Hepatocellular Carcinoma Tissue Specimens Research Articles

Prognostic role of macrophages and mast cells in the microenvironment of hepatocellular carcinoma after resection

BackgroundThe prognostic significance of mast cells and different phenotypes of macrophages in the microenvironment of hepatocellular carcinoma (HCC) following resection is unclear. We aimed in this study to assess the local distribution of infiltrating macrophages and mast cells of specific phenotypes in tissues of HCC and to evaluate their prognostic values for survival of post-surgical patients.MethodsThe clinicopathological and follow-up data of 70 patients with HCC, who underwent curative resection of tumor from 1997 to 2019, were collected. The infiltration of CD68+ and CD163+ macrophages and CD117+ mast cells was assessed immunohistochemically in representative resected specimens of HCC and adjacent tissues. The area fraction (AF) of positively stained cells was estimated automatically using QuPath image analysis software in several regions, such as tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells, individually and in associations, for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) was evaluated using Kaplan-Meier and Cox regression analyses.ResultsHigh AF of CD68+ macrophages in TC and IM and high AF of mast cells in IM and PT area were associated with a longer DFS. High AF of CD163+ macrophages in PT area correlated with a shorter DFS. Patients from CD163TChigh & CD68TClow group had a shorter DFS compared to all the rest of the groups, and cases with CD163IMlow & CD68IMhigh demonstrated significantly longer DFS compared to low AF of both markers. Patients from CD68IMhigh & CD163PTlow group, CD117IMhigh & CD163PTlow group, and CD117PThigh & CD163PTlow group had a significantly longer DFS compared to all other combinations of respective cells.ConclusionsThe individual prognostic impact of CD68+ and CD163+ macrophages and mast cells in the microenvironment of HCC after resection depends on their abundance and location, whereas the cumulative impact is built upon combination of different cell phenotypes within and between regions.

Correlation Between Low Cytoplasmic Expression of XBP1 and the Likelihood of Surviving Hepatocellular Carcinoma.

Our objectives in this study were to (i) evaluate the clinical significance of X-box-binding protein 1 (XBP1) expression in cases of hepatocellular carcinoma (HCC) and (ii) assess the potential of XBP1 to be used as a prognostic biomarker. The expression of XBP1 protein in 267 HCC tissue specimens was measured using immunohistochemistry in order to characterize the associations among XBP1 expression, clinicopathological factors and survival outcomes. Survival analysis using follow-up data was used to assess the prognostic value of XBP1 in cases of HCC. Immunohistochemistry revealed a significant decrease in cytoplasmic XBP1 protein expression in HCC tumor tissue. Immunoreactivity results showed that low cytoplasmic XBP1 expression was significantly associated with vascular invasion, as well as poor 5-year overall survival and long-term disease-specific (DSS) and disease-free (DFS) survival rates. Kaplan-Meier survival curves further confirmed a significant association between low cytoplasmic XBP1 protein expression and poor DSS and DFS. Univariate and multivariate analyses revealed that XBP1 expression, tumor differentiation, vascular invasion, tumor stage, and the rate of recurrence were linked to DSS, while low cytoplasmic XBP1 expression remained an independent predictor of poor DSS. Our analysis also revealed that XBP1 expression, tumor differentiation, vascular invasion, and T classification were linked to DFS, while low cytoplasmic XBP1 expression remained an independent predictor of poor DFS. Low cytoplasmic XBP1 protein expression may play an important role in the pathogenesis of HCC, which suggests that XBP1 could potentially be targeted to benefit therapeutic strategies for HCC.

SIRT1 Inhibition-Induced Mitochondrial Damage Promotes GSDME-Dependent Pyroptosis in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is a malignant tumor that affects the liver and poses a significant threat to human health. Further investigation is necessary to fully understand the role of SIRT1, a protein linked to tumorigenesis, in HCC development. To investigate the effect of SIRT1 on HCC and elucidate the underlying mechanism. Eight pairs of HCC and paracancerous normal tissue specimens were collected. The levels of SIRT1 and GSDME in tissue samples were assessed using immunohistochemistry and western blotting. SIRT1 levels were determined in HCC (Huh7, HepG2, SNU-423, SNU-398, and HCCLM3) and L-02 cells using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. SNU-423 and HCCLM3 cells were transfected with si-SIRT1 and/or si-GSDME to knock down SIRT1 or GSDME expression. RT-qPCR and western blotting were performed to measure the expression of SIRT1, pro-casp-3, cl-casp-3, GSDME, GSDME-N, PGC-1α, Bax, and cytochrome c (Cyto C). Cell proliferation, migration, invasion, and apoptosis were assessed using the cell counting kit-8 (CCK-8), wound healing assay, Transwell invasion assay, and flow cytometry, respectively. The release of lactate dehydrogenase (LDH) was evaluated using an LDH kit. SIRT1 was upregulated in HCC tissues and cells, and a negative correlation was observed between SIRT1 and GSDME-N. SIRT1 silencing suppressed the proliferation, migration, and invasion of HCC cells while also promoting apoptosis and inducing mitochondrial damage. Additionally, the silencing of SIRT1 resulted in the formation of large bubbles on the plasma membrane of HCC cells, leading to cellular swelling and aggravated GSDME-dependent pyroptosis, resulting in an increase in LDH release. Inhibition of GSDME reduced SIRT1 silencing-induced cell swelling, decreased LDH release rate, and promoted apoptosis. SIRT1 silencing promotes GSDME-dependent pyroptosis in HCC cells by damaging mitochondria.

High Indoleamine 2,3-Dioxygenase Expression along with Low Bridging Integrator-1 Expression in Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) adopts several tumor immune escape mechanisms; therefore, it has the potential to be targeted by immunotherapy. Indoleamine 2, 3-dioxygenase (IDO) is an immunosuppressive enzyme that has been observed to be overexpressed in HCC patients with poor prognoses. Bridging integrator 1 (Bin1) loss promotes immune escape in cancer by deregulating IDO. Our aim is to investigate IDO expression along with Bin1 expression to find evidence of immunosuppression in HCC patients. In this study, we investigated IDO and Bin1 expression in HCC tissue specimens and the correlation of IDO and Bin1 expression with clinicopathological characteristics and prognosis of HCC patients (n=45). Immunohistochemical analysis was performed to analyze the expression of IDO and Bin1. IDO was overexpressed in 38 (84.4%) out of 45 HCC tissue specimens. In addition, tumor size was significantly increased with an increase in the IDO expression (P=0.03). Low Bin1 expression was observed in 27(60%) HCC tissue specimens, whereas the remaining 18(40%) showed high Bin1 expression. Our data showed that expression of IDO along with Bin1 expression could be investigated for clinical evaluation in HCC. IDO might be used as an immunotherapeutic target for HCC. Therefore, further studies in larger patient cohorts are warranted.

Activation of ACLY by SEC63 deploys metabolic reprogramming to facilitate hepatocellular carcinoma metastasis upon endoplasmic reticulum stress

BackgroundTumor cells display augmented capability to maintain endoplasmic reticulum (ER) homeostasis and hijack ER stress pathway for malignant phenotypes under microenvironmental stimuli. Metabolic reprogramming is a well-known hallmark for tumor cells to provide specific adaptive traits to the microenvironmental alterations. However, it’s unknown how tumor cells orchestrate metabolic reprogramming and tumor progression in response to ER stress. Herein, we aimed to explore the pivotal roles of SEC63-mediated metabolic remodeling in hepatocellular carcinoma (HCC) cell metastasis after ER stress.MethodsThe expression levels of SEC63 in HCC tissues and adjacent non-cancerous tissues were determined by immunohistochemistry and western blot. The regulatory roles of SEC63 in HCC metastasis were investigated both in vitro and in vivo by RNA-sequencing, metabolites detection, immunofluorescence, and transwell migration/invasion analyses. GST pull-down, immunoprecipitation/mass spectrometry and in vivo ubiquitination/phosphorylation assay were conducted to elucidate the underlying molecular mechanisms.ResultsWe identified SEC63 as a new regulator of HCC cell metabolism. Upon ER stress, the phosphorylation of SEC63 at T537 by IRE1α pathway contributed to SEC63 activation. Then, the stability of ACLY was upregulated by SEC63 to increase the supply of acetyl-CoA and lipid biosynthesis, which are beneficial for improving ER capacity. Meanwhile, SEC63 also entered into nucleus for increasing nuclear acetyl-CoA production to upregulate unfolded protein response targets to improve ER homeostasis. Importantly, SEC63 coordinated with ACLY to epigenetically modulate expression of Snail1 in the nucleus. Consequently, SEC63 promoted HCC cell metastasis and these effects were reversed by ACLY inhibition. Clinically, SEC63 expression was significantly upregulated in HCC tissue specimens and was positively correlated with ACLY expression. Importantly, high expression of SEC63 predicted unfavorable prognosis of HCC patients.ConclusionsOur findings revealed that SEC63-mediated metabolic reprogramming plays important roles in keeping ER homeostasis upon stimuli in HCC cells. Meanwhile, SEC63 coordinates with ACLY to upregulate the expression of Snail1, which further promotes HCC metastasis. Metastasis is crucial for helping cancer cells seek new settlements upon microenvironmental stimuli. Taken together, our findings highlight a cancer selective adaption to ER stress as well as reveal the potential roles of the IRE1α-SEC63-ACLY axis in HCC treatment.

Expression and Correlation of MIF and ERK1/2 in Liver Cirrhosis and Hepatocellular Carcinoma Induced by Hepatitis B.

To detect expression and phosphorylation level of macrophage migration inhibitor (MIF) and extracellular-regulated kinases 1 and 2 (ERK1/2) in hepatitis B-induced liver cirrhosis (HBILC) and hepatocellular carcinoma (HCC) with a background of HBILC and analyze the correlation of MIF and ERK1/2 with HBILC and HCC. Twenty cases of normal liver tissues were collected as a control group, and 48 specimens of HBILC tissues and 48 specimens of HCC tissues were collected as the experimental group, which were assigned as the HBILC group and HCC group, respectively. All tissue specimens were processed into tissue chips. The expressions of MIF, ERK1/2, and their phosphorylated proteins were detected via immunohistochemistry, and MIF and ERK1/2 nucleic acid expressions were detected by in situ hybridization. The results were statistically analyzed using the chi-square test. Proteins and nucleic acids of MIF and ERK1/2 presented low expression in the control group and high expression in the HBILC group and HCC group. MIF expression in the three groups was 25.0%, 75.0%, and 79.17%, respectively, while that of the nucleic acids was 25.0%, 70.83%, and 68.75%, respectively. Expression of ERK1/2 in the three groups was 40.0%, 60.42%, and 81.25%, respectively, and that of nucleic acids was 40.0%, 79.17%, and 77.08%. Expression of pERK1/2 was low in the control and HBILC group and high in the HCC group. Expression of pERK1/2 in the three groups was 20%, 45.83%, and 75%, respectively. Expression of pERK1/2 in the HCC group was significantly different from that in the HBILC and control group (P<0.05), but the difference between the HBILC group and control group was not statistically significant (P>0.05). Occurrence and development of HBILC and HCC are not only related to the high expression of MIF but also closely related to the activation of the ERK1/2 signaling pathway.

CKLF as a Prognostic Biomarker and Its Association with Immune Infiltration in Hepatocellular Carcinoma.

The Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing (CMTM) family, comprising nine members, is involved in the tumorigenesis and progression of various cancers. However, the expression profiles and clinical significance of CMTM family members in hepatocellular carcinoma (HCC) are not fully clarified. In this study, the RNA-sequencing and clinical data were downloaded from The Cancer Genome Atlas (TCGA) databases. The Kaplan-Meier method and the Cox proportional hazards regression analysis were used to evaluate the prognostic significance of CMTM family members. Single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE algorithms were employed to explore the relationship between CMTM family genes and the tumor microenvironment in HCC. Finally, the prognostic CMTM family gene expression was further validated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical (IHC) staining in clinical HCC tissue specimens. The results indicated that, compared with normal tissues, the expression of CKLF, CMTM1, CMTM3, CMTM4, CMTM7, and CMTM8 were significantly upregulated in HCC, while the expression of CMTM2, CMTM5, and CMTM6 were significantly downregulated in HCC. Univariate and multivariate Cox regression analysis demonstrated that CKLF was an independent prognostic biomarker for the overall survival (OS) of HCC patients. In HCC, the expression of CKLF was found to be correlated with immune cell infiltration, immune-related functions, and immune checkpoint genes. The qRT-PCR and IHC confirmed that CKLF was highly expressed in HCC. Overall, this research suggested that CKLF is involved in immune cell infiltration and may serve as a critical prognostic biomarker, which provides new light on the therapeutics for HCC.

A novel risk score based on immune-related genes for hepatocellular carcinoma as a reliable prognostic biomarker and correlated with immune infiltration.

Immunological-related genes (IRGs) play a critical role in the immune microenvironment of tumors. Our study aimed to develop an IRG-based survival prediction model for hepatocellular carcinoma (HCC) patients and to investigate the impact of IRGs on the immune microenvironment. Differentially expressed IRGs were obtained from The Genomic Data Commons Data Portal (TCGA) and the immunology database and analysis portal (ImmPort). The univariate Cox regression was used to identify the IRGs linked to overall survival (OS), and a Lasso-regularized Cox proportional hazard model was constructed. The International Cancer Genome Consortium (ICGC) database was used to verify the prediction model. ESTIMATE and CIBERSORT were used to estimate immune cell infiltration in the tumor immune microenvironment (TIME). RNA sequencing was performed on HCC tissue specimens to confirm mRNA expression. A total of 401 differentially expressed IRGs were identified, and 63 IRGs were found related to OS on the 237 up-regulated IRGs by univariate Cox regression analyses. Finally, five IRGs were selected by the LASSO Cox model, including SPP1, BIRC5, STC2, GLP1R, and RAET1E. This prognostic model demonstrated satisfactory predictive value in the ICGC dataset. The risk score was an independent predictive predictor for OS in HCC patients. Immune-related analysis showed that the immune infiltration level in the high-risk group was higher, suggesting that the 5-IRG signature may play an important role in mediating immune escape and immune resistance in the TIME of HCC. Finally, we confirmed the 5-IRG signature is highly expressed in 65 HCC patients with good predictive power. We established and verified a new prognosis model for HCC patients based on survival-related IRGs, and the signature could provide new insights into the prognosis of HCC.

Secretome of senescent hepatoma cells modulate immune cell fate by macrophage polarization and neutrophil extracellular traps formation

Presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis which finally culminates in liver cancer. We now report the presence of senescent hepatocytes (p21 and p53 positive) in the vicinity of infiltrated immune cells in hepatocellular carcinoma tissue specimens by immunohistochemistry. Hence, we evaluated in vitro, the relevance of senescent hepatoma cells in altering the fate of monocytes and neutrophils by assaying for macrophage polarization and extracellular trap (NETs) formation, respectively. Premature senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by treating cells with doxorubicin. Senescent hepatoma cells showed strong inflammatory phenotype with induced expression of cytokines (IL1β, IL6, IL8 and IL13) as evaluated by flow cytometry. The senescent secretome from hepatoma cells when incubated with healthy monocytes caused it to differentiate predominantly towards M2 fate (CD80low CD86low CD163high CD206high) when analysed by flow cytometry. This was corroborated by the finding in clinical samples where human hepatocellular carcinoma harbouring senescent hepatocytes showed presence of M2 macrophages, while M1 macrophages were predominant in non-tumorous region. Additionally, the senescent secretome from Huh7 cells enhanced the NETs formation, while HepG2 secretome had an inhibitory effect. In conclusion, the "pro-inflammatory" senescent secretome drives non-inflammatory type M2 macrophage polarization and modulated neutrophil traps which in turn can influence the tumor microenvironment.

MicroRNA214 expression inhibits HCC cell proliferation through PTK2b/ Pyk2.

MicroRNAs (miRNAs/miRs) are crucial regulatory molecules that act as the most significantly downregulated microRNAs in hepatocellular carcinoma (HCC). PTK2b/Pyk2 is a non-receptor protein tyrosine kinase, which plays an important role in the development and metastasis of cancer. In this study, we explored the expression level and functional relationship between MicroRNA-214 (miR-214) and PTK2b/ Pyk2 in liver cancer cells. For this purpose, we analyzed the expression of miR-214 and PTK2b/Pyk2 in 38 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR. MTT, cell cycle and construct recombinant plasmids analysis were used to explore the effects of miR-214 and PTK2b/Pyk2 on liver cancer cell proliferation. Results showed that the expression level of mir-214 in liver cancer tissues and liver cancer cell lines was significantly lower than that in normal tissues and cells, while the expression of PTK2b/Pyk2 was significantly increased. The overexpression of mir-214 or inhibition PTK2b/Pyk2 inhibited the proliferation of HCC cells. This research showed that mir-214 has an inhibitory effect on liver cancer through the expression of PTK2b/Pyk2.

CDK1, CCNB1 and NDC80 are associated with prognosis and progression of hepatitis B virus-associated hepatocellular carcinoma: a bioinformatic analysis

To identify the key genes involved in the transformation of hepatitis B virus (HBV) into hepatocellular carcinoma (HCC) and explore the underlying molecular mechanisms. We analyzed the mRNA microarray data of 119 HBV-related HCC tissues and 252 HBV-related non-tumor tissues in GSE55092, GSE84044 and GSE121248 from the GEO database, and the "sva" R package was used to remove the batch effects. Integration analysis was performed to identify the differentially expressed genes (DEGs) in HBV-related liver cancer and liver tissues with HBV infection. The significant DEGs were functionally annotated using GO and KEGG analyses, and the most important modules and hub genes were explored with STRING analysis. Kaplan-Meier and Oncomine databases were used to verify the HCC gene expression data in the TCGA database to explore the correlations of the hub genes with the occurrence, progression and prognosis of HCC. We also examined the expressions of the hub genes in 17 pairs of surgical specimens of HCC and adjacent tissues using RT-qPCR. We identified a total of 121 DEGs and 3 genetic markers in HCC (P < 0.01). These DEGs included cyclin1 (CDK1), cyclin B1 (CCNB1), and nuclear division cycle 80 (NDC80), which participated in cell cycle, pyrimidine metabolism and DNA replication and were highly correlated (P < 0.05). Analysis of the UALCAN database confirmed high expressions of these 3 genes in HCC tissues, which were correlated with a low survival rate of the patients, as shown by Kaplan-Meier analysis of the prognostic data from the UALCAN database. CDK1, CCNB1 and NDC80 were all correlated with the clinical grading of HCC (P < 0.05). The results of RT-qPCR on the surgical specimens verified significantly higher expressions of CDK1, CCNB1 and NDC80 mRNA in HCC tissues than in the adjacent tissues. CDK1, CCNB1 and NDC80 genes can be used as prognostic markers of HBV-related HCC and may serve as potential targets in preclinical studies and clinical treatment of HCC.

MicroRNA-424 inhibits autophagy and proliferation of hepatocellular carcinoma cells by targeting ATG14

To determine whether miR-424 affects cancer cell proliferation and autophagy through ATG14 in hepatocellular carcinoma (HCC) cells. We detected miR-424-5p and ATG14 expression levels in surgical specimens of HCC and adjacent tissues and in different HCC cell lines (HepG2, SMMC-7721, Huh-7, MHCC97H, and HCCLM3) and normal human hepatocyte LO2 cells using qRT-PCR and Western blotting. In the cell transfection experiments, we observed the effects of miR-424-5p knockdown in Huh-7 cells and the effects of overexpression miR-424-5p and ATG14 in HCCLM3 cells on the proliferation, cell cycle, apoptosis and expression levels of autophagy-related proteins (LC3, Beclin1 and p62). Dual luciferase reporter assay was used to verify the possible interaction between miR-424-5p and ATG14. In HCC tissues and cells, ATG14 was highly expressed and miR-424-5p expression was downregulated. In HCC cells, overexpression of miR-424-5p obviously suppressed cell proliferation and promoted cell apoptosis (P < 0.05), while inhibiting miR-424-5p or overexpressing ATG14 significantly promoted cell proliferation and inhibited cell apoptosis (P < 0.05). Dual luciferase reporter assay indicated that miR-424-5p inhibits HCC cells by targeting ATG14. In addition, inhibition of miR-424-5p and overexpression of ATG14 both enhanced the expressions of LC3-ΙΙ/LC3-Ι and Beclin1 and decreased p62 expression (P < 0.05), but miR-424-5p overexpression reduced the expressions of LC3-ΙΙ/LC3-Ι and Beclin1 and increased p62 expression (P < 0.05). MiR-424 inhibits HCC cell autophagy and proliferation through regulating ATG14.

MicroRNA-4325 Suppresses Cell Progression in Hepatocellular Carcinoma via GATA-Binding Protein 6

MicroRNAs (miRs) are regulators of the formation and development of hepatocellular carcinoma (HCC). The biological role of miR-4325 in HCC has yet to be determined. This study is aimed at dissecting the role of miR-4325 in HCC and the underlying mechanism. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect miR-4325 expression in HCC tissue specimens and cells. Cell proliferation, migration, and invasion were assessed by using the MTT assay and Transwell assay, respectively. The miR-4325 target was predicted based on bioinformatics analysis and validated using the dual-luciferase reporter assay. Rescue experiments in the cells were utilized to functionally characterize the downstream molecular targets of miR-4325. We observed that miR-4325 expression levels were significantly reduced in both HCC tissue specimens and cell lines. Meanwhile, a lower miR-4325 level was associated with a poorer prognosis. Gain and loss of function assays revealed that miR-4325 markedly downregulated HCC cell growth, migration, and invasion. Moreover, we identified GATA-binding protein 6 (GATA6) as a miR-4325 target and found that GATA6 was abnormally expressed in HCC. Rescue assays demonstrated that the regulatory function of miR-4325 in HCC was mediated by GATA6. Taken together, miR-4325 suppresses HCC cell growth, migration, and invasion by targeting GATA6, suggesting that miR-4325 may potentially serve as a novel therapeutic target for HCC.

RETRACTED: Hepatocellular carcinoma cells-derived exosomal microRNA-378b enhances hepatocellular carcinoma angiogenesis

It has been already accepted that hepatocellular carcinoma (HCC) cells-derived exosomes mediate HCC development partially through transferring microRNAs (miRNAs). Illuminated by that, this work pivoting on HCC specifically starts from miR-378b in HepG2 cells-derived exosomes, involving with transforming growth factor β receptor III (TGFBR3). HCC tissue and normal tissue specimens were resected, in which miR-378b and TGFBR3 expression were tested. The connection between miR-378b and TGFBR3 was assessed. HepG2 cells were transfected with miR-378b and TGFBR3-related sequences to explore their functions in HCC cell progression. The extracted exosomes from HepG2 cells were identified and co-cultured with human umbilical vein endothelial cells to explore their roles in HCC cell progression and angiogenesis. Tumorigenesis in mice was conducted for further validation of the findings in cells. Up-regulated miR-378b and down-regulated TGFBR3 presented in HCC, and miR-378b targeted TGFBR3. Depleted miR-378b disturbed HCC cell migration and promoted apoptosis. Knockdown of TGFBR3 reversed the effects of down-regulated miR-378b on HCC cells. HepG2 cells-derived exosomes promoted angiogenesis in vitro and tumor growth in vivVo, which would be further enhanced by miR-378b overexpression while impaired by miR-378b down-regulation. It is elucidated that HepG2 cells-derived exosomal miR-378b enhances HCC cell progression and angiogenesis, which may be linked with TGFBR3, providing therapeutic agents for HCC curing.

Extracellular ubiquitin promotes hepatoma metastasis by mediating M2 macrophage polarization via the activation of the CXCR4/ERK signaling pathway.

BackgroundStored red blood cell (RBC) transfusion has been shown to enhance the risk of cancer recurrence. However, the underlying mechanism remains unknown. At our lab, we have demonstrated that the extracellular ubiquitin (eUb) released by aged RBCs could promote tumor metastasis in a melanoma mouse model. This study aimed to confirm the pro-tumor effect of eUb on hepatocellular carcinoma (HCC) and explore the related immunoregulatory mechanisms.MethodsForty HCC tissue specimens and the corresponding adjacent nontumor and normal liver tissues were collected. Two human hepatoma cell lines (MHCC-97H and HepG2.2.15), one murine hepatoma cell line (Hepa1-6), and one human monocyte cell line (THP-1) were adopted in this study. The coculture of hepatoma cells with macrophages was initiated with Transwell inserts. Cell migration in vitro was detected by Transwell and wound-healing assays, while in vivo tumor metastasis was measured by luciferase assay and H&E staining. Macrophage polarization was measured by flow cytometry, immunofluorescence, ELISA, qPCR, and Western blot. Protein expression was detected by Western blot, and immunoprecipitation was used to confirm the interaction between Ub and CXCR4 (CXC chemokine receptor type 4).ResultsUb and CXCR4 were significantly upregulated in HCC tissues, and a positive correlation existed between them. In vitro, the migration of hepatoma cells was not affected by eUb directly, but their metastatic abilities were enhanced after coculture with the macrophages pretreated with eUb. Meanwhile, eUb promoted hepatoma cell metastasis in the lung in vivo and increased the ratio of M2 macrophages in the lung tissues and peripheral blood of tumor-bearing mice. Furthermore, the eUb-induced M2 macrophage polarization was related to the activation of the CXCR4/ERK (extracellular regulated protein kinase) signaling pathway.ConclusionsExtracellular ubiquitin promoted hepatoma metastasis through M2 macrophage polarization via the activation of the CXCR4/ERK signaling pathway, indicating that a personalized transfusion strategy is needed for the treatment of HCC patients. Neutralizing Ub in stored RBC units could lessen the detrimental clinical outcomes induced by the transfusion of stored RBCs.

MTA1 Promotes Hepatocellular Carcinoma Progression by Downregulation of DNA-PK-Mediated H1.2T146 Phosphorylation.

Global incidence and mortality associated with hepatocellular carcinoma (HCC) is steadily increasing. Metastasis-associated 1 (MTA1) can induce tumorigenesis and metastatic progression in HCC. However, the mechanistic details of MTA1-mediated regulation of HCC has not been completely defined. Epigenetic histone modification is closely related to tumor development. Histone cluster 1 H1 family member c (H1.2) is important for epigenetic histone modification and chromatin remodeling; however, whether it has a role in HCC tumorigenesis is not known. In the current study, we confirmed that MTA1 promoted HCC cell growth and migration. Our results further show that MTA1 inhibited the phosphorylation of histone cluster 1 H1 family member c (H1.2) at threonine-146 residue (T146) (H1.2T146ph). MTA1 inhibited H1.2T146ph by mediating proteasomal degradation of the DNA protein kinase (DNA-PK). Pharmacological inhibition of proteasomal degradation of DNA-PK or genetic ablation of E3 ligase mouse double minute 2 (MDM2) rescued expression of DNA-PK, and subsequent phosphorylation of H1.2. MTA1's role in HCC was inhibited by ectopic expression of H1.2T146ph in HCC cell lines. Our results showed that H1.2T146ph can bind to MTA1 target genes. Collectively, our study confirms that MTA1 functions as an oncogene and promotes HCC progression. The epigenetic histone modifier H1.2T146ph exerts critical role in the regulation of MTA1-induced tumorigenesis. MTA1 regulates posttranslational activation of H1.2 by regulating the cognate kinase, DNA-PK, via the ubiquitin proteasome system. MTA1 expression was inversely correlated to both DNA-PK and phosphorylated H1.2 in HCC tissue specimens compared to tumor adjacent normal hepatic tissue, revealing that the MTA1/MDM2/DNA-PK/H1.2 is an important therapeutic axis in HCC.

HIF1A activates the transcription of lncRNA RAET1K to modulate hypoxia-induced glycolysis in hepatocellular carcinoma cells via miR-100-5p

Hepatocellular carcinoma (HCC) remains the primary cause of cancer-related death. Metabolic change is the major characteristic of cancer. The present study attempted to investigate the regulatory mechanisms of HCC energy metabolism from the perspective of noncoding RNA regulation of HIF1A and LDHA. The expression of miR-100-5p expression was significantly suppressed in HCC tissue samples and HCC cell lines under 1% O2-induced hypoxia. miR-100-5p overexpression significantly suppressed hypoxia-induced increases in lactate concentration and glucose uptake. Exposure to 1% O2 induced HIF1A protein and reduced miR-100-5p expression, while HIF1A silencing dramatically rescued miR-100-5p expression upon 1% O2 exposure. In addition, 1% O2-induced increases in lactate concentration and glucose uptake were also suppressed by HIF1A silencing. Next, by analyzing available data in TCGA, we found that lncRNA RAET1K was correlated with HIF1A and miR-100-5p.LncRNA RAET1K could downregulate the expression of miR-100-5p by acting as a sponge, while HIF1A bound the lncRNA RAET1K promoter region to activate its transcription. LncRNA RAET1K silencing significantly suppressed HCC cell proliferation and invasion and also suppressed hypoxia-induced increases in lactate concentration and glucose uptake, while miR-100-5p inhibition reversed the effects of lncRNA RAET1K silencing on hypoxia-induced glycolysis in HCC cells. Finally, the expression of HIF1A, lncRNA RAET1K, and LDHA was upregulated in HCC tissue specimens; the expression of miR-100-5p was negatively related to HIF1A, lncRNA RAET1K, and LDHA; and HIF1A, lncRNA RAET1K, and LDHA were positively correlated with each other. In conclusion, the HIF1A/lncRNA RAET1K/miR-100-5p axis modulates hypoxia-induced glycolysis in HCC cells and might affect HCC progression.

STYXL1 promotes malignant progression of hepatocellular carcinoma via downregulating CELF2 through the PI3K/Akt pathway.

This study was aimed to investigate the expression characteristics of STYXL1 in hepatocellular carcinoma (HCC), and to further analyze its regulatory role in promoting HCC development by targeting CELF2 to activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Expression levels of STYXL1 in 25 pairs of HCC tissue specimens and paracancerous normal ones collected from HCC patients were examined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Meanwhile, qRT-PCR was also performed to further verify the expression of STYXL1 in HCC cell lines. In addition, after STYXL1 knockdown model was constructed by lentivirus transfection in HCC cell lines Hep3B and Huh7, the Cell Counting Kit-8 (CCK-8), cell colony formation, 5-Ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays were performed to analyze the influence of STYXL1 on HCC cell functions. Furthermore, an in-depth study of the relationship between STYXL1 and CELF2 was conducted to figure out the underlying mechanism. The results of qRT-PCR revealed that the expression level of STYXL1 in HCC samples was remarkably higher than that in adjacent ones, and the difference was statistically significant. Compared with HCC patients with low expression of STYXL1, patients with high expression of STYXL1 had a higher overall survival rate. Similarly, the proliferation ability of HCC cells in sh-STYXL1 group remarkably decreased compared with controls, while the apoptosis ability was oppositely enhanced. In addition, Western Blotting results indicated that STYXL1 could elevate the expressions of PI3K/Akt pathway-related proteins. Meanwhile, a negative correlation between CELF2 and STYXL1 was identified in HCC tissues. Finally, the result of cell reverse experiments demonstrated that STYXL1 could affect the malignant progression of HCC via modulating CELF2 expression. STYXL1 expression was remarkably upregulated in HCC tissues, as well as in cell lines. Its level was closely related to the poor prognosis of HCC patients. In addition, STYXL1 might be able to accelerate HCC proliferation rate and inhibit cell apoptosis via downregulating CELF2 through the PI3K/Akt pathway.

CCAT2 contributes to hepatocellular carcinoma progression via inhibiting miR-145 maturation to induce MDM2 expression.

Long noncoding RNA colon cancer-associated transcript 2 (CCAT2) has been recently found to function as an oncogene in hepatocellular carcinoma (HCC). However, the mechanisms of CCAT2 in HCC development remain to be further explored. In the present study, we found that CCAT2 was abnormally upregulated in HCC cells and tissue specimens, exhibiting an inverse correlation with microRNA (miR)-145 expression. Mechanistic investigation showed that CCAT2 selectively blocked miR-145 processing, leading to decreased mature miR-145 presence. Both the in vitro and in vivo effects of CCAT2 knockdown on the proliferation and metastasis of HCC cells were reversed by miR-145 inhibitor, indicating that miR-145 modulation accounts for CCAT2-meditated HCC progression. Furthermore, miR-145 mimic dramatically suppressed HCC cells' proliferation and metastasis, revealing a tumor suppressor role of miR-145 in HCC. Mechanistically, MDM2 was predicted to be a potential target of miR-145. The luciferase and western blot assay demonstrated that miR-145 mimic largely inhibited MDM2 3'-untranslated region luciferase activity and MDM2 expression, followed by the upregulation of p53/p21 expression. Finally, the coexpression of MDM2 in miR-145 mimic-transfected HCC cells was able to largely compromise the inhibitory effects of miR-145 mimic on HCC cells' proliferation and metastasis in vitro and tumor formation in a xenograft model, confirming MDM2 is the critical mediator of miR-145 in HCC. In summary, our findings indicated that CCAT2 selectively blocks the miR-145 maturation process and plays an oncogene in HCC. Furthermore, a novel CCAT2/miR-145/MDM2 axis was revealed in HCC development and might provide a new target in the molecular treatment of HCC.

Frequency of somatic mutations in TERT promoter, TP53 and CTNNB1 genes in patients with hepatocellular carcinoma from Southern Italy.

Somatic mutations in the TERT promoter and in the TP53 and CTNNB1 genes are considered drivers for hepatocellular carcinoma (HCC) development. They show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. TP53, CTNNB1 and TERT genetic mutations were investigated in tumor and non-tumor liver tissues from 67 patients with HCC and liver tissue specimens from 41 control obese subjects from Southern Italy. Furthermore, TERT expression was assessed by reverse transcription-quantitative PCR. Neither CTNNB1 mutations or TP53 R249S substitution were detected in any case. The TP53 R72P polymorphism was found in 10/67 (14.9%) tumors, but was not found in either non-tumor tissues (P=0.001) or controls (P=0.009). TERT gene promoter mutations were found in 29/67 (43.3%) tumor tissues but were not found in either non-tumor (P<0.0001) or control liver specimens (P<0.0001). The most frequent mutation in the tumors was the known hot spot at −124 bp from the TERT ATG start site (−124G>A, 28 cases, 41.8%; P<0.0001). A new previously never reported TERT promoter mutation (at −297 bp from the ATG, −297C>T) was found in 5/67 (7.5%) tumors, in 0/67 (0%) non-tumor (P<0.0001), and in 0/41 (0%) controls (P=0.07). This mutation creates an AP2 consensus sequence, and was found alone (1 case) or in combination (4 cases) with the −124 bp mutation. The mutation at −124 and −297 bp induced a 33-fold (P<0.0001) and 40-fold increase of TERT expression levels, respectively. When both mutations were present, TERT expression levels were increased >300-fold (P=0.001). A new TERT promoter mutation was identified, which generates a de novo binding motif for AP2 transcription factors, and which significantly increases TERT promoter transcriptional activity.