024;15:2287. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in RCA: 35] [Reference Citation Analysis (0)] |
| 24. | Tarighat SS, Santhanam R, Frankhouser D, Radomska HS, Lai H, Anghelina M, Wang H, Huang X, Alinari L, Walker A, Caligiuri MA, Croce CM, Li L, Garzon R, Li C, Baiocchi RA, Marcucci G. The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation. Leukemia. 2016;30:789-799. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 95] [Cited by in RCA: 144] [Article Influence: 13.1] [Reference Citation Analysis (0)] |
| 25. | Yang L, Xia H, Smith K, Gilbertsen AJ, Jbeli AH, Abrahante JE, Bitterman PB, Henke CA. Tumor suppressors RBL1 and PTEN are epigenetically silenced in IPF mesenchymal progenitor cells by a CD44/Brg1/PRMT5 regulatory complex. Am J Physiol Lung Cell Mol Physiol. 2024;327:L949-L963. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 1] [Cited by in RCA: 3] [Article Influence: 1.5] [Reference Citation Analysis (0)] |
| 26. | Cao L, Wu G, Zhu J, Tan Z, Shi D, Wu X, Tang M, Li Z, Hu Y, Zhang S, Yu R, Mo S, Wu J, Song E, Li M, Song L, Li J. Genotoxic stress-triggered β-catenin/JDP2/PRMT5 complex facilitates reestablishing glutathione homeostasis. Nat Commun. 2019;10:3761. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 24] [Cited by in RCA: 39] [Article Influence: 5.6] [Reference Citation Analysis (0)] |
| 27. | Lu X, Fernando TM, Lossos C, Yusufova N, Liu F, Fontán L, Durant M, Geng H, Melnick J, Luo Y, Vega F, Moy V, Inghirami G, Nimer S, Melnick AM, Lossos IS. PRMT5 interacts with the BCL6 oncoprotein and is required for germinal center formation and lymphoma cell survival. Blood. 2018;132:2026-2039. [RCA] [PubMed] [DOI] [Full Text] [Cited by in Crossref: 34] [Cited by in RCA: 58] [Article Influence: 7.3] [Reference Citation Analysis (0)] |
| 28. | Tolomeo M, Cascio A. The Multifaced Role of STAT3 in Cancer and Its Implication for Anticancer Therapy. Int J Mol Sci. 2021;22:603. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 38] [Cited by in RCA: 242] [Article Influence: 48.4] [Reference Citation Analysis (0)] |
| 29. | Igelmann S, Neubauer HA, Ferbeyre G. STAT3 and STAT5 Activation in Solid Cancers. Cancers (Basel). 2019;11:1428. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 44] [Cited by in RCA: 82] [Article Influence: 11.7] [Reference Citation Analysis (0)] |
| 30. | Jung YY, Ha IJ, Um JY, Sethi G, Ahn KS. Fangchinoline diminishes STAT3 activation by stimulating oxidative stress and targeting SHP-1 protein in multiple myeloma model. J Adv Res. 2022;35:245-257. [RCA] [PubMed] [DOI] [Full Text] [Full Text (PDF)] [Cited by in Crossref: 7] [Cited by in RCA: 50] [Article Influence: 12.5] [Reference Citation Analysis (0)] |