Hepatocellular Carcinoma Cell Migration Research Articles

Prognostic, oncogenic roles, and pharmacogenomic features of AMD1 in hepatocellular carcinoma

Background AMD1 is the gene encoding S-adenosylmethionine decarboxylase 1. How AMD1 affects the prognosis of hepatocellular carcinoma (HCC) patients is unclear.MethodsUsing the Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma datasets, gene enrichment and immunological traits were compared between groups with high and low AMD1 expression. After altering AMD1 expression in HCC cells, cell viability, the clonal formation rate, and migration and invasion ability were detected. Univariate Cox regression analysis and Pearson correlation were used to screen for AMD1-related genes (ARGs). Multidimensional bioinformatic algorithms were utilized to establish a risk score model for ARGs.Results AMD1 expression was notably increased in the majority of cancer types. High AMD1 expression was associated with adverse outcomes and poorer immunotherapy response in HCC patients. AMD1 exhibited higher expression levels in HCC cell lines. The efficient inhibition of HCC cell proliferation, migration, and invasion in vitro can be achieved through the downregulation of AMD1. The AMD1-related risk score was constructed with the expression of 9 ARGs, and demonstrated high predictive efficacy in multiple validation cohorts. Patients with high risk scores exhibited greater resistance to classical chemotherapy drugs. The nomogram, which consists of age, stage, and the AMD1-related risk score, was used to calculate the probability of survival for each individual.ConclusionThe present study indicates that AMD1 functions as a potential role in HCC progression and may serve as a therapeutic target in HCC. This study constructed a novel AMD1-related scoring system for predicting the prognosis and treatment responsiveness of patients with HCC, enabling the prediction of prognosis and identification of potential treatment targets.

Activated hepatic stellate cell-derived small extracellular vesicles facilitate M2 macrophage polarization and hepatoma progression via miR-27a-3p

The progression of hepatoma is heavily influenced by the microenvironment. Tumor-associated macrophages (TAMs) are considered to play a critical role in the tumor microenvironment (TME) and increase the aggressiveness of hepatoma. The activation of hepatic stellate cells (HSCs) is involved in hepatoma progression, and accumulating evidence demonstrates a change in microRNA (miRNA) expression during HSC activation. Therefore, the potential roles of HSCs-related miRNAs in macrophage differentiation and hepatoma progression deserve to be explored. The present study aimed to investigate the effects of miRNAs carried by small extracellular vesicles (sEVs) released by activated HSCs on hepatoma progression. The results indicated that miR-27a-3p was significantly upregulated in cells and corresponding sEVs during the activation of primary rat HSCs and human HSC line-LX2 cells. Furthermore, miR-27a-3p contributed to the proliferation and migration of hepatoma cells and promoted M2 polarization of macrophage. HSC-sEVs overexpressing miR-27a-3p can directly facilitate tumor progression and modulate macrophage polarization, indirectly contributing to hepatoma progression. Finally, Sprouty2 (SPRY2) was verified to be the target gene of miR-27a-3p. In conclusion, activated HSC-derived sEVs with high levels of miR-27a-3p might induce M2 macrophage polarization and promote hepatoma progression, providing new insights into the mechanism of hepatoma progression.

MiR-145-5p regulates hepatocellular carcinoma malignant advancement and immune escape via down-regulation of AcylCoA synthase ACSL4

Hepatocellular carcinoma (HCC) exhibits a subtle onset, high incidence rates, and low survival rates, becoming a substantial threat to human health. Hence, it is crucial to discover fresh biomarkers and treatment targets for the early detection and management of HCC. CCK-8, scratch-wound, and transwell assays were used to evaluate the biological properties of HCC cell lines (Huh-7 and Hep3B). Bioinformatics analysis identified the downstream target mRNA of miR-145-5p as acyl-CoA synthetase long-chain family member 4 (ACSL4). RT-qPCR was used to test miR-145-5p and ACSL4 levels. Transwell chambers were used to co-incubate purified CD8+ T cells and HCC cells for 48 h, and the effect of CD8+ T cells on apoptosis in HCC cells was detected by flow cytometry. A subcutaneous graft tumor model was constructed, and ELISA kits were used to assess cytokine levels and CD8+ T cell activation markers. HCC cells showed a decline in miR-145-5p levels and a rise in ACSL4 levels. Overexpression of miR-145-5p hindered HCC cell proliferation, migration, and invasion, while stimulating CD8+ T cell activation. Conversely, overexpression of ACSL4 enhanced the malignant biological properties of HCC cells and reduced the effect of CD8+ T cells, while silencing ACSL4 had the opposite effect. miR-145-5p targeted and downregulated ACSL4, while overexpression of miR-145-5p weakened the promotion of HCC malignant progression caused by ACSL4 overexpression. Additionally, overexpression of miR-145-5p and silencing ACSL4 were effective in inhibiting tumor growth in vivo. In conclusion, miR-145-5p targets and downregulates ACSL4, leading to the inhibition of HCC malignant progression and preventing immune escape in HCC cells.

Expression of ALG8 in hepatocellular carcinoma and its diagnostic and prognostic significance

Background Alpha-1,3-glucosyltransferase (ALG8), a key enzyme in protein glycosylation, is implicated in the oncogenesis and progression of several human malignancies. This study aimed to define the role of ALG8 in hepatocellular carcinoma (HCC) and uncover its mechanisms of action. Methods ALG8 expression in HCC and normal tissues was analyzed using the TCGA and GEO databases, validated by RT-qPCR and western blot. Survival outcomes were evaluated via Cox analyses, and ALG8’s impact on HCC behavior was examined through functional assays. GO, KEGG, and GSEA identified ALG8-related pathways, validated by biochemical assays. Results In bioinformatics analyses, ALG8 was overexpressed in HCC tissues (p < 0.05 for all comparisons) and correlated with poorer survival (p = 0.006 and p = 0.025, respectively), establishing its role as an independent prognostic factor. In vitro experiments showed that knockdown of ALG8 reduced HCC cell proliferation, migration, and invasion. Using the STRING platform and TCGA-LIHC dataset, we identified ALG8-interacting genes and their associated differentially expressed genes (DEGs). GO and KEGG analyses further linked ALG8 to genes involved in glycosylation, signal release, and other processes, as well as pathways including neuroactive ligand-receptor interaction and N-Glycan biosynthesis. GSEA, corroborated by western blot and immunofluorescence, points to the Wnt/β-catenin signaling cascade as a probable mechanistic pathway through which ALG8 may modulate HCC progression. Conclusion Elevated ALG8 expression in HCC is linked to worse outcomes and increased tumor aggressiveness, with silencing ALG8 reducing Wnt/β-catenin signaling, highlighting ALG8 as a potential therapeutic target.

The ubiquitination degradation of KLF15 mediated by WSB2 promotes lipogenesis and progression of hepatocellular carcinoma via inhibiting PDLIM2 expression.

Krüppel-like factors15 (KLF15) is a cancer suppressor in many cancers. However, its precise function in the development of hepatocellular carcinoma (HCC) remains unclear. Lipogenesis is necessary for the development of HCC. This research aims to investigate the role of KLF15 in the regulation of hepatic lipid production and HCC progression. The binding relationships among genes were confirmed by ChIP, dual luciferase assays, and Co-IP. Lipogenesis was examined by oil red O staining. Triglyceride and cholesterol levels were measured through commercial kits. The effect of treatment on HCC cell viability, proliferation, migration, and invasion were assessed using CCK-8, clone formation, or transwell assays. A subcutaneous tumorigenic model was utilized to explore the effects of PDLIM2 in HCC in vivo. KLF15 were downregulated in human HCC tissues. KLF15 overexpression reduced lipid droplet production, suppressed the expression of genes associated with lipogenesis, and promoted cell proliferation, migration, and invasion. KLF15 suppressed the NF-κB pathway through transcriptional activation of PDLIM2. PDLIM2 knockdown attenuated the effect of KLF15 overexpression on HCC. WSB2 degraded KLF15 through ubiquitination to promote HCC lipogenesis and development. The ubiquitination degradation of KLF15 was mediated by WSB2, which led to transcriptional repression of PDLIM2 and further activation of the NF-κB pathway, ultimately promoting HCC lipogenesis and development.

Identification of the Roles of Coagulation-related Signature and its Key Factor RABIF in Hepatoma Cell Malignancy.

Hepatoma is a high morbidity and mortality cancer, and coagulation is a potential oncogenic mechanism for hepatoma development. In this study, we aimed to reveal the role of coagulation in hepatoma. We applied the LASSO to construct a coagulation-related risk score (CRS) and a clinical nomogram with independent validation. The heterogeneity of various aspects, including functional enrichment, SNV, CN, immunocyte infiltration, immune pathways, immune checkpoint, and genomic instability indexes, was evaluated. Besides, the prognostic value of the CRS genes was tested. We selected the critical risky gene related to coagulation from the LASSO coefficients, for which we applied transwell and clone formation assays to confirm its roles in hepatoma cell migration and clone formation ability, respectively. The CRS and the nomogram predicted patients' survival with good accuracy in both two datasets. The high-CRS group was associated with higher cell cycle, DNA repair, TP53 mutation rates, amplification, and lower deletion rates at chromosome 1. For immunocyte infiltration, we noticed increased Treg infiltration and globally upregulated immune checkpoints and genomic instability indexes. Additionally, every single CRS gene affected the patient's survival. Finally, we observed that RABIF was the riskiest gene in the CRS. Its knockdown suppressed hepatoma cell migration and clone formation capability, which could be rescued by RABIF overexpression. We built a robust CRS with great potential as a prognosis and immunotherapeutic indicator. Importantly, we identified RABIF as an oncogene, promoting hepatoma cell migration and clone formation, revealing underlying pathological mechanisms, and providing novel therapeutic targets for hepatoma treatment.

KLF7 Promotes Hepatocellular Carcinoma Progression Through Regulating SLC1A5-Mediated Tryptophan Metabolism.

Krüppel-like factor 7 is a transcriptional activator and acts as an oncogene in human cancers, including hepatocellular carcinoma (HCC). Tryptophan metabolism is important for HCC cell proliferation, metastasis, and invasion. It is unclear whether KLF7 could regulate Trp metabolism in HCC. In this study, we found that Trp metabolism was suppressed in HCC cells with KLF7 knockdown. The mRNA and protein levels of SLC1A5, SLC7A5, and TPH1, as well as the content of Trp and serotonin, were reduced after KLF7 knockdown, and were potentiated following KLF7 overexpression. Increasing the content of serotonin could restore the malignancy of tumour cells invitro and tumour growth invivo. Conversely, decreasing the content of serotonin suppressed HCC cell proliferation. The binding activity of KLF7 was on the promoter of SLC1A5, and KLF7 positively regulated the expression of SLC1A5. KLF7 contributed to the proliferation and migration of HCC cells by up-regulation of SLC1A5. Collectively, KLF7 promotes the progression of HCC through regulating Trp metabolism. The newly identified axis of KLF7/ SLC1A5 in HCC could represent a potential target for HCC therapy.

Farrerol suppresses epithelial-mesenchymal transition in hepatocellular carcinoma via suppression of TGF-β1/Smad2/3 signaling

BackgroundEpithelial-mesenchymal transition (EMT) is an essential process for the metastasis of multiple malignancies, including hepatocellular carcinoma (HCC). Farrerol is a plant-derived flavonoid and has significant pharmacological effects. However, the anticancer activities of farrerol have not been fully elucidated. Here, we investigated the effects of farrerol on HCC progression. MethodsThe potential of farrerol to prevent HCC cell migration and invasiveness was evaluated by wound healing and transwll matrix assays. Immunoblotting, immunofluorescence, and qPCR were used to detect the levels of EMT-related proteins. Transforming growth factor beta (TGF-β) (10ng/ml) was used to stimulate HCC cells, followed by measurement of cell migration, invasiveness, and the EMT. TGF-β1/Smads signaling was examined by immunoblotting. A xenograft mouse model was used to assess the anticancer efficacy of farrerol in vivo. The expression levels of EMT- and angiogenesis-related proteins in xenograft tumors were evaluated by immunoblotting or immunohistochemistry. ResultsWe found that farrerol blocked HCC cell migration and invasiveness. Farrerol upregulated E-cadherin levels and reduced N-cadherin and vimentin levels. Farrerol also downreuglated the expression levels of EMT-related transcription factors including slug, snail, twist, and zeb1. Furthermore, farrerol suppressed TGF-β-stimulated migration, invasiveness, and the EMT in HCC cells. The phosphorylation of Smad 2/3 induced by TGF-β was inhibited by farrerol. Importantly, farrerol suppressed HCC growth and the EMT in vivo. Farrerol also inhibited tumor angiogenesis by inhibiting hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in vivo. ConclusionOverall, farrerol suppresss HCC by inhibiting migration, invasiveness, the EMT, and angiogenesis, implying that farrerol could be a promising antimetastasis agent for HCC.

METTL3 and HERC4: Elevated Expression and Impact on Hepatocellular Carcinoma Progression.

Background: Methyltransferase-like 3 (METTL3) and HECT and RLD domain containing E3 ubiquitin protein ligase 4 (HERC4) have been studied in the field of oncology; howbeit, their roles and interaction in hepatocellular carcinoma (HCC) await elucidation. Methods: Initially, METTL3 and HERC4 expressions in normal and HCC samples were predicted employing the UALCAN database, and the targeting relationship between these two was explored via coimmunoprecipitation assay. Following the quantification on N6-methyladenosine (m6A) enrichment, the localization of METTL3 and HERC4 on HCC cells was visualized via immunofluorescence assay. The effects of METTL3 and HERC4 on HCC cells proliferation and migration were determined in vitro assays. METTL3 and HERC4 expressions were quantified via quantitative polymerase chain reaction, and those of metastasis-related proteins N-cadherin and vimentin were calculated with immunoblotting assay. Furthermore, the levels of angiogenic factors such as vascular endothelial growth factor and basic fibroblast growth factor were measured by enzyme-linked immunosorbent assay. Results: METTL3 and HERC4 expressed highly in HCC and their expressions were positively correlated with tumor grade. METTL3 overexpression enhanced the expression of HERC4 and promoted the proliferation and migration abilities of HCC cells. Specifically, METTL3 overexpression increased vimentin and N-cadherin expressions, while its silencing did conversely. Besides, HERC4 overexpression reversed the effects of METTL3 silencing on the proliferation and migration as well as the levels of angiogenic factors in HCC cells. Conclusion: This study reveals the upregulation of METTL3 and HERC4 expression in HCC and their role in HCC by enhancing the proliferation, migration, and angiogenesis potential of HCC cells.

Crispr-Cas9-based long non-coding RNA interference and activation identified that the aberrant expression of Myc-regulated ST8SIA6 antisense RNA 1 promotes tumorigenesis and metastasis in hepatocellular carcinoma

Objective: Long non-coding RNAs (lncRNAs) participate in the formation, progression, and metastasis of cancer. This study aimed to explore the roles of the lncRNA ST8SIA6 antisense RNA 1 (ST8SIA6-AS1) in tumorigenesis and elucidate the underlying molecular mechanism of its upregulation in hepatocellular carcinoma (HCC). Material and Methods: A total of 56 in-house pairs of HCC tissues were examined, and ST8SIA6-AS1 levels were determined through real-time polymerase chain reaction (PCR). The biological behavior of ST8SIA6-AS1 by Crispr-Cas9-based gene repression and activation was determined in vitro and in vivo. The binding sites and biological behavior of Myc proto-oncogene and forkhead box A on chromatin were investigated through luciferase reporter assays, chromatin immunoprecipitation–quantitative PCR, and co-immunoprecipitation (co-IP) assays. The regulatory mechanisms of ST8SIA6-AS1 expression were analyzed with encyclopedia of DNA elements and gene expression profiling interactive analysis. Results: The expression of ST8SIA6-AS1 significantly increased in multiple HCC cell lines and the 56 in-house pairs of HCC tissues (P = 0.0018). Functionally, high-efficiency Crispr-Cas9-based knockdown of ST8SIA6-AS1 revealed that ST8SIA6-AS1 knockdown attenuated the proliferation, migration, and infiltration of HCC cells and considerably reduced the growth rate of subcutaneous and orthotopic HCC tumors. Conversely, ST8SIA6-AS1 overexpression considerably improved the oncogenic characteristics of the HCC cells. Furthermore, ST8SIA6-AS1 upregulation was regulated by the direct binding of transcription factor Myc to the −260 bp to+155 bp and +1003 bp to +1312 bp regions of the ST8SIA6-AS1 transcription start site, which is a segment with high level of H3K27 acetylation. Myc knockdown or treatment with the BET bromodomain inhibitor JQ-1 considerably reduced ST8SIA6-AS1 RNA expression in the HCC cells. Conclusion: Our study has established the oncogenic role of ST8SIA6-AS1 and elucidated the Myc-dependent upregulation mechanism of ST8SIA6-AS1 in HCC, providing a profound theoretical molecular basis for the carcinogenic function of ST8SIA6-AS1 in HCC.

KRT80 in hepatocellular carcinoma plays oncogenic role via epithelial-mesenchymal transition and PI3K/AKT pathway.

Hepatocellular carcinoma (HCC), a globally prevalent form of cancer, is featured by aggressive growth and early metastasis. Elucidating the underlying mechanism and identifying the effective therapy are critical for advanced HCC patients. In the study, we detect that KRT80 was upregulated in HCC samples. HCC patients with higher KRT80 are associated with worse overall survival after surgery. Gain-of and loss-of function studies show that KRT80 enhanced HCC cells proliferation, migration, invasion, and angiogenesis, whereas its silencing abolishes the effects in vivo and in vitro. Mechanistic investigation shows that KRT80 may function as an independent prognostic risk factor and act as an oncogene by influencing EMT and modulating the PI3K/AKT signaling pathway. Together, these findings suggest that KRT80 may be a potential oncogene and a good indicator in predicting prognosis. Targeting KRT80 can offer new insights into the prevention and treatment of HCC.

TMSB4X is a regulator of inflammation-associated ferroptosis, and promotes the proliferation, migration and invasion of hepatocellular carcinoma cells

BackgroundFerroptosis and inflammation are involved in cancer progression. The aim of this study was to identify inflammation-associated ferroptosis regulators in hepatocellular carcinoma (HCC).MethodsFerrDb database was searched for ferroptosis-related genes. RNA sequencing data and clinicopathologic information of HCC patients were downloaded from the Cancer Genome Atlas (TCGA) database. Weighted gene co-expression network analysis was applied to obtain the genes probably involved in inflammation-associated ferroptosis. Univariate Cox regression analysis was conducted to screen prognostic genes, and 10 machine learning algorithms were combined to find the optimal strategy to evaluate the prognosis of the patients based on the prognosis-related genes. The patients were divided into high risk group and low risk group, and the differentially expressed genes were obtained. Thymosin beta 4 X-linked (TMSB4X) was overexpressed or knocked down in HCC cell lines, and then qPCR, CCK-8, Transwell, flow cytometery assays were performed to detect the change of HCC cells’ phenotypes, and Western blot was used to detect the change of ferroptosis markers.Results157 genes related to inflammation and ferroptosis in HCC were obtained by WGCNA. rLasso algorithm, with the highest C-index, screened out 29 hub genes, and this model showed good efficacy to predict the prognosis of HCC patients. The patients in high risk group and low risk groups showed distinct molecular characteristics. TMSB4X was the most important gene which dominated the classification, and it was highly expressed in HCC samples. TMSB4X promoted the viability, migration and invasion, and repressed ferroptosis of HCC cells.ConclusionThe risk model constructed based on the inflammation-associated ferroptosis regulators is effective to predict the clinical outcome of HCC patients. TMSB4X, involved in inflammation-associated ferroptosis, is a potential biomarker and therapeutic target for HCC.

USP11 promotes lipogenesis and tumorigenesis by regulating SREBF1 stability in hepatocellular carcinoma

BackgroundThe relationship between hepatocellular carcinoma (HCC) metastasis and cancer metabolism reprogramming is becoming increasingly evident. Ubiquitin-specific protease 11 (USP11), a member of the deubiquitinating enzyme family, has been linked to various cancer-related processes. While USP11 is known to promote HCC metastasis and proliferation, the precise mechanisms, especially those related to cancer metabolism, remain unclear.MethodsThrough mass spectrometry, co-immunoprecipitation, immunofluorescence, and ubiquitination assays, we identified USP11 as the key deubiquitinase for SREBF1.Lipogenesis was evaluated using Oil Red O and Nile Red staining, along with the detection of triglycerides and cholesterol. To assess HCC cell proliferation, migration, and invasion in vitro, Transwell assays, EDU, colony formation, and CCK-8 were conducted. Xenograft models in nude mice were developed to verify the role of the USP11/SREBF1 axis in lipogenesis and tumor growth in vivo.ResultsUSP11 directly interacts with SREBF1, and its silencing leads to the disruption of SREBF1 stabilization through K48-linked deubiquitination and degradation. Importantly, the truncated mutant USP11 (503–938 aa) interacts with the truncated mutant SREBF1 (569–1147aa), with K1151 playing a crucial role in this interaction. Higher levels of USP11 enhance lipogenesis, proliferation, and metastasis in HCC cells. Importantly, the knockdown of SREBF1 weakened the effects of USP11 in enhancing lipogenesis and tumorigenesis. Futhermore, the elevated expression of USP11 and SREBF1 in HCC tissue serves as an indicator of poor prognosis in HCC patients.ConclusionsIn summary, our study reveals that USP11 promotes HCC proliferation and metastasis through SREBF1-induced lipogenesis. These findings provide a foundation for novel therapies targeting lipid metabolism in HCC.

RPS21 Enhances hepatocellular carcinoma development through GPX4 stabilization

The study highlights that RPS21, a gene encoding a component of the 40S ribosomal subunit, plays an oncogenic role in hepatocellular carcinoma (HCC) and may influence tumor aggressiveness by affecting antioxidant capacity. RPS21 was found to be upregulated in HCC through RNA-sequencing of clinical samples and analysis of the TCGA database. Kaplan-Meier survival analyses linked higher RPS21 expression to lower survival rates across multiple metrics (OS, PFS, RFS, DSS). Mutation analysis via the cBioPortal showed that primarily amplifications in RPS21 are associated with a poorer prognosis. Tissue microarrays confirmed higher RPS21 levels in tumor samples, which were associated with more advanced clinical stages and grades. Experimental interventions involving lentiviral knockdown or overexpression of RPS21 significantly altered HCC cell proliferation and migration. These findings were supported by mouse models, which showed impacts on tumor growth and metastasis. Further mechanistic studies indicated that RPS21 modulates the ubiquitination and stability of GPX4, a key player in ferroptosis and oxidative stress regulation in HCC cells. This comprehensive study, which merges bioinformatic analysis with laboratory research, positions RPS21 as a viable target for HCC therapy and opens new pathways for understanding and treating liver cancer.

Gallein, G protein βγ subunits inhibitor, suppresses the TGF-α-induced migration of hepatocellular carcinoma cells via inhibition of the c-Jun N-terminal kinase.

G protein-coupled receptor (GPCR) signaling regulates a wide range of pathophysiological cell functions via G protein α and βγ subunits. Small molecules targeting the subunits of Gα and Gβγ have been developed as cancer therapeutics. We have previously reported that transforming growth factor-α (TGF-α) induces the migration of human hepatocellular carcinoma (HCC) HuH7 cells through the activation of AKT, p38 mitogen-activated protein kinase (MAPK), Rho-kinase and c-Jun N-terminal kinase (JNK). This study aims to determine whether Gβγ subunits regulate the TGF-α-induced migration of HCC HuH7 cells using gallein, a Gβγ subunits inhibitor. The Janus family of tyrosine kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway was also involved in the regulation of the migration. Gallein significantly reduced the TGF-α-induced cell migration. In contrast, fluorescein, a gallein-related compound that has no effect on Gβγ subunits, failed to affect the cell migration. Gallein suppressed the TGF-α-stimulated phosphorylation of JNK without affecting the phosphorylation of epidermal growth factor receptor, AKT, p38 MAPK, target protein of Rho-kinase and STAT3. Conversely, fluorescein did not attenuate the phosphorylation of JNK. These results strongly suggest that Gβγ subunits act as positive regulators in TGF-α-induced migration of HCC cells via the JNK signalling pathway.

CAF-derived miR-642a-3p supports migration, invasion, and EMT of hepatocellular carcinoma cells by targeting SERPINE1.

Cancer-associated fibroblasts (CAFs) and hepatocellular carcinoma (HCC) cells interact to promote HCC progression, but the underlying mechanisms remain unclear. Serpin family E member 1 (SERPINE1) has conflicting roles in HCC, and microRNAs (miRNAs) are known to regulate tumor progression through intercellular communication. Therefore, we investigated the potential involvement of miRNA/SERPINE1 axis in crosstalk between CAFs and HCC cells. In this study, candidate miRNAs targeting SERPINE1 3' UTR were predicted using multiple miRNA databases. The miRNAs and SERPINE1 mRNA expression in Huh7 cells was assessed after co-culture with CAFs using RT-qPCR. Huh7 cell proliferation and invasion were detected after SERPINE1 siRNA. The functions of the CAF-derived miR-642a-3p/SERPINE1 axis in HCC cells were examined using CCK-8, wound healing, transwell assays, western blot, and dual-luciferase reporter assays. Moreover, a orthotopic xenograft model was used to investigate the contribution of miR-642a-3p knockdown in HCC. SERPINE1 mRNA expression decreased, while miR-642a-3p expression increased in Huh7 cells co-cultured with CAFs. SERPINE1 knockdown enhanced Huh7 cell proliferation and invasion as well as miR-642a-3p expression. miR-642a-3p overexpression promoted migration, invasion, and epithelial-mesenchymal transition (EMT) in Huh7 cells by targeting SERPINE1, while miR-642a-3p knockdown yielded the opposite effect. Rescue experiments confirmed that SERPINE1 knockdown attenuated the inhibitory effects of miR-642a-3p knockdown on migration, invasion, and EMT in Huh7 cells. Importantly, miR-642a-3p knockdown suppressed growth and EMT in orthotopic liver tumors. CAF-derived miR-642a-3p/SERPINE1 axis facilitated migration, invasion, and EMT in the HCC cells, suggesting miR-642a-3p/SERPINE1 axis can be a potential therapeutic target for HCC.

Comprehensive analysis of GPN1 in human cancer and its effects on the migration of hepatocellular carcinoma cells.

To investigate the prognostic value of GPN1 in cancer and its role in the migration of hepatocellular carcinoma (HCC or LIHC) cells, we used several databases to assess GPN1 expression levels and effects in human tumors. Furthermore, experiments were conducted to verify changes in GPN1 expression in HCC cell lines and explore its biological function. We found that GPN1 gene and protein expression were significantly increased in several tumor tissues. Higher GPN1 expression was associated with unfavorable overall survival. Additionally, there was a strong association between GPN1 expression and several clinicopathological features, according to multivariate Cox regression analysis. Moreover, GPN1 gene mutation and methylation were present in some tumors. A relationship was also found between GPN1 expression and immune infiltration. Notably, immune checkpoint analysis showed that GPN1 expression was correlated with PD-1/PDL-1 and CTLA-4, suggesting it may serve as a biomarker for predicting immune subtypes and response to immunotherapy in HCC. Enrichment analysis in HCC indicated that GPN1 is primarily involved in RNA metabolism. Additionally, drug sensitivity analysis revealed that GPN1 appeared to be responsive to 16 drugs. Finally, GPN1 upregulation was confirmed to promote the migration of HCC cells. This study provides a comprehensive overview of GPN1 in human cancer and demonstrates that GPN1 contributes to the migration of HCC cells, potentially serving as a prognostic and immunotherapy biomarker.

Fibroblast Heterogeneity in Hepatocellular Carcinoma and Identification of Prognostic Markers Based on Single-cell Transcriptome Analysis.

HCC is a malignant tumor with high morbidity and mortality. Fibroblasts play a key role in the tumor microenvironment (TME). However, the transcriptional regulatory mechanisms of fibroblasts remained unclear in HCC. The aim of this study was to explore the complex role of fibroblasts in hepatocellular carcinoma (HCC) and to reveal their transcriptional regulatory mechanisms. The goal of this study was to discover potential prognostic markers for HCC by analyzing the genetic variations and differentiation process of fibroblasts. Single-cell transcriptome data from the non-tumor liver site and primary tumor site of HCC were acquired from GSE149614, processed, and clustered using the Seurat pipeline. The inferCNV algorithm was applied to infer copy number variations (CNVs) in fibroblasts. Subsequently, the mechanism underlying the interaction between fibroblasts and other cells in the TME of HCC was analyzed using CellChat software. The trajectory of cellular differentiation of fibroblasts from normal state to malignant state was examined using Monocle 2. SCENIC analysis was performed to identify key transcription factors (TFs) in fibroblasts and assess their correlation with HCC prognosis. Finally, qRT-PCR and Transwell assays were carried out to analyze the mRNA expression and cell metastasis. We identified a total of nine different cell types (B cells, cycling cells, endothelial cells, epithelial cells, fibroblasts, hepatocytes, macrophages, plasma cells, and T cells) based on the single-cell transcriptomic data of HCC. Among them, fibroblasts were highly enriched at the primary tumor site, and their number increased with advanced stages. In addition, significant deletions were detected on chromosome 6p of fibroblasts, and genes in this region were remarkably enriched in pathways associated with antigen processing and presentation. Intercellular communication showed that epithelial cells regulated fibroblasts the most. The differentiation of fibroblasts was mainly accompanied by a transition from normal to malignant state. Importantly, CEBPD and FOSB, the TFs most associated with the putative timing of fibroblasts, were under-expressed in human hepatocytes and showed a significant correlation with HCC prognosis. Overexpressed CEBPD inhibited HCC cell migration and invasion. In conclusion, our study revealed that fibroblast recruitment and differentiation, as well as copy number loss at chromosome 6p, were associated with a higher degree of malignancy and immune dysfunction in HCC. The current discoveries provided new insights into the clinical treatment and diagnosis of HCC.

FKBP4 promotes glycolysis and hepatocellular carcinoma progression via p53/HK2 axis.

FKBP4, a member of the FK506-binding protein (FKBP) family, is a promising target for a variety of disorders, including cancer. However, its underlying molecular mechanism and potential function in hepatocellular carcinoma (HCC) are largely elusive. Therefore, we aimed to investigate the expression status, functional implications and underlying mechanisms of FKBP4 in HCC. Our bioinformatics analysis of TCGA LIHC datasets, ICGC LIRI-JP datasets and GEO datasets results showed FKBP4 was upregulated in HCC tissues. We also confirmed the elevated FKBP4 in clinical HCC samples. Additionally, quantitative RT-PCR results revealed FKBP4 was highly expressed in all five tested HCC cell lines. We also observed a correlation between elevated FKBP4 expression and poor prognosis in HCC patients. Loss of FKBP4 can inhibit the proliferation and migration in HCC cells. Furthermore, we found that silencing FKBP4 suppressed glucose uptake, lactic acid production and18F-FDG uptake compared with the control group. Mechanistically, our funding indicated that FKBP4 participates in glycolysis through p53 mediated HK2 signaling pathway, specially, FKBP4 knockdown promotes the expression and stability of p53 protein rather than affecting the transcription level. Finally, rescue experiments revealed that simultaneous knockdown of both FKBP4 and p53 partially reversed the inhibitory effects on HK2 protein levels and18F-FDG uptake. Our study elucidates a novel role of FKBP4 in promoting HCC development and glycolysis by modulating the p53/HK2 signaling pathway. Given the critical role of aerobic glycolysis in the progression of HCC, targeting FKBP4 may offer a new therapeutic strategy for treating this malignancy.

Integrative analysis based on ATAC-seq and RNA-seq reveals a novel oncogene PRPF3 in hepatocellular carcinoma.

Assay of Transposase Accessible Chromatin Sequencing (ATAC-seq) is a high-throughput sequencing technique that detects open chromatin regions across the genome. These regions are critical in facilitating transcription factor binding and subsequent gene expression. Herein, we utilized ATAC-seq to identify key molecular targets regulating the development and progression of hepatocellular carcinoma (HCC) and elucidate the underlying mechanisms. We first compared chromatin accessibility profiles between HCC and normal tissues. Subsequently, RNA-seq data was employed to identify differentially expressed genes (DEGs). Integrating ATAC-seq and RNA-seq data allowed the identification of transcription factors and their putative target genes associated with differentially accessible regions (DARs). Finally, functional experiments were conducted to investigate the effects of the identified regulatory factors and corresponding targets on HCC cell proliferation and migration. Enrichment analysis of DARs between HCC and adjacent normal tissues revealed distinct signaling pathways and regulatory factors. Upregulated DARs in HCC were enriched in genes related to the MAPK and FoxO signaling pathways and associated with transcription factor families like ETS and AP-1. Conversely, downregulated DARs were associated with the TGF-β, cAMP, and p53 signaling pathways and the CTCF family. Integration of the datasets revealed a positive correlation between specific DARs and DEGs. Notably, PRPF3 emerged as a gene associated with DARs in HCC, and functional assays demonstrated its ability to promote HCC cell proliferation and migration. To the best of our knowledge, this is the first report highlighting the oncogenic role of PRPF3 in HCC. Furthermore, ZNF93 expression positively correlated with PRPF3, and ChIP-seq data indicated its potential role as a transcription factor regulating PRPF3 by binding to its promoter region. This study provides a comprehensive analysis of the epigenetic landscape in HCC, encompassing both chromatin accessibility and the transcriptome. Our findings reveal that ZNF93 promotes the proliferation and motility of HCC cells through transcriptional regulation of a novel oncogene, PRPF3.