Adjacent Normal Liver Tissues Research Articles

Nucleolar spindle associated protein 1 (NUSAP1) facilitates proliferation of hepatocellular carcinoma cells.

BackgroundThe most fundamental characteristic of cancer cells is abnormal proliferation, which is partly due to deregulation of cell cycle. Nucleolar spindle associated protein 1 (NUSAP1) is originally identified as microtubule and chromosome binding protein and takes an important role in chromosome segregation fidelity. Recent studies have been documented that NUSAP1 is associated with the progression of several cancers. However, its expressions and functions are unclear in hepatocellular carcinoma (HCC).MethodsqPCR and western blotting were used to examine NUSAP1 levels in fresh paired HCC tissues and corresponding adjacent normal liver tissues (ANT). MTT and colony formation assay were performed to evaluate whether NUSAP1 can influence the proliferation of HCC cells. Anchorage-independent growth assay was implemented to detect the effect of NUSAP1 on in vitro tumorigenicity capacity. Flow cytometry, qPCR and western blotting assay were used to clarify the detailed mechanisms that NUSAP1 accelerates the capability for cell proliferation and in vitro tumorigenicity.ResultsFirstly, we analyzed the availably public dataset The Cancer Genome Atlas (TCGA), and discovered that NUSAP1 is dramatically upregulated in HCC tissues and closely correlated with poor prognosis of patients suffering HCC, which was confirmed in fresh HCC tissues and ANT. Subsequently, cell function assay illustrated that the upregulation of NUSAP1 facilitates proliferation and in vitro tumorigenicity capacity of HCC cells. Besides, molecular experiments suggested that NUSAP1 promotes cell cycle transition of HCC cells.ConclusionsIn summary, the above results inferred that NUSAP1 takes an indispensable role in cell cycle progression. And it might be served as a novel prognostic factor in HCC.

MicroRNA‑373 exerts anti‑tumor functions in human liver cancer by targeting Rab22a

Liver cancer is a one of the most frequent types of tumor worldwide. It has long been recognized that microRNAs are important participants in the progression of various types of cancer. The present study explored the role of microRNA‑373 (miR‑373) in liver cancer development. Reverse transcription‑quantitative polymerase chain reaction was performed to evaluate the transcription level of miR‑373 in 96 liver cancer tissues and adjacent normal liver tissues. The association of miR‑373 with clinicopathological characteristics was analyzed using the χ2 test. Kaplan‑Meier univariate analysis and multivariate hazard analysis were performed to identify the clinical potential of miR‑373 in the prognosis of liver cancer patients. Transfection of miR‑373 mimics into Hep3B and HepG2 liver cancer cell lines was conducted to reveal the underlying mechanism in regulating liver cancer progression. The functional assays included proliferation, migration, invasion and luciferase assays. The findings of the present study demonstrated that miR‑373 transcription level was markedly downregulated in liver cancer tissues compared with the adjacent normal tissues and was associated with the clinical prognosis of liver cancer patients. Overexpressing miR‑373 mimics in liver cancer cell lines decreased cell proliferation and invasion, suggesting that miR‑373 exerts anti‑tumor effects in liver cancer. In addition, data from the present study demonstrated the direct effect of miR373 on inhibiting the expression and signaling of Ras‑related protein Rab22a, a well‑known oncoprotein. Taken together, the results from the present study suggested that miR‑373 suppresses liver cancer progression and may serve as a promising prognosis prediction biomarker.

Decreased Expression of Long Non-Coding RNA GMDS Divergent Transcript (GMDS-DT) is a Potential Biomarker for Poor Prognosis of Hepatocellular Carcinoma.

BackgroundIncreasing evidence suggests that long non-coding RNA (lncRNA) is closely related to the development of cancer. The present study investigated the potential predictive value of lncRNA GMDS divergent transcript (GMDS-DT) in the prognosis of patients with hepatocellular carcinoma (HCC) after hepatectomy.Material/MethodsGMDS-DT was acquired by microarray data in 3 pairs of M1 and M2 macrophage duplicate samples. Real-time polymerase chain reaction (PCR) was performed to evaluate expression levels of GMDS-DT in liver cancer relative to normal tissue of 198 patients. The significance of GMDS-DT in prognosis after hepatectomy was examined via Kaplan-Meier test and Cox regression analysis.ResultsThe expression of GMDS-DT in liver cancer tissue was significantly lower than that in adjacent normal liver tissue (P<0.001), and was significantly associated with drinking history and metastasis (both P<0.05). The Kaplan-Meier test suggested that patients with lower expression levels of GMDS-DT in liver cancer tissue had significantly shorter disease-free survival and overall survival times after hepatectomy (P=0.028 and P=0.003, respectively). Cox regression analysis further indicated that GMDS-DT was an independent risk factor for disease-free survival and overall survival times of patients after hepatectomy (P=0.015 and P=0.001, respectively).ConclusionsLncRNA GMDS-DT might be a potential biomarker for the prognosis of patients with liver cancer after hepatectomy.

Increased Expression of Serine Hydroxymethyltransferase 2 (SHMT2) is a Negative Prognostic Marker in Patients with Hepatocellular Carcinoma and is Associated with Proliferation of HepG2 Cells.

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) is a key enzyme in one-carbon cell metabolism, including in liver cancer. However, the associations between SHMT2 expression at the gene and protein level and prognosis in patients with hepatocellular carcinoma (HCC) remains unknown. This study aimed to investigate the expression levels of SHMT2 in tumor tissue samples from patients with HCC and clinical outcome and the effects of silencing the expression of the SHMT2 gene in HepG2 cells.Material/MethodsExpression levels of SHMT2 were evaluated in 144 cases of HCC using immunohistochemistry and correlated with clinicopathological factors using the chi-squared (χ2) test. The prognostic significance of SHMT2 expression was analyzed by univariate analysis and multivariate analysis. Twenty pairs of HCC tissue and adjacent normal liver tissue were compared for SHMT2 expression levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR). HepG2 cells underwent SHMT2 gene silencing and MTT and transwell assays investigated cell proliferation and migration. Western blot was used to detect the expression of markers of epithelial-mesenchymal transition (EMT).ResultsExpression levels of SHMT2 in HCC tissues were significantly correlated with tumor grade and hepatitis B virus (HBV) infection, and increased expression was an independent negative prognostic factor in patients with HCC (P=0.003). Increased expression of the SHMT2 gene promoted the proliferation and migration of the HepG2 HCC cell line.ConclusionsIncreased expression of SHMT2 was a negative prognostic biomarker in patients with HCC. Expression of the SHMT2 gene promoted the proliferation and migration of HepG2 HCC cells.

MiR-155, miR-96 and miR-99a as potential diagnostic and prognostic tools for the clinical management of hepatocellular carcinoma.

Increasing evidence has demonstrated that circulating microRNAs (miRNAs) can be utilized as potential biomarkers for the diagnosis of cancer, as well as a prognostic tool for the management of the disease. Therefore, the present study aimed to evaluate the predictive ability of miRNA (miR)-155, miR-96 and miR-99a for the diagnosis and prognosis of hepatocellular carcinoma (HCC). Tissues were collected from 30 patients with HCC and their matched adjacent normal liver tissues, as well as from serum samples from 30 patients with HCC and 30 healthy controls. Reverse transcription-quantitative PCR was used to measure the expression levels of miR-155, miR-96 and miR-99a. The expression levels of miR-155 and miR-96 were upregulated in the tissues and serum of patients with HCC, whereas miR-99a expression levels were decreased. Receiver operating characteristics (ROC) curve analysis revealed that circulating miR-155, miR-96, miR-99a and a combination of these three miRNAs could serve as diagnostic biomarkers for HCC with areas under the curve (AUC) of 0.84, 0.824, 0.799 and 0.931, respectively. Serum α-fetoprotein (AFP) was detected using electrochemiluminescence immunoassay analyzer. The addition of AFP with the combination of these three miRNAs offered a higher accuracy of HCC diagnosis (AUC, 0.979; sensitivity, 90.0%; specificity, 100.0%). In addition, elevated expression levels of miR-155 and miR-96 were associated with poor survival time of patients with HCC. The panel of miR-155, miR-96, miR-99a and AFP had a higher sensitivity and specificity for the diagnosis of HCC when compared with a single marker. Furthermore, the present data suggested that miR-155 and miR-96 may be potential prognostic markers for the clinical management of patients with HCC.

MicroRNA-106b-5p promotes hepatocellular carcinoma development via modulating FOG2.

Background: A recent study has revealed that miR-106b-5p might promote hepatocellular carcinoma (HCC) stemness maintenance and metastasis by targeting PTEN via PI3K/Akt pathway based on HCC cell lines and animal models. Its clinical relevance remains unknown.Purpose: Herein, we aimed to evaluate associations of miR-106b-5p dysregulation with various clinicopathological features of HCC patients and investigate its functions during HCC progression.Patients and methods: At first, miR-106b-5p expression in 130 pairs of HCC and adjacent normal liver tissues was detected by quantitative PCR. Chi-square test was then performed to determine clinical significance. Further investigations on its functions were performed by miRNA target prediction and validation, as well as cellular experiments.Results: miR-106b-5p levels in HCC tissues were significantly higher than those in the adjacent normal liver tissues (P<0.001). High miR-106b-5p expression was significantly associated with advanced tumor stage (P=0.02) and high tumor grade (P=0.03). In addition, Friend of GATA 2 (FOG2) was identified as a direct target of miR-106b-5p in HCC cells. Moreover, the clinical relevance to HCC progression of the combined high miR-106b-5p and low FOG2 expression was more significant than high miR-106b-5p alone. Functionally, enforced expression of miR-106b-5p reduced FOG2 expression and promoted the proliferation and invasion of HCC cells. Furthermore, co-transfection of FOG2 restored the oncogenic roles of miR-106b-5p over-expression.Conclusion: Our data offer the convincing evidence that miR-106b-5p upregulation may promote the aggressive progression of HCC. miR-106b-5p overexpression may promote HCC cell proliferation and invasion by suppressing FOG2, implying its potentials as a promising therapeutic target for HCC patients.

Abstract 2837: Fusobacterium nucleatum and T cells in colorectal cancer liver metastasis

Abstract Background: Accumulating evidence links the intestinal microbiota and colorectal carcinogenesis. Fusobacterium nucleatum has been shown to promote colorectal tumor growth and inhibit antitumor immune responses. Emerging evidence demonstrates an enrichment of Fusobacterium species in colorectal cancer live metastasis (CRLM). Aim: To test the hypothesis that higher amount of Fusobacterium nucleatum is associated with lower density of CD8+ T-cells in CRLM. Method: Genomic DNA was extracted from CRLM and adjacent normal liver tissues. We measured the amount of Fusobacterium nucleatum DNA in 164 CRLM tissues using a quantitative polymerase chain reaction assay. The density of CD8+ T-cells in CRLM was determined by immunohistochemical staining. Result: Fusobacterium nucleatum was detected in 8 (4.9%) of 164 CRLM cases. A higher density of CD8+ T-cells in CRLM was significantly associated with better relapse-free survival (P =0.036). Fusobacterium nucleatum-negative cases, Fusobacterium nucleatum-positive cases were inversely associated with the density of CD8+T-cells (P =0.020). Conclusions: The presence of Fusobacterium nucleatum is inversely associated with CD8+ T-cells density in CRLM. Upon validation, our data may have implications for preventing the formation of liver metastases in colorectal cancer through targeting intestinal microflora. Citation Format: Yuki Sakamoto, Kosuke Mima, Nobuya Daitoku, Yukiharu Hiyoshi, Katsunori Imai, Masaaki Iwatsuki, Takatsugu Ishimoto, Yoshifumi Baba, Shiro Iwagami, Yuji Miyamoto, Yoichi Yamashita, Naoya Yoshida, Hideo Baba. Fusobacterium nucleatum and T cells in colorectal cancer liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2837.

Abstract 4321: Epigenetic regulation of TP73 expression in HCC and GI cancer

Abstract Tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. TP73 is the homologue of the master tumor suppressor TP53 involved in cellular responses to stress and development. The TP73 gene encodes two different protein isoforms, TAp73 and ΔNp73, and maps to a region on chromosome 1p36 that is frequently deleted in neuroblastoma and other tumors, and is thought to contain multiple tumor suppressor genes. However, the analysis of p73-knockout mice yielded conflicting results with respect to tumor suppression. World-wide efforts in sequencing the TP73 gene in patient tumor samples have not provided evidence for genetic alterations as a common cause of TP73 inactivation in human cancer. The role of TP73 in tumorigenesis has remained elusive to date. In this study, we isolated two stem cell lines from normal young and old human liver tissues to determine TP73 expression in normal human liver stem cell and hepatocellular cancer (HCC) cell lines (HepG2, SNU398, SNU449 &amp; SNU475), gastrointestinal cancer (GI) cell lines (Caco2 &amp; HCT116) and a normal skin fibroblast cell line (HS27). The results show that TP73 only expresses in cancer cell lines. Further immunohistochemistry studies on human normal liver tissues as well as adjacent normal and cancerous liver tissues from HCC patients confirm that TP73 only expresses in the cancerous tissues. Moreover, methylation-specific PCR and bisulfite sequencing studies revealed that TP73 promoter is activated only in cancer cell lines by DNA methylation. Furthermore, ChIP assay results demonstrated that CTCF (a chromosomal networking protein CCCTC binding factor) binds to TP73 promoter and regulates TP73 expression. Our observation may prove significant for the development of future therapeutic and diagnostic applications. Citation Format: Zhixing Yao, Cristina Di Poto, Habtom W. Ressom, Zaki A. Sherif. Epigenetic regulation of TP73 expression in HCC and GI cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4321.

&lt;p&gt;HOXA10 knockdown inhibits proliferation, induces cell cycle arrest and apoptosis in hepatocellular carcinoma cells through HDAC1&lt;/p&gt;

BackgroundHomeobox A10 (HOXA10) has been implicated in the development and progression of various human cancers. However, the precise biological functions of HOXA10 in hepatocellular carcinoma (HCC) have not been defined.MethodsIn this study, we examined mRNA expression by quantitative real-time PCR (qRT-PCR) of HOXA10 as well as histone deacetylase (HDAC) and protein levels by Western blot of HOXA10, HDAC1, Cyclin D1, proliferating cell nuclear antigen (PCNA), Survivin and p53 acetylation in HCC tissues and cell lines. We also assessed cell proliferation using Cell Counting Kit-8 (CCK-8) and analyzed cell cycle by flow cytometry. Furthermore, tumor growth of HCC cells in vivo was monitored using the nude mouse xenograft model. Finally, HDAC1 promoter activity and binding in HCC cell lines were detected by luciferase reporter assay and chromatin immunoprecipitation (ChIP), respectively.ResultsWe uncovered the elevated expression of HOXA10 in HCC tissues compared to adjacent normal liver tissues. RNA interference-mediated knockdown of HOXA10 inhibited HCC cell proliferation both in vitro and in vivo. HOXA10 knockdown also induced cell cycle arrest at G0/G1 phase and apoptosis, which were accompanied with the reduced expression of Cyclin D1, PCNA and Survivin. Notably, HOXA10 knockdown enhanced p53 acetylation (Lys382), which is crucial to the activation of p53. Likewise, HOXA10 knockdown suppressed the transcription of HDAC1, a potential deacetylase for p53. In line with these observations, HDAC1 downregulation abrogated the effects of HOXA10 overexpression on proliferation, cell cycle progression, apoptosis and p53 acetylation, indicating the role of HDAC1 in mediating HOXA10 functions.ConclusionOur results demonstrate that HOXA10 knockdown inhibits proliferation, induces cell cycle arrest and apoptosis in HCC cells by regulating HDAC1 transcription.

MicroRNA-450b-3p inhibits cell growth by targeting phosphoglycerate kinase 1 in hepatocellular carcinoma.

Dysregulation of microRNAs frequently contributes to the occurrence and progression of human diseases, including hepatocellular carcinoma (HCC). In this study, the role of miR-450b-3p in HCC was investigated. Gene Expression Omnibus database and HCC specimens were used to evaluate the expression level of miR-450b-3p and the patient's prognosis. Cell functional analyses and tumor xenograft model were used to assess the role of miR-450b-3p in HCC. Bioinformatics was used to predict the downstream target gene of miR-450b-3p, which was verified by dual-luciferase reporter assay. MiR-450b-3p was found to be downregulated in HCC cell lines and tissues, compared with nontransformed immortal hepatic cells and adjacent normal liver tissues, respectively. Lower expression of miR-450b-3p was associated with poor overall survival and disease-free survival in patients with HCC. Ectopic expression of miR-450b-3p inhibited HCC cell viability, colony formation, and cell-cycle progression in vitro, and suppressed the growth of HCC xenograft tumors in vivo. Interestingly, a negative correlation between miR-450b-3p and phosphoglycerate kinase 1 (PGK1) protein was observed among HCC specimens. Additionally, miR-450b-3p inhibited PGK1 expression and phosphorylation of protein kinase B in HCC cell lines. Further experiments confirmed that PGK1 was a direct target of miR-450b-3p. Moreover, restoration of PGK1 abrogated the inhibitory effect of miR-450b-3p on HCC proliferation and cell division. In conclusion, miR-450b-3p is downregulated in human HCC and exerts tumor suppressive effects at least in part by inhibiting PGK1.

Human hepatocellular adenoma as a promising cell source for bioartificial liver systems.

The present study assessed human hepatocellular adenoma (HCA) as a potential source of biological material for bioartificial liver (BAL) systems. The histological characteristics of HCA tissues from 8 patients were examined using hematoxylin and eosin staining. The glycogen synthesis capacity of HCA cells was assessed using Periodic Acid-Schiff (PAS) staining and the expression of genes involved in liver function were examined using immunohistochemical staining (IHC) and reverse transcription-quantitative PCR analysis. Primary cells from HCA tissues were subsequently isolated and cultured in vitro. Cells within HCA tissues from 8 patients exhibited a polygonal shape, similar to that of cells in adjacent normal liver tissues. PAS staining of HCA tissues indicated the capacity of these cells to synthesize and store glycogen. Furthermore, IHC and PCR analyses revealed that the expression of liver function genes in HCA tissues were similar to those observed within normal adjacent liver tissues. Primary cells isolated from HCA tissues exhibited an irregular polygonal shape and positive in vitro growth. The current study demonstrated that HCA tissues exhibit histological and functional characteristics matching those of normal human liver tissue and may therefore be a promising alternative to hepatocytes as a source of biological material for BAL systems.

PGM5: a novel diagnostic and prognostic biomarker for liver cancer.

BackgroundLiver cancer is a common malignancy and a significant public health problem worldwide, but diagnosis and prognostic evaluation remain challenging for clinicians. Metabolic reprogramming is a hallmark of cancer, and we therefore examined the diagnostic and prognostic value of a metabolic enzyme, phosphoglucomutase-like protein 5 (PGM5), in liver cancer.MethodsAll data were from The Cancer Genome Atlas database. R and related statistical packages were used for data analysis. Hepatic PGM5 expression was determined in different groups, and the chi-squared test and Fisher’s exact test were used to determine the significance of differences. The pROC package was used to determine receiver operating characteristic (ROC) curves, the survival package was used to for survival analysis and development of a Cox multivariable model, and the ggplot2 package was used for data visualization.ResultsPGM5 expression was significantly lower in cancerous than adjacent normal liver tissues, and had modest diagnostic value based on ROC analysis and calculations of area under the curve (AUC). Hepatic PGM5 expression had positive associations with male sex and survival, but negative associations with advanced histologic type, advanced histologic grade, advanced stage, and advanced T classification. Patents with low PGM5 levels had poorer overall survival and relapse-free survival. PGM5 was independently associated with patient prognosis.ConclusionPGM5 has potential use as a diagnostic and prognostic biomarker for liver cancer.

Genomic and transcriptional heterogeneity of multifocal hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) often presents with multiple nodules within the liver, with limited effective interventions. The high genetic heterogeneity of HCC might be the major cause of treatment failure. We aimed to characterize genomic heterogeneity, infer clonal evolution, investigate RNA expression pattern and explore tumour immune microenvironment profile of multifocal HCC. Whole-exome sequencing and RNA sequencing were carried out in 34 tumours and 6 adjacent normal liver tissue samples from 6 multifocal HCC patients. Protein expression of Ki67, AFP, P53, Survivin and CD8 was detected by immunohistochemistry. Fluorescence in situ hybridization was carried out to validate the amplification status of sorafenib-targeted genes. We deciphered genomic and transcriptional heterogeneity among tumours in each multifocal HCC patient including mutational profiles, copy number alterations, tumour evolutionary trajectory and tumour immune microenvironment profiles. Of note, sorafenib-targeted alterations were identified in the trunk of phylogenetic tree in only one out of the six patients, which may explain the relative low treatment response rate to sorafenib in clinical practice. Moreover, we demonstrated RNA expression patterns and tumour immune microenvironment profiles of all nodules. We found that RNA expression pattern was associated with Edmondson-Steiner grading. Based on the differential expression of 66 reported immune markers, unsupervised hierarchical clustering analysis of 34 nodules identified immune subsets: one low expression cluster with seven nodules and one high expression cluster with 11 nodules. CD8+ T cells were more enriched in nodules of the high expression cluster. Our study provided a detailed view of genomic and transcriptional heterogeneity, clonal evolution and immune infiltration of multifocal HCC. The heterogeneity of druggable targets and immune landscape might help interpret the clinical responsiveness to targeted drugs and immunotherapy for multifocal HCC patients.

FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression

BackgroundForkhead box M1 (FOXM1) is a proliferation-associated transcription factor of the forkhead box proteins superfamily, which includes four isoforms FOXM1a, b, c, and d. FOXM1 has been implicated in hepatocellular carcinoma (HCC) progression, but the underlying molecular mechanism remains elusive. In this study, we aim to clarify the molecular basis for FOXM1-mediated HCC progression.MethodsBioinformatic analysis was used to explore the differentially expressed genes predicting HCC proliferation. The expression of FOXM1 and kinesin family member (KIF)4A was confirmed by western blotting and immunohistochemistry in HCC tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of FOXM1 and KIF4A on HCC. The effect of FOXM1 on the regulation of KIF4A expression was studied in cell biology experiments. The interaction between KIF4A and FOXM1 was analyzed by chromatin immunoprecipitation and luciferase experiments. A series of experiments was performed to explore the functions of FOXM1/KIF4A in HCC progression, such as cell proliferation, cell growth, cell viability, and cell cycle. A xenograft mouse model was used to explore the regulatory effect of FOXM1-KIF4A axis on HCC tumor growth.ResultsFOXM1 and KIF4A were overexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and are significant risk factors for HCC recurrence and shorter survival. We found that KIF4A was dominantly regulated by FOXM1c among the four isoforms, and further identified KIF4A as a direct downstream target of FOXM1c. Inhibiting FOXM1 decreased KIF4A expression in HCC cells, whereas its overexpression had the opposite effect. FOXM1-induced HCC cell proliferation was dependent on elevated KIF4A expression as KIF4A knockdown abolished FOXM1-induced proliferation of HCC cells both in vitro and in vivo.ConclusionThe FOXM1–KIF4A axis mediates human HCC progression and is a potential therapeutic target for HCC treatment.

The expression of nuclear factor of activated T cells in hepatocellular carcinoma and its relationship with clinicopathologic factors

Objective To analyze the expression of activated T cell nuclear factor (NFAT) in hepatocellular carcinoma (HCC) tissues and its correlation with clinicopathological factors. Methods Data of 105 patients including 87 males and 18 females, aged 55.1±10.8 years old, diagnosed with HCC who underwent hepatectomy in hepatobiliary surgery department of the first central hospital of Tianjin from September 2014 to December 2016 were retrospectively analyzed. Immunohistochemical staining was used to detect the expression of NFAT subtypes in HCC tissues and adjacent normal liver tissues, and the differences in expression of NFAT subtypes and related factors were analyzed. Results HCC tissues had higher expression of NFAT4 and lower expression of NFAT1 compared to adjacent tissues (P<0.05). NFAT1 positive group had higher HBV infected rate (93.1% vs. 78.7%) and lower microvascular invasion rate than that in NFAT1 negative group (24.1% vs.46.8%) (P<0.05). NFAT3 positive group had more younger patients (≤60 years old) (80.0% vs. 60.0%) and higher microvascular invasion rate (46.2% vs. 15.0%) (P<0.05). NFAT4 positive group had higher microvascular invasion rate (43.3% vs. 22.2%) (P<0.05). Conclusion HCC tissues had different expressions of NFATs. The expressions of NFAT1, NFAT3 and NFAT4 are related to microvascular invasion. Key words: Carcinoma, hepatocellular; NFATC transcription factors; Immunohistochemistry; Microvascular invasion

Expression and clinical significance of protein kinase CK2β in hepatocellular carcinoma

Objective To investigate the expression and clinical significance of protein kinase CK2β in hepatocellular carcinoma ( HCC) tissue and the association of CK2β expression with patient prognosis. Methods HCC tissue and adjacent tissue samples were collected from127 HCC patients Who were diagnosed in Department of Hepatopancreatobiliary Surgery, The first Affiliated Hospital of Zhengzhou Universi-ty from Januany 2012 to June 2013, and immunohistochemistry was used to measure the expression of CK2β. The association of CK2β ex-pression with clinical features of HCC patients was analyzed. A total of 20 HCC tissue samples, 20 adjacent tissue samples, 20 cirrhotic tis-sue samples, and 20 normal liver tissue samples were collected from March to June 2018 in our hospital, and Western blot and real-timePCR were used to measure the protein and mRNA expression of CK2β in these tissue samples. A one-way analysis of variance was used forcomparison of means between multiple groups, and Bonferroni test was used for further comparison between two groups; the chi-square testwas used for comparison of categorical data between groups. The Mann-Whitney U test or the Kruskal-Wallis H test was used to investi-gate the association of CK2β expression with clinical features. The Kaplan-Meier method was used for survival analysis, and the log-ranktest was used for comparison between groups. Results Western blot and real-time PCR showed that the expression rate of CK2β in HCCtissue was significantly higher than that in adjacent tissue, cirrhotic tissue, and normal liver tissue ( P 0. 05) , and the expression of CK2β in adjacent tissueand cirrhotic tissue was significantly higher than that in normal liver tissue ( P < 0. 05) . Immunohistochemical staining showed that there wasa significant difference in the positive rate of CK2β between HCC tissue and adjacent tissue in the 127 HCC patients ( 85. 8% vs 63. 0%, P < 0. 001) . There was a significant difference in CK2β expression distribution between HCC patients with different ages ( Z =-2. 277, P = 0. 023) , presence or absence of liver cirrhosis ( Z =-2. 144, P = 0. 032) , different tumor sizes ( Z =-2. 289, P = 0. 004) , or differ-ent Edmondson-Steiner pathological grades ( χ2= 8. 210, P = 0. 016) . The Kaplan-Meier survival curve analysis showed that the stronglypositive CK2β expression group had a significantly shorter postoperative survival time than the moderately positive, weakly positive, and neg-ative CK2β expression groups ( all P < 0. 001) . Conclusion CK2β may be involved in the development and progression of HCC, and itspositive expression is associated with the prognosis of HCC patients.

Overexpression of HexCer and LacCer containing 2-hydroxylated fatty acids in cholangiocarcinoma and the association of the increase of LacCer (d18:1-h23:0) with shorter survival of the patients.

Alteration of glycosphingolipid (GSL) synthesis is observed in many types of cancer. In this study, we have analyzed the expression of sphingolipids and GSLs in cholangiocarcinoma (CCA) tissues and adjacent normal liver tissues. Neutral lipids were extracted from tissue samples using mild-alkaline treatment method followed by TLC and LC-MS analysis. The expression of ceramides, hexosylceramides (HexCer), and lactosylceramides (LacCer) was altered in CCA tissues, 61.1% (11/18) of them showing an increase whereas 38.9% (7/18) showing a decrease, compared with the adjacent normal tissue. Cers and GSLs containing 2-hydroxylated fatty acids except one LacCer molecular species were overexpressed in CCA tissues, and the increase of LacCer (d18:1-h23:0) was correlated with shorter survival of CCA patients, suggesting the involvement of GSL synthesis and fatty acid hydroxylation in progression of CCA. Taken together, we have demonstrated in this study the increase of GSL synthesis and fatty hydroxylation in CCA, which probably be used as a target for CCA treatment.

Thymosin β 10 is overexpressed and associated with unfavorable prognosis in hepatocellular carcinoma.

Thymosin β 10 (TMSB10) has been demonstrated to be overexpressed and function as an oncogene in most types of human cancer including hepatocellular carcinoma (HCC). In our study, we present more evidence about the clinical significance and biological function of TMSB10 in HCC. First, we observed levels of TMSB10 expression were obviously increased in HCC tissues compared with normal liver tissues at The Cancer Genome Atlas (TCGA) datasets. Furthermore, we confirmed that TMSB10 mRNA and protein levels were also increased in HCC tissue samples compared with normal adjacent normal liver tissue samples. In addition, we found high TMSB10 expression was remarkably associated with the advanced tumor stage, large tumor size, distant metastasis, and poor prognosis, and acted as an independent factor for predicting poor overall survival in HCC patients. Loss-of-function studies suggested silencing of TMSB10 expression dramatically reduced cell proliferation, migration, and invasion in HCC. In conclusion, TMSB10 may hold promise as a tumor biomarker for predicting prognosis and a potential target for developing a novel therapeutic strategy.

Clinicopathological significance of miR-27b targeting Golgi protein 73 in patients with hepatocellular carcinoma.

Using five bioinformatics analysis software, we identified Golgi protein 73 (GP73) as a putative target of microRNA-27b (miR-27b), which is closely related to various biological processes or diseases such as bone metabolism disease, adipose cell and muscle cell development, pulmonary hypertension, cervical cancer, and breast cancer. However, the clinical significance of miR-27b in hepatocellular carcinoma (HCC) is still unclear. The differential expression of miR-27b in HCC and adjacent normal liver tissues was measured by quantitative reverse transcription PCR. Our results showed that the expression of miR-27b in tumor tissues is lower than that in adjacent nontumor tissues. The expression of miR-27b was significantly lower in HCC tissues with high expression of GP73, when compared with adjacent nontumor tissues. Moreover, down-regulated expression of miR-27b was closely correlated with serum GP73, tumor-node-metastasis stage, tumor size, and portal vein thrombosis. GP73 mRNA might be a target of miR-27b. The 5-year overall survival rate of the low miR-27b expression group was significantly lower than that of the high miR-27b expression group. Moreover, multivariate analysis of prognostic factors, with a Cox proportional hazards model, showed that low miR-27b expression was a significant and independent predictor of poor survival in HCC. Hence, the abnormal expression of miR-27b might be related to the occurrence and development of tumors. Similarly, a study in the Cancer Genome Atlas database demonstrated that the expression of miR-27b in 50 normal individuals was 1.6 times higher than that of 372 patients with liver cancer. The overall survival rate of the low GP73 expression group (275 liver cancer patients) was significantly longer than that of the high GP73 expression group (90 normal individuals). MiR-27b suppresses the expression of GP73 and is therefore a potential prognostic biomarker and therapy target in HCC.

Peroxiredoxin2 downregulation enhances hepatocellular carcinoma proliferation and migration, and is associated with unfavorable prognosis in patients.

It has been revealed by our previous proteomic study that the expression profile is different between well-differentiated and poorly differentiated hepatocellular carcinoma (HCC). Among those differently expressed proteins, peroxiredoxin2 (PRDX2) was our protein of interest. The present study aimed to further investigate the value of PRDX2 as a prognostic factor in HCC. Tissue microarrays were used to investigate the expression difference between HCC tissues and their adjacent normal liver tissues. The expression of PRDX2 at both mRNA and protein levels was examined by q-RT-PCR, western blotting and immunohistochemical assessment in HCC tissues and cell line HCCLM3. Silencing of PRDX2 in HCCLM3 was achieved usingpGMLV-SC1 lentiviral vectors. Cell Counting Kit-8 (CCK-8) and Transwell migration assays were used to assess cell proliferation and migration, respectively. Categorical variables were assessed using the Chi-square test, and ordinal variables were examined using the Mann-Whitney U test. The difference of continuous variables between groups were compared with t-tests. The Kaplan-Meier method was used to calculate the overall survival (OS) and disease-free survival (DFS) of patients, and the log-rank test was used to analyze the differences between groups. The results revealed that the expression of PRDX2 was decreased at both the mRNA and protein levels in an HCC cell line compared to that of a normal human liver cell line. PRDX2 protein expression levels were significantly downregulated in HCC tissues and were positively linked to overall survival (OS) and disease-free survival (DFS) of HCC patients. Patients with high PRDX2 expression levels had longer OS and DFS times than those with lower PRDX2 expression. Silencing of PRDX2 in the HCC cell line HCCLM3 promoted cancer cell proliferation and migration. Our findings indicated that PRDX2 may play an important role in HCC development; PRDX2 may serve as a useful prognostic factor and a therapeutic target.