Hepatocellular Carcinoma Stem Cells Research Articles

FBW7-Mediated Degradation of CHD3 Suppresses Hepatocellular Carcinoma Metastasis and Stemness to Enhance Oxaliplatin Sensitivity.

Ubiquitination plays a key role in various cancers, and F-box and WD repeat domain containing 7 (FBW7) is a tumor suppressor that targets several cancer-causing proteins for ubiquitination. This paper set out to pinpoint the role of FBW7 in hepatocellular carcinoma (HCC). The target proteins of FBW7 and the expression of hromodomain helicase DNA binding protein 3 (CHD3) were analyzed in liver HCC (LIHC) samples using the BioSignal Data website. The effects of CHD3 and FBW7 on HCC cell viability, migration, invasion and stemness were investigated through cell counting kit (CCK)-8, wound healing, transwell and sphere formation assays. Detection on CHD3 and FBW7 expressions as well as their relationship was performed employing quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunoprecipitation, ubiquitination and western blot analyses. The prediction of Ubibrowser revealed CHD3 as a target protein of FBW7. The data of starBase exhibited a higher expression level of CHD3 in LIHC samples relative to normal samples. CHD3 was upregulated in HCC cells. CHD3 knockdown inhibited HCC cell proliferation, migration, invasion, stemness and oxaliplatin sensitivity. FBW7 targeted CHD3 for ubiquitination. FBW7 overexpression restrained HCC cell migration, invasion and stemness, and attenuated the effects of overexpressed CHD3 on promoting migration, invasion, stemness and oxaliplatin resistance in HCC cells. FBW7 overexpression suppresses HCC cell metastasis, stemness and oxaliplatin resistance via targeting CHD3 for ubiquitylation and degradation.

The p-MYH9/USP22/HIF-1α axis promotes lenvatinib resistance and cancer stemness in hepatocellular carcinoma

Lenvatinib is a targeted drug used for first-line treatment of hepatocellular carcinoma (HCC). A deeper insight into the resistance mechanism of HCC against lenvatinib is urgently needed. In this study, we aimed to dissect the underlying mechanism of lenvatinib resistance (LR) and provide effective treatment strategies. We established an HCC model of acquired LR. Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were used to detect the stemness of HCC cells. Molecular and biochemical strategies such as RNA-sequencing, immunoprecipitation, mass spectrometry and ubiquitination assays were used to explore the underlying mechanisms. Patient-derived HCC models and HCC samples from patients were used to demonstrate clinical significance. We identified that increased cancer stemness driven by the hypoxia-inducible factor-1α (HIF-1α) pathway activation is responsible for acquired LR in HCC. Phosphorylated non-muscle myosin heavy chain 9 (MYH9) at Ser1943, p-MYH9 (Ser1943), could recruit ubiquitin-specific protease 22 (USP22) to deubiquitinate and stabilize HIF-1α in lenvatinib-resistant HCC. Clinically, p-MYH9 (Ser1943) expression was upregulated in HCC samples, which predicted poor prognosis and LR. A casein kinase-2 (CK2) inhibitor and a USP22 inhibitor effectively reversed LR in vivo and in vitro. Therefore, the p-MYH9 (Ser1943)/USP22/HIF-1α axis is critical for LR and cancer stemness. For the diagnosis and treatment of LR in HCC, p-MYH9 (Ser1943), USP22, and HIF-1α might be valuable as novel biomarkers and targets.

Nrf2/ASPM axis regulated vasculogenic mimicry formation in hepatocellular carcinoma under hypoxia.

Hypoxic microenvironment is a common feature of most solid tumors including hepatocellular carcinoma (HCC). Vasculogenic mimicry (VM) formation by tumor cells could provide blood supply to tumor cells under hypoxia. NFE2 like basic leucine zipper (bZIP) transcription factor 2 (Nrf2), a regulator of cellular homeostasis, may promote tumor progression in the hypoxic conditions. However, the role and regulatory mechanisms of Nrf2 in HCC are not fully elucidated. Nrf2 and assembly factor for spindle microtubules (ASPM) expression modulations were conducted by lentiviral transfections. Western blot, immunofluorescence, ChIP-qPCR, dual-luciferase reporter gene assay, flow cytometry, RNA sequencing, multiple bioinformatics databases analysis, cell function assays in vitro, mouse model in vivo and human HCC tissues were employed to assess the effect of Nrf2/ASPM axis on HCC progression under hypoxia. Nrf2 and ASPM expression facilitated epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) feature, and VM formation of HCC cells under hypoxia. Furthermore, Nrf2-regulated ASPM expression, via binding directly to the promoter region of ASPM and transcriptionally promoting ASPM expression. ASPM re-expression in Nrf2 knockdown cells or ASPM knockdown in Nrf2 overexpression cells reversed the cellular function caused by Nrf2. Meantime, retinol metabolism pathway was disrupted following abnormal ASPM expression. Nrf2/ASPM axis in murine models accelerated tumor growth and VM, corroborating in vitro findings. All-trans retinoic acid treatment reversed stemness and VM of HCC cells in vitro and in vivo. Clinically, Nrf2 and ASPM expressions were related to poor prognosis of HCC patients. Nrf2 drives EMT, CSCs characteristics and VM in HCC under hypoxia through the modulation of ASPM. Retinol metabolism pathway was dysregulated in HCC cells with ASPM overexpression. Nrf2/ASPM axis and related pathway provided potential therapeutic target for HCC.

Cancer-associated fibroblasts-secreted exosomal miR-92a-3p promotes tumor growth and stemness in hepatocellular carcinoma through activation of Wnt/β-catenin signaling pathway by suppressing AXIN1.

Cancer-associated fibroblasts (CAFs) are a major cellular component in the tumor microenvironment and have been shown to exhibitprotumorigenic effects in hepatocellular carcinoma (HCC). This study aimed to delve into the mechanisms underlying the tumor-promoting effects of CAFs in HCC. Small RNA sequencing was conducted to screen differential expressed microRNAs in exosomes derived from CAFs and normal fibroblasts (NFs). The miR-92a-3p expression was then measured using reverse transcriptase quantitative real-time PCR in CAFs, NFs, CAFs-derived exosomes (CAFs-Exo), and NF-derived exosomes (NFs-Exo). Compared to NFs or NF-Exo, CAFs and CAFs-Exo significantly promoted HCC cell proliferation, migration, and stemness. Additionally, compared to NFs or NF-Exo, miR-92a-3p level was notably higher in CAFs and CAFs-Exo, respectively. Exosomal miR-92a-3p was found to enhance HCC cell proliferation, migration, and stemness. Meanwhile, AXIN1 was targeted by miR-92a-3p. Exosomal miR-92a-3p could activate β-catenin/CD44 signaling in HCC cells by inhibiting AXIN1 messenger RNA. Furthermore, in vivo studies verified that exosomal miR-92a-3p notably promoted tumor growth and stemness through targeting AXIN1/β-catenin axis. Collectively, CAFs secreted exosomal miR-92a-3p was capable of promoting growth and stemness in HCC through activation of Wnt/β-catenin signaling pathway by suppressing AXIN1. Therefore, targeting CAFs-derived miR-92a-3p may be a potential strategy for treating HCC.

Ovatodiolide inhibited hepatocellular carcinoma stemness through SP1/MTDH/STAT3 signaling pathway

Hepatocellular carcinoma (HCC) is characterized with high recurrence and mortality, and the clinical treatments for HCC are very limited. Hepatocellular carcinoma stem cells are the root of HCC progress, recurrence, and multidrug resistance. Ovatodiolide (OVA) is a bioactive diterpenoid served as an inflammatory and immunotherapeutic responses modulator. In this research, we found OVA inhibited HCC stemness through inhibiting MTDH gene transcription. Moreover, we firstly discovered transcription factor SP1 bound to the promoter region of MTDH to transcriptionally regulate MTDH level. Mechanically, we demonstrated OVA decreased SP1 protein stability to transcriptionally inhibit MTDH gene, and inhibited the nuclear translocation of p65, and then diminished IL-6 level to suppress JAK/STAT3 signaling pathway, eventually decreases CD133 level and the stemness of HCC. Furthermore, we demonstrated ACT004, OVA derivative with high metabolic stability towards cytochrome P450 enzymes, showed no genotoxicity and no accumulative or delayed toxicities after long-term administration in rats. And the in vivo efficacy experiments indicated ACT004 inhibited tumor growth of hepatocellular carcinoma. In conclusion, we revealed the mechanism of OVA in regulating HCC stemness, detected the toxicity of OVA derivative and evaluated the in vivo efficacy which lays a foundation for further discovery of anti-HCC stem cell agents and provide a new strategy for the application of OVA in clinical treatment.

Downregulation of GPX8 in hepatocellular carcinoma: impact on tumor stemness and migration

PurposeGPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown.MethodsImmunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry.ResultsThe GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8.ConclusionThe findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

Role of CCR1/5/7 in hepatocellular carcinoma: a study on prognostic evaluation, molecular subtyping, and association with immune infiltration.

This study aims to assess the prognostic value of the C-C motif chemokine receptor (CCR) gene family in hepatocellular carcinoma (HCC) and its relationship with immune infiltration and molecular subtypes of HCC. The evaluation of the GSE14520 dataset and TCGA database confirmed the prognostic significance of CCR. Building upon the correlation between CCR1, CCR5, and CCR7 and favorable prognosis, we further validated the prognostic importance of CCR1, CCR5, and CCR7 in ICGC database and an independent cohort from Guangxi autonomous region. Then, we constructed a risk prognosis model. Additionally, we observed significant positive correlations between CCR1, CCR5, and CCR7 and the infiltration of B cells, T cells, and macrophages in HCC. Subsequently, we conducted CCK assays, Transwell assays, and colony formation assays to evaluate the molecular biological functions of CCR1, CCR5, and CCR7. These experiments further confirmed that upregulation of CCR1, CCR5, and CCR7 can individually inhibit the proliferation, migration, and stemness of HCC cells. By analyzing the relationship between expression levels and tumor mutation frequency, we discovered that patients with high CCR1 expression were more likely to be classified as non-proliferative HCC. Similar conclusions were observed for CCR5 and CCR7. The association of CCR1, CCR5, and CCR7 with the molecular subtypes of HCC suggests that they may serve as intermediary molecules linking immune status and molecular subtypes in HCC. In summary, CCR1, CCR5, and CCR7 have the potential to serve as prognostic biomarkers for HCC and regulate HCC progression by influencing immune cell infiltration.

High expression of UBE2S promotes progression of hepatocellular carcinoma by increasing cancer cell stemness

To investigate the expression of the ubiquitination enzyme UBE2S in different cell types in hepatocellular carcinoma (HCC) microenvironment and its impact on proliferation and stemness of HCC cells. TCGA and CPTAC database were used to analyze the transcriptional and promoter methylation levels and protein expressions of UBE2S in HCC. Specific expression patterns of UBE2S, intercellular communication and key transcription factors in different cell types were analyzed based on single-cell sequencing data from TISCH website. We further examined UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells using immunohistochemistry and immunofluorescence staining. We also tested the effects of UBE2S knockdown on stemness of HCC-LM3 and HepG2 cells using clone formation experiments and sphere formation assay. Analysis based on TCGA database suggested significant overexpression of UBE2S in both paired and non-paired tumor tissues (P < 0.001), and its transcriptional level increased with tumor grades. The methylation level of UBE2S promoter was significantly decreased in HCC (P < 0.001), and its transcription level increased obviously in HCC with TP53 mutation (P < 0.001). Analysis of CPTAC database also demonstrated overexpression of UBE2S protein in HCC tissues (P < 0.001). Three prognostic models suggested that HCC patients with high UBE2S expression had poorer prognosis (P < 0.001). Single-cell sequencing data analysis revealed high expressions of UBE2S in T cells and high intensities of interaction between endothelial cells, epithelial cells and fibroblasts in HCC microenvironment. Immunohistochemistry and immunofluorescence staining demonstrated high UBE2S expressions in clinical samples of HCC tissues, HCC cells and T cells. In HCC-LM3 and HepG2 cells, UBE2S knockdown significantly inhibited cell clone formation and tumor sphere formation (P < 0.05). UBE2S is highly expressed in T cells in HCC microenvironment in close correlation with a poor prognosis. High UBE2S expression promotes the stemness of HCC cells.

FEN1 upregulation mediated by SUMO2 via antagonizing proteasomal degradation promotes hepatocellular carcinoma stemness

PurposeMetastasis of hepatocellular carcinoma (HCC) critically impacts the survival prognosis of patients, with the pivotal role of hepatocellular carcinoma stem cells in initiating invasive metastatic behaviors. The Flap Endonuclease 1 (FEN1) is delineated as a metallonuclease, quintessential for myriad cellular processes including DNA replication, DNA synthesis, DNA damage rectification, Okazaki fragment maturation, baseexcision repair, and the preservation of genomic stability. Furthermore, it has been recognized as an oncogene in a diverse range of malignancies. Our antecedent research has highlighted a pronounced overexpression of protein FEN1 in hepatocellular carcinoma, where it amplifies the invasiveness and metastatic potential of liver cancer cells. However, its precise role in liver cancer stem cells (LCSCs) remains an enigma and requires further investigation. MethodsTo rigorously evaluate the stemness attributes of LCSCs, we employed sphere formation assays and flow cytometric evaluations. Both CD133+ and CD133- cell populations were discerningly isolated utilizing immunomagnetic bead separation techniques. The expression levels of pertinent genes were assayed via real-time quantitative PCR (RT-qPCR) and western blot analyses, while the expression profiles in hepatocellular carcinoma tissues were gauged using immunohistochemistry. Subsequent immunoprecipitation, in conjunction with mass spectrometry, ascertained the concurrent binding of proteins FEN1 and Small ubiquitin-related modifier 2 (SUMO2) in HCC cells. Lastly, the impact of SUMO2 on proteasomal degradation pathway of FEN1 was validated by supplementing MG132. ResultsOur empirical findings substantiate that protein FEN1 is profusely expressed in spheroids and CD133+ cells. In vitro investigations demonstrate that the upregulation of protein FEN1 unequivocally augments the stemness of LCSCs. In a congruent in vivo context, elevation of FEN1 noticeably enhances the tumorigenic potential of LCSCs. Conversely, inhibiting protein FEN1 resulted in a marked reduction in LCSC stemness. From a mechanistic perspective, there exists a salient positive correlation between the protein expression of FEN1 and SUMO2 in liver cancer tissues. Furthermore, the level of SUMO2-mediated modification of FEN1 is pronouncedly elevated in LCSCs. Interestingly, SUMO2 has the ability to bind to FEN1, leading to a inhibition in the proteasomal degradation pathway of FEN1 and an enhancement in its protein expression. However, it is noteworthy that this interaction does not affect the mRNA level of FEN1. ConclusionIn summation, our research elucidates that protein FEN1 is an effector in augmenting the stemness of LCSCs. Consequently, strategic attenuation of protein FEN1 might proffer a pioneering approach for the efficacious elimination of LCSCs.

LAPTM4B-mediated hepatocellular carcinoma stem cell proliferation and MDSC migration: implications for HCC progression and sensitivity to PD-L1 monoclonal antibody therapy

In hepatocellular carcinoma (HCC), immunotherapy is vital for advanced-stage patients. However, diverse individual responses and tumor heterogeneity have resulted in heterogenous treatment outcomes. Our mechanistic investigations identified LAPTM4B as a crucial gene regulated by ETV1 (a transcription factor), especially in liver cancer stem cells (LCSCs). The influence of LAPTM4B on LCSCs is mediated via the Wnt1/c-Myc/β-catenin pathway. CXCL8 secretion by LAPTM4B drove myeloid-derived suppressor cell (MDSC) migration, inducing unfavorable patient prognosis. LAPTM4B affected PD-L1 receptor expression in tumor microenvironment and enhanced tumor suppression induced by PD-L1 monoclonal antibodies in HCC patients. LAPTM4B up-regulation is correlated with adverse outcomes in HCC patients, sensitizing them to PD-L1 monoclonal antibody therapy.

Interferon-α induces differentiation of cancer stem cells and immunosuppression in hepatocellular carcinoma by upregulating CXCL8 secretion

Interferon-alpha (IFN-α) is widely used in the clinical treatment of patients with chronic hepatitis B and hepatocellular carcinoma (HCC). However, high levels of CXCL8 are associated with resistance to IFN-α therapy and poorer prognosis in advanced cancers. In this study, we investigated whether IFN-α could directly induce the production of CXCL8 in HCC cells and whether CXCL8 could antagonize the antitumor activity of IFN-α. We found that IFN-α not only upregulated the expression of the inducible genes CXCL9, CXCL10, CXCL11 and PD-L1, but also significantly stimulated CXCL8 secretion in HCC cells. Mechanically, IFN-α induces CXCL8 expression by activating the AKT and JNK pathways. In addition, our results demonstrate that IFN-α exposure significantly increases the differentiation of HCC stem cells, but this effect is reversed by the addition of the CXCL8 receptor CXCR1/2 inhibitor Reparixin and STAT3 inhibitor Stattic. Besides, our study reveals that the cytokine CXCL8 secreted by IFN-α-induced HCC cells inhibits T-cell function. Conversely, inhibition of CXCL8 promotes TNF-α and IFN-γ secretion by T cells. Finally, liver cancer patients who received anti-PD-1/PD-L1 immunotherapy with high CXCL8 expression had a lower immunotherapy efficacy. Overall, our findings clarify that IFN-α triggers immunosuppression and cancer stem cell differentiation in hepatocellular carcinoma by upregulating CXCL8 secretion. This discovery provides a novel approach to enhance the effectiveness of HCC treatment in the future.

N6-Methyladenosine Reader YTHDF1 Promotes Stemness and Therapeutic Resistance in Hepatocellular Carcinoma by Enhancing NOTCH1 Expression.

Inhibition of YTHDF1 expression suppresses stemness of hepatocellular carcinoma cells and enhances sensitivity to targeted therapies, indicating that targeting YTHDF1 may be a promising therapeutic strategy for liver cancer.

Study on the Mechanism of Notch Pathway Mediates the Role of Lenvatinib-resistant Hepatocellular Carcinoma Based on Organoids.

The emergence of treatment resistance has hindered the efficacy of targeted therapies used to treat patients with hepatocellular carcinoma (HCC). This study aimed to explore the mechanism of organoids constructed from lenvatinib-resistant HCC cells. Hep3B cell and human HCC organoids were cultured and identified using hematoxylin and eosin staining and Immunohistochemistry. Lenvatinib-sensitive/ resistant Hep3B cells were constructed using lenvatinib (0, 0.1, 1, and 10 μM) and lenvatinib (0, 1, 10, and 100 μM). qRT-PCR and flow cytometry were utilized to determine HCC stem cell markers CD44, CD90, and CD133 expressions. Transcriptome sequencing was performed on organoids.-Western blot evaluated Notch pathwayrelated proteins (NOTCH1 and Jagged) expressions. Furthermore, DAPT, an inhibitor of the Notch pathway, was used to investigate the effects of lenvatinib on resistance or stemness in organoids and human HCC tissues. The organoids were successfully cultivated. With the increase of lenvatinib concentration, sensitive cell organoids were markedly degraded and ATP activity was gradually decreased, while there was no significant change in ATP activity of resistant cell organoids. CD44 expressions were elevated after lenvatinib treatment compared with the control group. KEGG showed that lenvatinib treatment of organoids constructed from Hep3B cells mainly activated the Notch pathway. Compared with the control group, NOTCH1 and Jagged expressions elevated, and ATP activity decreased after lenvatinib treatment. However, ATP activity was notably decreased after DAPT treatment. Moreover, DAPT inhibited lenvatinib resistance and the increase in the expressions of CD44 caused by lenvatinib. Besides, 100 μM lenvatinib significantly inhibited the growth and ATP activity of human HCC organoids, and DAPT increased the inhibitory effect of lenvatinib. Lenvatinib regulated resistance and stemness in organoids via the Notch pathway.

Transcription Factor E2F1 Enhances Hepatocellular Carcinoma Cell Proliferation and Stemness by Activating GINS1.

Present studies report that high expression of GINS complex subunit 1 (GINS1) is notably pertinent to poor survival for hepatocellular carcinoma (HCC), but it remains unclear how GINS1 affects the progression of HCC. This study aims at investigating the mechanism by which GINS1 affects HCC cell proliferation and stemness. We performed bioinformatics analysis for determining GINS1 expression in HCC tissues, as well as the HCC patients' survival rate with different expression levels of GINS1. E2F transcription factor 1 (E2F1) was predicted as the upstream transcription factor of GINS1, and the binding relation between the two was verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Quantitative real-time polymerase chain reaction was adopted to evaluate the expression of GINS1 and E2F1. The protein expression levels of GINS1, E2F1, and cell stemness-related genes (SOX-2, NANOG, OCT4, and CD133) were detected by Western blot. Afterward, the proliferative capacity and stemness of HCC tumor cells were determined through colony formation, cell counting kit-8, and sphere formation assays. Our study found the high expression of GINS1 and E2F1 in HCC, and overexpressed GINS1 markedly enhanced the sphere formation and proliferation of HCC cells, while silencing GINS1 led to the opposite results. Besides, E2F1 promoted the transcription of GINS1 by working as an upstream transcription factor. The results of the rescue experiment suggested that overexpressed E2F1 could offset the suppressive effect of GINS1 silencing on HCC cell stemness and proliferation. We demonstrated that the transcription factor E2F1 accelerated cell proliferation and stemness in HCC by activating GINS1 transcription. The results can provide new insight into the GINS1-related regulatory mechanism in HCC, which suggest that it may be an effective way for HCC treatment by targeting the E2F1/GINS1 axis.

HNF4A-AS1 inhibits the progression of hepatocellular carcinoma by promoting the ubiquitin-modulated degradation of PCBP2 and suppressing the stability of ARG2 mRNA.

Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor with a poor prognosis. Extensive research has revealed the significant role of long noncoding RNAs (lncRNAs) in the regulation of tumor development. In this study, high-throughput sequencing analysis was used to assess the expression levels of lncRNAs in three pairs of HCC tissues and their corresponding noncancerous tissues. Through quantitative real-time polymerase chain reaction (qRT-PCR) analysis and clinicopathological analysis, it was discovered that HNF4A-AS1 was downregulated in HCC tissues. Furthermore, its expression levels were found to be positively correlated with the prognosis of HCC patients. Subsequent in vitro and in vivo functional studies demonstrated that HNF4A-AS1 inhibits the proliferation, invasion, and stemness of HCC cells. Mechanistically, it was observed that HNF4A-AS1 physically interacts with the KH3 domain of PCBP2 through a specific segment (491-672 nt). This interaction facilitates the recruitment of PCBP2 by AIP4, leading to the ubiquitination and subsequent degradation of PCBP2. Furthermore, HNF4A-AS1 was found to regulate the stability of AGR2 mRNA by modulating PCBP2, thereby influencing the malignant phenotype of HCC. Overall, our study demonstrated a positive association between the decrease in HNF4A-AS1 expression and the prognosis of patients with HCC in a clinical setting. HNF4A-AS1 can suppress the stability of ARG2 mRNA by promoting the ubiquitin-modulated degradation of PCBP2, which suppresses HCC progression. HNF4A-AS1 may serve as a potential therapeutic target for HCC.

Integration of single-cell RNA-seq and bulk RNA-seq to construct liver hepatocellular carcinoma stem cell signatures to explore their impact on patient prognosis and treatment.

Liver hepatocellular carcinoma (LIHC) is a prevalent form of primary liver cancer. Research has demonstrated the contribution of tumor stem cells in facilitating tumor recurrence, metastasis, and treatment resistance. Despite this, there remains a lack of established cancer stem cells (CSCs)-associated genes signatures for effectively predicting the prognosis and guiding the treatment strategies for patients diagnosed with LIHC. The single-cell RNA sequencing (scRNA-seq) and bulk RNA transcriptome data were obtained based on public datasets and computerized firstly using CytoTRACE package and One Class Linear Regression (OCLR) algorithm to evaluate stemness level, respectively. Then, we explored the association of stemness indicators (CytoTRACE score and stemness index, mRNAsi) with survival outcomes and clinical characteristics by combining clinical information and survival analyses. Subsequently, weighted co-expression network analysis (WGCNA) and Cox were applied to assess mRNAsi-related genes in bulk LIHC data and construct a prognostic model for LIHC patients. Single-sample gene-set enrichment analysis (ssGSEA), Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) and Tumor Immune Estimation Resource (TIMER) analysis were employed for immune infiltration assessment. Finally, the potential immunotherapeutic response was predicted by the Tumor Immune Dysfunction and Exclusion (TIDE), and the tumor mutation burden (TMB). Additionally, pRRophetic package was applied to evaluate the sensitivity of high and low-risk groups to common chemotherapeutic drugs. A total of four genes (including STIP1, H2AFZ, BRIX1, and TUBB) associated with stemness score (CytoTRACE score and mRNAsi) were identified and constructed a risk model that could predict prognosis in LIHC patients. It was observed that high stemness cells occurred predominantly in the late stages of LIHC and that poor overall survival in LIHC patients was also associated with high mRNAsi scores. In addition, pathway analysis confirmed the biological uniqueness of the two risk groups. Personalized treatment predictions suggest that patients with a low risk benefited more from immunotherapy, while those with a high risk group may be conducive to chemotherapeutic drugs. The current study developed a novel prognostic risk signature with genes related to CSCs, which provides novel ideas for the diagnosis, prognosis and treatment of LIHC.

Circular RNA cFAM210A, degradable by HBx, inhibits HCC tumorigenesis by suppressing YBX1 transactivation

Hepatitis B protein x (HBx) has been reported to promote tumorigenesis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), but the mechanism awaits further investigation. In this study, we found that cFAM210A (a circular RNA derived from the third exon of transcript NM_001098801 of the FAM210A gene; CircBase ID: hsa_circ_0003979) can be silenced by HBx. cFAM210A expression was downregulated and negatively correlated with tumorigenesis in patients with HBV-related HCC. Furthermore, cFAM210A reduced the proliferation, stemness, and tumorigenicity of HCC cells. Mechanistically, HBx increased the N6-methyladenosine (m6A) level of cFAM210A by promoting the expression of RBM15 (an m6A methyltransferase), thus inducing the degradation of cFAM210A via the YTHDF2-HRSP12-RNase P/MRP pathway. cFAM210A bound to YBX1 and inhibited its phosphorylation, suppressing its transactivation function toward MET. These findings suggest the important role of circular RNAs in HBx-induced hepatocarcinogenesis and identify cFAM210A a potential target in the prevention and treatment of HBV-related HCC.

SETD1A drives stemness by reprograming epigenetic landscape in hepatocellular carcinoma stem cells.

Cancer stem cells (CSCs) are responsible for tumor progression and recurrence. However, the mechanisms regulating hepatocellular carcinoma (HCC) stemness remain unclear. Applying a genome-scale CRISPR knockout screen, we identify that the H3K4 methyltransferase SETD1A and other members of Trithorax group proteins drive cancer stemness in HCC. SETD1A is positively correlated with poor clinical outcome in HCC patients. Combination of SETD1A and serum AFP significantly improves the accuracy of predicting HCC relapse. Mechanistically, SETD1A mediates transcriptional activation of various histone-modifying enzymes, facilitates deposition of H3K4me3 and H3K27me3 and activates oncogenic enhancers and super-enhancers, leading to activation of oncogenes and inactivation of tumor suppressor genes simultaneously in HCC CSCs. In addition, SETD1A cooperates with PABPC1 to regulate H3K4me3 modification on oncogenes. Our data pinpoint SETD1A as a key epigenetic regulator driving HCC stemness and progression, highlighting the potential of SETD1A as a candidate target for HCC intervention and therapy.

Deep dissection of stemness-related hierarchies in hepatocellular carcinoma

BackgroundIncreasing evidence suggests that hepatocellular carcinoma (HCC) stem cells (LCSCs) play an essential part in HCC recurrence, metastasis, and chemotherapy and radiotherapy resistance. Multiple studies have demonstrated that stemness-related genes facilitate the progression of tumors. However, the mechanism by which stemness-related genes contribute to HCC is not well understood. Here, we aim to construct a stemness-related score (SRscores) model for deeper analysis of stemness-related genes, assisting with the prognosis and individualized treatment of HCC patients.Further, we found that the gene LPCAT1 was highly expressed in tumor tissues by immunohistochemistry, and sphere-forming assay revealed that knockdown of LPCAT1 inhibited the sphere-forming ability of hepatocellular carcinoma cells.MethodsWe used the TCGA-LIHC dataset to screen stemness-related genes of HCC from the MSigDB database. Prognosis, tumor microenvironment, immunological checkpoints, tumor immune dysfunction, rejection, treatment sensitivity, and putative biological pathways were examined. Random forest created the SRscores model. The anti-PD-1/anti-CTLA4 immunotherapy, tumor mutational burden, medication sensitivity, and cancer stem cell index were compared between the high- and low-risk score groups. We also examined risk scores for different cell types using single-cell RNA sequencing data and correlated transcription factor activity in cancer stem cells with SRscores genes. Finally, we tested core marker expression and biological functions.ResultsPatients can be divided into two subtypes (Cluster1 and Cluster2) based on the TCGA-LIHC dataset's identification of 11 stemness-related genes. Additionally, a SRscores was developed based on subtypes. Cluster2 and the group with the lowest SRscores had superior survival and immunotherapy response than Cluster1 and the group with the highest SRscores. The group with a high SRscores was significantly more enriched in classical tumor pathways than the group with a low SRscores. Multiple transcription factors and SRscores genes are correlated. The core gene LPCAT1 is highly expressed in rat liver cancer tissues and promotes tumor cell sphere formation.ConclusionA SRscores model can be utilized to predict the prognosis of HCC patients as well as their response to immunotherapy.

YY1-regulated lncRNA SOCS2-AS1 suppresses HCC cell stemness and progression via miR-454-3p/CPEB1

BackgroundCancer stem cells are one fundamental reason for the high recurrence rate of hepatocellular carcinoma (HCC) and its resistance to treatment. This study explored the mechanism by which SOCS2-AS1 affects HCC cell stemness. MethodsStem cells of HCC cell lines Huh7 and SNU-398 were sorted as NANOG-positive by flow cytometry. Stem cell sphere formation ability was detected. Stem cell viability, migration, invasion, and apoptosis were assessed by colony formation assays, Transwell assays, wound-healing assays, and TUNEL assays, respectively. The binding sites for SOCS2-AS1, miR-454-3p, miR-454-3p, and CPEB1 mRNA were assessed by dual-luciferase reporter assays. Quantitative real-time PCR (qPCR) and Western blot studies were performed to evaluate gene expression levels. ChIP and EMSA assays were conducted to confirm that YY1 binds with the SOCS2-AS1 promoter. A subcutaneous xenograft model was used to verify results in vivo. Tumor tissues were analyzed by H&E and TUNEL staining. ResultsSOCS2-AS1 was expressed at low levels in NANOG+ HCC stem cells, and HCC patients with a high level of SOCS2-AS1 expression had a higher survival rate. SOCS2-AS1 inhibited HCC cell stemness, migration, and invasion, and increased the cisplatin sensitivity of HCC cells by regulating miR-454-3p/CPEB1. YY1 was confirmed as a transcription factor of SOCS2-AS1, and served to inhibit SOCS2-AS1 transcription. YY1 knockdown suppressed HCC stemness via SOCS2-AS1. The role of SOCS2-AS1 was confirmed in a subcutaneous xenograft model, and SOCS2-AS1 overexpression enhanced the inhibitory effect of cisplatin on HCC in vivo. ConclusionsYY1-regulated lncRNA SOCS2-AS1 suppresses HCC cell stemness and progression via miR-454-3p/CPEB1.