CCNB1 promotes the development of hepatocellular carcinoma by mediating DNA replication in the cell cycle

In our studies, cyclin B1 (CCNB1) mRNA and protein were overexpressed in hepatocellular carcinoma (HCC) tissues compared with non-HCC tissues. Moreover, CCNB1 was overexpressed in the serum of HCC patients. The expression of CCNB1 was associated with several crucial clinicopathologic characteristics, and the HCC patients with overexpressed CCNB1 had worse overall survival outcomes. In the screening of interactional genes, a total of 266 upregulated co-expression genes, which were positively associated with CCNB1, were selected from the datasets, and 67 downregulated co-expression genes, which were negatively associated with CCNB1, were identified. The key genes might be functionally enriched in DNA replication and the cell cycle pathways. CDC20, CCNA2, PLK1, and FTCD were selected for further research because they were highly connected in the protein-protein interaction networks. Upregulated CDC20, CCNA2, and PLK1 and downregulated FTCD might result in undesirable overall survival outcomes for HCC patients. The univariate Cox analysis results showed that CDC20 and PLK1 might be two independent risk factors, while FTCD might be protective in HCC. Therefore, CCNB1 may participate in the cell cycle of HCC by regulating DNA replication, and CCNB1 may provide a direction for the diagnosis of early-stage HCC and targeted HCC therapy.     

Construction of a competing endogenous RNA network to analyse glucose-6-phosphate dehydrogenase dysregulation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumour with high rates of morbidity and mortality worldwide. Therefore, it is of great significance to find new molecular markers for HCC diagnosis and treatment. G6PD is known to be dysregulated in a variety of tumours. In addition, the ceRNA network plays a crucial role in the occurrence and development of HCC. However, the mechanism by which the ceRNA network regulates G6PD in HCC remains unclear. We used TCGA-LIHC data to analyse the possibility of using G6PD as an independent prognostic marker. Univariate Cox proportional hazards regression, multivariate Cox proportional hazards regression, and receiver operating characteristic curve analysis were used to analyse the influence of G6PD overexpression on the prognosis of HCC patients. We also analysed the biological function of G6PD, its effect on the immune microenvironment, and drug sensitivity. Finally, we constructed a ceRNA network of lncRNAs/miR-122-5p/G6PD to explore the regulatory mechanism of G6PD. G6PD was highly expressed in HCC, was related to pathological stage and poor prognosis, and could be used as an independent prognostic indicator of HCC. The expression of G6PD was closely related to the immune microenvironment of HCC. In addition, the expression of G6PD in HCC could be regulated by the ceRNA network. Therefore, G6PD can be used as an immunotherapy target to improve the survival and prognosis of HCC patients, and the ceRNA regulatory network of G6PD has potential diagnostic and therapeutic value for HCC.     

CXCL2/10/12/14 are prognostic biomarkers and correlated with immune infiltration in hepatocellular carcinoma

Background: C-x-C motif chemokine ligands (CXCLs) are critical regulators of cancer immunity and angiogenesis, which affect disease progression and treatment responses. The character of each CXCL in the prognosis and immune infiltration of hepatocellular carcinoma (HCC) patients is unclear yet. Methods: Differentially expressed CXCLs between HCC and normal control were screened by Oncomine and GEPIA2. Genetic alternations of CXCLs in HCC were analyzed by cBioPortal. Clinicopathological relevance of CXCLs in HCC patients was analyzed using UALCAN. The prognostic value of CXCLs was evaluated using univariate and multivariate analyses. Correlations of CXCLs’ expression with immune infiltration, chemokines and their receptors were assessed integrating TIMER, TISIDB, and GEPIA2. The co-expressed genes of CXCLs were discovered, and functional enrichment analysis was performed for them. Results: CXCL9/10 was significantly higher expressed while CXCL2/12/14 was lower expressed in HCC than normal tissues, but they didn’t show significant clinicopathological relevance in HCC patients. High-expression of CXCL2/10/12/14 indicated favorable outcomes of HCC patients. The expression of CXCL9/10/12/14 was significantly positively correlated with not only the infiltration and biomarkers’ expression of various tumor-infiltrating immune cells but also the abundance of chemokines and their receptors. The co-expressed genes of the five CXCLs were extracellular components and regulated immune or inflammatory responses and signaling pathways of chemokine, Toll-like receptor and tumor necrosis factor might be involved. Conclusion: The present study proposed CXCL2/10/12/14 might predict outcomes of HCC patients and were extensively related with the immune microenvironment in HCC. It would be a prospective therapeutic strategy for HCC to enhance effective immunity surveillance through intervening in these CXCLs.     

CFHR3 is a potential novel biomarker for hepatocellular carcinoma

Complement factor H-related 3 (CFHR3) is a protein-coding gene acting in various diseases. However, its prognostic values of CFHR3 in hepatocellular carcinoma (HCC) are not understandable. Therefore, we present a further study on CFHR3 in HCC. CFHR3 expression data were acquired from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). We compared the differential expression of CFHR3 between the low-stage (stage I and II) and high-stage (stage III and IV) patients with HCC in the TCGA and ICGC cohorts. Furthermore, we assessed the CFHR3 expression as a prognostic marker using the Kaplan-Meier survival analysis, univariate, and multivariate analysis. The Kaplan-Meier analysis declared that CFHR3 overexpression was correlated with a good prognosis for HCC patients. Multivariate analysis proved the prognostic significance of CFHR3 expression levels (P < .001 and .003 for TCGA and ICGC, respectively). Immune-related scores in low-risk cohorts were higher than high-risk cohorts. Gene set enrichment analysis implied that the low CFHR3 expression phenotype was significantly enriched in critical biological functions and pathways and was associated with tumorigenesis, such as regulation of cell activation cycle, and the WNT and NOTCH signal pathway. Above all, CFHR3 could be a novel prognostic biomarker and therapeutic target for HCC.     

SLC7A2 deficiency promotes hepatocellular carcinoma progression by enhancing recruitment of myeloid-derived suppressors cells

The main reason for poor prognosis in hepatocellular carcinoma (HCC) patients is high metastasis and recurrence. Cancer progression depends on a tumor-supportive microenvironment. Therefore, illustrating the mechanisms of tumor immunity in underlying HCC metastasis is essential. Here, we report a novel role of solute carrier family 7 member 2 (SLC7A2), a member of the solute carrier family, in HCC metastasis. The reduction of SLC7A2 was an independent and significant risk factor for the survival of HCC patients. Upregulation of SLC7A2 decreased HCC invasion and metastasis, whereas downregulation of SLC7A2 promoted HCC invasion and metastasis. We further found that deficient SLC7A2 medicated the upregulation of CXCL1 through PI3K/Akt/NF-kκB pathway to recruit myeloid-derived suppressor cells (MDSCs), exerting tumor immunosuppressive effect. Moreover, we found that G9a-mediated di-methylation of H3K9 (H3K9me2) silenced the expression of SLC7A2 to suppress HCC metastasis and immune escape. In conclusion, G9a-mediated silencing of SLC7A2 exerts unexpected functions in cancer metastasis by fostering a tumor-supportive microenvironment through CXCL1 secretion and MDSCs recruitment. Thus, SLC7A2 may provide new mechanistic insight into the cancer-promoting property of MDSCs.Subject terms: Cancer microenvironment, Cancer immunotherapy, Metastasis     

Non-classical MHC-Ι genes in chronic hepatitis B and hepatocellular carcinoma

The non-classical human leukocyte antigens (HLA)-E, HLA-G, and HLA-F have been shown to modulate immune responses. We examined whether non-classical HLA polymorphisms are associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). Fifteen Single-nucleotide polymorphisms (SNPs) in these non-classical class I alleles were investigated by ligase detection reaction. A fragment of 650 bp located in the 3' untranslated region of HLA-G was investigated. Four SNPs (rs17875380, rs41557518, rs114465251, and rs115492845) were associated with altered susceptibility to HBV or HCC, and HLA-F*01:04, HLA-G*01:05N, and HLA-E*01:01 were associated with hepatitis B or hepatitis B complicated with HCC. Six of 16 designated HLA-E, -G, and -F haplotypes were associated with risk of hepatitis B or HCC. Our study provides healthy reference and detailed analyses of non-classical HLA class Ι polymorphisms that provide insight into immune mechanisms involved in susceptibility to hepatitis B and HCC.     

An intronic polymorphism rs2237062 in the CXCL14 gene influences HBV-related HCC progression in Chinese population

CXCL14 (C-X-C motif chemokine ligand 14) is a conserved member of chemokine family and functions as a chemoattractant for multiplicate immunocytes. CXCL14 expression is constitutive in normal tissues, but absent in wide range of epithelial tumors. Many reports have claimed its important role in tumorigenesis and vascularization. An association between rs2237062 polymorphism and hepatocellular carcinoma (HCC) susceptibility was found in patients with chronic HCV infection in Japanese population. Here we analyzed, by using a polymerase chain reaction-ligation detection reaction (PCR-LDR), the polymorphism in 202 non-HCC patients with HBV infection, 361 HBV-related HCC patients and 407 healthy controls. The aim was to detect the possible association of this single-nucleotide polymorphism (SNP) with HBV-related HCC susceptibility and progression. However, no association was found between rs2237062 polymorphism and susceptibility to HBV infection or HBV-related HCC. Intriguingly, our stratification analysis revealed that HBV-related HCC patients in advanced phase (TNM-II–IV stage) had significantly higher C allele frequency at this polymorphism than patients at early stage (TNM-I stage) (33.5% vs. 25.7%), and its odds ratio reached 1.47 (95% CI 1.06–2.04, P = 0.021). These results suggest that the rs2237062 polymorphism in the CXCL14 gene might influence HBV-related HCC progression in Chinese population.     

Down-regulation of RBP4 indicates a poor prognosis and correlates with immune cell infiltration in hepatocellular carcinoma

Recent research has indicated that metabolically related genes play crucial roles in the pathogenesis of hepatocellular carcinoma (HCC). We evaluated the associations between novel biomarkers and retinol-binding protein 4 (RBP4) for predicting clinical HCC outcomes, hub-related genes, pathway regulation, and immune cells infiltration. Bioinformatic analyses based on data from The Cancer Genome Atlas were performed using online analysis tools. RBP4 expression was low in HCC and was also down-regulated in pan-cancers compared with normal tissues. RBP4 expression was also significantly different based on age (41–60 years old versus 61–80 years old), and low RBP4 expression levels were associated with advanced tumor stages and grades. Higher RBP4 expression was associated with better overall survival time in HCC patients, and we identified a deletion-mutation rate of 1.4% in RBP4. We also identified ten co-expressed genes most related to RBP4 and explored the relationships between six hub genes (APOB, FGA, FGG, SERPINC1, APOA1, and F2) involved in RBP4 regulation. A pathway enrichment analysis for RBP4 indicated complement and coagulation cascades, metabolic pathways, antibiotic biosynthesis pathways, peroxisome proliferator-activated receptor signaling pathways, and pyruvate metabolism pathways. These results suggest that RBP4 may be a novel biomarker for HCC prognosis, and an indicator of low immune response to the disease.     

Prognostic evaluation and immune infiltration analysis of five bioinformatic selected genes in hepatocellular carcinoma

Despite the development in hepatocellular carcinoma (HCC) treatment in recent years, the therapeutic outcome of HCC remains unfavourable. This study examines the prognosis of HCC from a genetic level using clinical databases and single-cell data to identify genes with a high prognostic value. Three up-regulated genes (UBE2S, PTTG1, and CDC20) and two down-regulated genes (SOCS2 and DNASE1L3) in HCC tissues were identified. Various analyses confirmed its correlation with tumour stage (p < 0.01) and patient survival time (log-rank p < 0.001). Immune analysis, single-cell analysis, and gene set enrichment analysis (GSEA) were employed to provide insight on how they affect cancer progression, and we observed a close relation between these genes and tumour immune infiltration. Eventually, we constructed a risk score system that risk score = (0.0465) × UBE2S + (0.1851) × CDC20 + (−0.0461) × DNASE1L3 + (−0.2279) × SOCS2 (5-year area under curve = 0.706). The risk score system may serve as an effective novel prognostic system for HCC patients. This study might provide novel ideas for prognostic or therapeutic biomarkers for HCC.     

Changes in intra-abdominal fat in early postmenopausal women: effects of hormone use

Objective: Intra‐abdominal fat (IAF) accumulates with age, is greater among postmenopausal vs. premenopausal women, and is linked to risk for both type 2 diabetes and cardiovascular disease. Whether hormone replacement therapy (HRT) prevents or attenuates changes in IAF and related risk factors is not clear. The objectives of this observational study were to 1) determine whether HRT attenuated the expected age‐related increase in IAF and 2) identify the independent effects of HRT and fat distribution on changes in disease risk factors. Research Methods and Procedures: Subjects were early postmenopausal white women 45 to 55 years of age. Women either used HRT at the time of enrollment (n = 33) or did not (n = 17). Subjects were evaluated at baseline and 2 years for body composition (DXA), body fat distribution (computed tomography), insulin sensitivity (Si; minimal model), and serum lipids. Results: IAF increased significantly over 2 years, and this increase was not attenuated by HRT. HRT users had less IAF throughout the study. HRT users showed an increase in Si, whereas non‐users showed a decrease. Superficial subcutaneous adipose tissue was significantly and independently related to total cholesterol, whereas IAF was related to high‐density lipoprotein cholesterol, triglycerides, and Si. Discussion: HRT users had less IAF at baseline and throughout the study. Whether HRT altered the relationship between total body fat and IAF or whether differences between groups existed before the study should be addressed through a randomized, interventional study design. HRT had a significant effect on Si; IAF and superficial subcutaneous adipose tissue were significant determinants of disease risk factors.     

Two common nonsynonymous paraoxonase 1 (<Emphasis Type="Italic">PON1</Emphasis>) gene polymorphisms and brain astrocytoma and meningioma

Background  

Human serum paraoxonase 1 (PON1) plays a major role in the metabolism of several organophosphorus compounds. The enzyme is encoded by the polymorphic gene PON1, located on chromosome 7q21.3. Aiming to identify genetic variations related to the risk of developing brain tumors, we investigated the putative association between common nonsynonymous PON1 polymorphisms and the risk of developing astrocytoma and meningioma.     

Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.Subject terms: Tumour biomarkers, Cancer     

High-throughput assessment of CpG site methylation for distinguishing between HCV-cirrhosis and HCV-associated hepatocellular carcinoma

Methylation of promoter CpG islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. Therefore, some postulate that aberrantly methylated CpG regions may be important biomarkers indicative of cancer development. In this study we used the Illumina GoldenGate Methylation BeadArray Cancer Panel I for simultaneously profiling methylation of 1,505 CpG sites in order to identify methylation differences in 76 liver tissues ranging from normal to pre-neoplastic and neoplastic states. CpG sites for ESR1, GSTM2, and MME were significantly differentially methylated when comparing the pre-neoplastic tissues from patients with concomitant hepatocellular carcinoma (HCC) to the pre-neoplastic tissues from patients without HCC. When comparing paired HCC tissues to their corresponding pre-neoplastic non-tumorous tissues, eight CpG sites, including one CpG site that was hypermethylated (APC) and seven (NOTCH4, EMR3, HDAC9, DCL1, HLA-DOA, HLA-DPA1, and ERN1) that were hypomethylated in HCC, were identified. Our study demonstrates that high-throughput methylation technologies may be used to identify differentially methylated CpG sites that may prove to be important molecular events involved in carcinogenesis.     

Biochemical genetics of alcohol dehydrogenase isozymes in the gray short-tailed opossum (Monodelphis domestica)

Polyacrylamide gel-isoelectric focusing (PAGE-IEF) methods were used to examine the multiplicity, tissue distribution, and biochemical genetics of alcohol dehydrogenase (ADH) isozymes among gray short-tailed opossums (Monodelphis domestica). Seven ADH isozymes were resolved and distinguished on the basis of their isoelectric points, tissue distributions, and substrate and inhibitor specificities. ADH1 and ADH2 exhibited Class I properties and were observed in liver (and intestine) extracts. ADH3, ADH4, and ADH5 showed “high-K _m ” (possibly Class IV) properties, with ADH3 and ADH4 exhibiting high activity in cornea, ear, stomach, and esophagus extracts. ADH6 and ADH7 exhibited Class III properties, including activities as formaldehyde dehydrogenases, with each showing different tissue distribution characteristics; ADH6 was widely distributed, and ADH7 was restricted to prostate extracts. An additional form of formaldehyde dehydrogenase (FDH) was observed, which was inactive with hexenol and ethanol as substrates. Isoelectric point variants were observed for ADH3 (three forms) and for ADH4 (two forms), and the inheritance of ADH3 was studied in 15 families ofM. domestica. The data were consistent with codominant inheritance of two alleles (ADH3*A andADH3*B) at a single autosomal locus (designatedADH3) and with a model involving a dimeric ADH isozyme: ADH3 (γ₂ isozyme, forming three dimers designated γ ₂ ¹ , γ¹ γ², and γ ₂ ² in heterozygous individuals).     

Calcium and Magnesium: Low Passive Permeability and Tubular Secretion in the Mouse Medullary Thick Ascending Limb of Henle's Loop (MTAL)

Recent studies from our laboratory have shown that in the mouse and rat nephron Ca²⁺ and Mg²⁺ are not reabsorbed in the medullary part of the thick ascending limb (mTAL) of Henle's loop. The aim of the present study was to investigate whether the absence of transepithelial Ca²⁺ and Mg²⁺ transport in the mouse mTAL is due to its relative low permeability to divalent cations. For this purpose, transepithelial ion net fluxes were measured by electron probe analysis in isolated perfused mouse mTAL segments, when the transepithelial potential difference (PD_te.) was varied by chemical voltage clamp, during active NaCl transport inhibition by luminal furosemide. The results show that transepithelial Ca²⁺ and Mg²⁺ net fluxes in the mTAL are not driven by the transepithelial PD_te. At zero voltage, a small but significant net secretion of Ca²⁺ into the tubular lumen was observed. With a high lumen-positive PD_te generated by creating a transepithelial bath-to-lumen NaCl concentration gradient, no Ca²⁺ and Mg²⁺ reabsorption was noted; instead significant and sustained Ca²⁺ and Mg²⁺ net secretion occurred. When a lumen-positive PD_te was generated in the absence of apical furosemide, but in the presence of a transepithelial bath-to-lumen NaCl concentration gradient, a huge Ca²⁺ net secretion and a lesser Mg²⁺ net secretion, not modified by ADH, were observed. Replacement of Na⁺ by K⁺ in the lumen perfusate induced, in the absence of PD_te changes, important but reversible net secretions of Ca²⁺ and Mg²⁺. In conclusion, our results indicate that the passive permeability of the mouse mTAL to divalent cations is very low and not influenced by ADH. This nephron segment can secrete Ca²⁺ and Mg²⁺ into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. Given the impermeability of this epithelium to Ca²⁺ and Mg²⁺, the secretory processes would appear to be of cellular origin. Received: 30 January 1996/Revised: 24 April 1996     

CELF2 is a candidate prognostic and immunotherapy biomarker in triple-negative breast cancer and lung squamous cell carcinoma: A pan-cancer analysis

CUGBP Elav-like family member 2(CELF2) plays crucial roles in the development and activation of T cell. However, the impacts of CELF2 on tumour-infiltrating immune cells (TIICs) and clinical outcomes of tumours remain unclear. In this study, we found that elevated CELF2 expression was markedly correlated with prolonged survival in multiple tumours, particularly in breast and lung cancers. Notably, CELF2 only impacted the prognosis of triple-negative breast cancer (TNBC) with lymph node metastasis. Further investigation showed CELF2 expression was positively correlated with the infiltration abundance of dendritic cells (DCs), CD8+ T cells and neutrophils in breast invasive carcinoma (BRCA) and DCs in lung squamous cell carcinoma (LUSC). CELF2 also had strong correlations with markers of diverse TIICs such as T cells, tumour-associated macrophages and DCs in BRCA and LUSC. Importantly, CELF2 was significantly associated with plenty of immune checkpoint molecules (ICMs) and outperformed five prevalent biomarkers including PD-1, PD-L1, CTLA-4, CD8 and tumour mutation burden in predicting immunotherapeutic responses. Immunohistochemistry also revealed lower protein levels of CELF2 in TNBC and LUSC compared to normal tissues, and patients with high expression showed significantly prolonged prognosis. In conclusion, we demonstrated that increased CELF2 expression was closely related to better prognosis and superior TIIC infiltration and ICM expression, particularly in BRCA and LUSC. CELF2 also performed well in evaluating the immunotherapeutic efficacy, suggesting CELF2 might be a promising biomarker.     

Novel multiplex method to assess insulin,leptin and adiponectin regulation of inflammatory cytokines associated with colon cancer

The role of altered levels of insulin, leptin and adiponectin in contributing to the observed increased risk of colon cancer associated with obesity remains to be determined. Elevated insulin and leptin associated with obesity are linked to inflammatory responses. Conversely, adiponectin levels are reduced in obese individuals and this hormone is generally associated with anti-inflammatory responses. Inflammatory cytokines are key components of processes linked with carcinogenesis. Insulin, leptin and adiponectin receptor expression profiles were assessed in human normal, adenomatous polyp and tumour tissue. Insulin, leptin and adiponectin regulation of inflammatory cytokines previously identified as being associated with early events in colon carcinogenesis were further investigated here using a surrogate colon epithelial cell line and a custom designed GeXP assay of the inflammatory cytokines (CCL20, CXCL1, CXCL2, CXCL3, CXCL11, IL1RN, CXCL4, IL8, CCL19, CCL21, CCL23, CCL5, IL10RB and TNFRSF1A). Mean insulin, leptin and adiponectin receptor expression levels were lower in adenomatous polyp samples in comparison with normal and tumour tissue. In contrast to leptin, insulin significantly reduced CCL20 and CXCL11 and increased CXCL3 expression. Full length adiponectin, but not globular adiponectin, induced CCL5, CXCL1, CXCL3 and CCL20 gene expression. GeXP assay permitted measurement of changes in gene expression of cytokines in response to insulin and adiponectin, indicating the potential for insulin and adiponectin regulation of mediators of inflammation associated with early events in colon carcinogenesis.