Leukemia inhibitory factor receptor (LIFR) is detected as a novel suppressor gene of hepatocellular carcinoma using double-combination array

Hepatocellular carcinoma (HCC) is one of the world’s top five causes of cancer-related deaths and treatment options are limited in this type of cancer. In the present study, we attempt to identify the novel suppressor genes of HCC using a double-combination array we designed. Leukemia inhibitory factor receptor (LIFR) is one of suppressor genes using this method. We found that 23 of 48 (47.9%) tumor tissues showed promoter hypermethylation of LIFR gene, and the expression level was clearly reduced in tumor tissues (P < 0.0001). The present study suggests that our method is a meaning technique able to detect novel genes and that LIFR gene is a new suppressor gene of HCC.     

Silencing of Hint1, a novel tumor suppressor gene,by promoter hypermethylation in hepatocellular carcinoma

The Hint1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. Previous studies in mice provided evidence that Hint1 may be haploinsufficient with respect to its function as a tumor suppressor. In the present study, we investigated the aberrant methylation of Hint1 and explored possible relationships between aberrant methylation and clinicopathological features in hepatocellular carcinoma (HCC). Hypermethylation of Hint1 was evaluated by the methylation specific PCR (MSP) method in 40 patients with HCC (tumor and paired adjacent non-tumor tissues) from Taiwan, 22 cases of normal liver tissue (14 from Taiwan and 8 from the US). HINT1 expression in tissues was detected by immunohistochemistry. The frequencies of hypermethylation of Hint1 in tumor, paired adjacent non-tumor and normal liver tissue were 55.0%, 37.5% and 9.1%, respectively. A statistically significant inverse association was found between Hint1 methylation status and expression of the HINT1 protein in tumor tissues (p = 0.003). The relationship between Hint1 methylation status and clinical features and other, previously measured biomarkers was also analyzed. p16 hypermethylation was statistically significantly associated with Hint1 methylation status (p = 0.035). There were no correlations between Hint1 methylation and hepatitis B (HBV) or hepatitis C (HCV) infection status or aflatoxin B₁ (AFB₁-) and polycyclic aromatic hydrocarbons (PAHs)-DNA adduct levels. These results suggest that promoter hypermethylation of Hint1 may play a role in hepatocarcinogenesis.     

HTPAP gene on chromosome 8p is a candidate metastasis suppressor for human hepatocellular carcinoma

Our previous studies suggested that chromosome 8p deletion is associated with metastasis of hepatocellular carcinoma (HCC), in which some novel metastasis suppressor genes might be harbored. The present study aimed to identify the metastatic suppressor gene(s). A cDNA chip was constructed with the expressed sequence tags (ESTs) from chromosome 8p and used to compare the difference of expression profiling between the MHCC97-H and MHCC97-L cell lines with different metastatic potentials and similar genetic backgrounds. In all, 10 ESTs were significantly downregulated in MHCC97-H cell line with higher metastatic potential. One full-length gene, HTPAP (phosphatidic acid phosphatase type 2 domain containing 1B), was identified at chromosome 8p12. Sequencing and bioinformatic analyses revealed that HTPAP has 826 bp and encodes a putative protein of 175 amino acids with a transmembrane segment at the NH2 terminus, two protein kinase C phosphorylation site and one tyrosine kinase phosphorylation site. Its expression level in metastatic tumor tissues was much lower than that of primary HCC tissues. Both in vitro and in vivo assays suggested that HTPAP could suppress the invasion and metastasis of HCC. These suggested that HTPAP is a novel metastatic suppressor gene for HCC. The mechanism of the effect of HTPAP on HCC metastasis is not clear yet and deserves further investigation.     

NDRG1 as a biomarker for metastasis,recurrence and of poor prognosis in hepatocellular carcinoma

N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.     

MicroRNA-107: a novel promoter of tumor progression that targets the CPEB3/EGFR axis in human hepatocellular carcinoma

MicroRNAs (miRNAs) are dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC). MiR-107 has been implicated in several types of cancer regulation; however, relatively little is known about miR-107 in human HCC. In the present study, we showed that the overexpression of miR-107 accelerates the tumor progression of HCC in vitro and in vivo through its new target gene, CPEB3. Furthermore, our results demonstrated that CPEB3 is a newly discovered tumor suppressor that acts via the EGFR pathway. Therefore, our study demonstrates that the newly discovered miR-107/CPEB3/EGFR axis plays an important role in HCC progression and might represent a new potential therapeutic target for HCC treatment.     

一个肝癌相关基因的生物信息学及其基因表达分析

目的 :筛选并克隆肝癌相关基因 ,探讨肝癌发病机制。方法 :在国家人类基因组南方研究中心肝癌基因表达谱及其功能研究的工作基础上 ,发现 1个未知功能基因 (暂命名LLC基因 ) ,其DNA拷贝数在肝癌基因组中呈降低的趋势。本研究利用生物信息学和RT PCR的方法 ,对其进行功能预测并在转录组水平检测该基因的表达。结果 :LLC基因定位于染色体8p2 3 3区段 ,全长为 670 6bp ,含有 2个外显子 ,1个内含子 ,其开放阅读框长为 1871bp ,编码 1个含有 62 3个氨基酸的蛋白。该基因在 67%( 16/2 4)肝癌组织中呈现下调趋势。同时其在人体内多种组织均有表达。结论 :LLC基因有可能是一个新的肝癌相关抑癌基因     

CpG island hypermethylation of SOCS-1 gene is inversely associated with HBV infection in hepatocellular carcinoma

The CpG island hypermethylation of the suppressor of cytokine signaling-1 (SOCS-1) gene is frequently methylated in hepatocellular carcinoma (HCC), but its clinicopathological significance and the potential risk factors for the epigenetic change in HCC remain poorly understood. The methylation status of SOCS-1 CpG island was evaluated in fresh-frozen tissues from 284 HCC patients using the methylation-specific polymerase chain reaction. The expression of SOCS-1 protein was analyzed by immunohistochemical staining. SOCS-1 methylation was found in 163 (57%) of 284 HCCs. SOCS-1 methylation was positively associated with patient age (P = 0.002) and HCV infection status (P = 0.004), and was inversely associated with HBV infection (P = 0.0002). In the multivariate logistic regression analysis, the HB_sAg-negative HCCs showed a 2.78 (95% CI = 1.31–5.89, P = 0.007) times greater risk of SOCS-1 methylation than the HB_sAg-positive HCCs. SOCS-1 methylation also occurred at a 4.34 times (95% CI = 1.24–14.25, P = 0.02) higher prevalence in antiHCV-positive cases than in antiHCV-negative cases. No prognostic effect of SOCS-1 methylation was observed in the HCCs. In conclusion, the present study suggests that SOCS-1 methylation in HCC may be negatively associated with HB_sAg status.     

Glypican-5 is a novel metastasis suppressor gene in non-small cell lung cancer

Glypican-5 (GPC5) may be a potential tumor suppressor gene in non-small cell lung cancer (NSCLC). The present study aimed to clarify the GPC5 expression pattern and to explore its potential functions in NSCLC. The expression of GPC5 gene was lower in lung cancer tissues compared with adjacent noncancerous tissues. The GPC5 gene expression in the lymph node metastasis group was remarkably lower than that in the non-metastasis group. The tissue microarray (TMA) study found that the overall survival rate of GPC5-positive group was significantly higher than that of GPC5-negative group in AC subgroup. Overexpressing GPC5 in NSCLC cell lines significantly suppressed their migration, invasion, and proliferation activities and also induced G1/S phase arrest of the cells in vitro. Our data suggest that GPC5 is a novel metastasis suppressor gene in NSCLC and may be a potential biomarker that predicts NSCLC metastasis.     

Role of ZIC1 methylation in hepatocellular carcinoma and its clinical significance

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in humans, and its prognosis is generally poor even after surgery. The zinc finger of the cerebellum (ZIC1) gene is a novel tumor suppressor gene that plays a crucial role in vertebrate development. Altered expression of ZIC1 is observed in various types of human cancers. The aims of the present study were to investigate the methylation status of ZIC1 in HCC and evaluate its clinical implication. The methylation status of ZIC1 was analyzed in 132 pairs of HCC and corresponding noncancerous tissues by methylation-specific polymerase chain reaction (PCR) (MSP). The expression of ZIC1 messenger RNA (mRNA) in HCC tissues was examined by real-time PCR. Methylation frequency of ZIC1 in HCC was significantly higher than that in the corresponding noncancerous tissues (P?P?=?0.022), histological differentiation (P?=?0.033), and tumor stage (P?=?0.009). The downregulation of the ZIC1 mRNA expression in HCC was correlated with the ZIC1 methylation (P?P?mRNA and associated with poor survival in HCC. Overall, aberrant methylation is an important mechanism for ZIC1 inactivation in HCC, and ZIC1 methylation may be a promising biomarker for the diagnosis and prognosis of HCC.     

Hepatitis B Virus X Protein Inhibits Tumor Suppressor miR-205 through Inducing Hypermethylation of miR-205 Promoter to Enhance Carcinogenesis

The infection of hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma (HCC), in which HBV X protein (HBx) plays crucial roles. MicroRNAs are involved in diverse biologic functions and in carcinogenesis by regulating gene expression. In the present study, we aim to investigate the underlying mechanism by which HBx enhances hepatocarcinogenesis. We found that miR-205 was downregulated in 33 clinical HCC tissues in comparison with adjacent noncancerous hepatic tissues. The expression levels of miR-205 were inversely correlated with those of HBx in abovementioned tissues. Then, we demonstrated that HBx was able to suppress miR-205 expression in hepatoma and liver cells. We validated that miR-205 directly targeted HBx mRNA. Ectopic expression of miR-205 downregulated HBx, whereas depletion of endogenous miR-205 upregulated HBx in hepatoma cells. Notably, our data revealed that HBx downregulated miR-205 through inducing hypermethylation of miR-205 promoter in the cells. In terms of function, the forced miR-205 expression remarkably inhibited the HBx-enhanced proliferation of hepatoma cells in vitro and in vivo, suggesting that miR-205 is a potential tumor-suppressive gene in HCC. HBx-transgenic mice showed that miR-205 was downregulated in the liver. Importantly, HBx was able to abrogate the effect of miR-205 on tumor suppression in carcinogenesis. Therefore, we conclude that HBx is able to inhibit tumor suppressor miR-205 to enhance hepatocarcinogenesis through inducing hypermethylation of miR-205 promoter during their interaction. Therapeutically, miR-205 may be useful in the treatment of HCC.     

Aberrant TIG1 methylation associated with its decreased expression and clinicopathological significance in hepatocellular carcinoma

Recently, it has been reported that tazarotene-induced gene 1 (TIG1) methylation was frequently detected in a variety of human cancers. However, the relationship between the TIG1 methylation and the characteristics of hepatocellular carcinoma (HCC) remains unknown. The aim of present study was to observe the promoter methylation of TIG1 in HCC tissues and assess its prognostic significance for HCC. Real-time quantitative polymerase chain reaction and methylation-specific polymerase chain reaction were used, respectively, to examine the mRNA expression and methylation status of TIG1 in 91 pairs of HCC and adjacent noncancerous tissues. The mRNA expression level of TIG1 was significantly lower in HCC tissues than in adjacent noncancerous tissues. The rate of TIG1 promoter methylation was significantly higher in HCC tissues than in adjacent noncancerous tissues (P?<?0.001). A strong correlation between downregulation and promoter methylation was found in these tumors (P?<?0.001). More importantly, TIG1 methylation status was related to tumor size (P?=?0.015), histological differentiation (P?=?0.004), and tumor stage (P?<?0.001). Kaplan–Meier survival analysis showed that TIG1 promoter hypermethylation was associated with a worse outcome in patients with HCC. Further, Cox multivariate analysis indicated that TIG1 methylation status was an independent prognostic factor for the overall survival rate of HCC patients. In conclusion, our data suggested that epigenetic silencing of TIG1 gene expression by promoter hypermethylation may play an important role in HCC.     

肝癌中具有高突变率位于染色体17p13.3区的新抑癌基因

目的　以往研究表明肝癌中染色体 1 7p1 3 .3区有高频率的杂合性缺失。其最小杂合性缺失范围已被确定在D1 7S643至D1 7S1 574位点间 ,而且其中的D1 7S92 6位点有最高的杂合性缺失率。含有该位点的基因组克隆P579已被测序分析 ,在P579范围共有 1 3个新基因。这里报告其中的一个新基因 (命名为肝癌抑癌基因 1 ,HCCS1 )的克隆和特性研究结果。方法　利用直接杂交筛选方法获得基因组克隆P579中的基因克隆。根据HCCS1基因克隆的cDNA序列与基因组序列进行比较确定基因的外显子与内含子。应用RT PCR扩增组织中的HCCS1基因 ,序列测定检查突变。应用免疫组化检测HCCS1在组织中的表达。应用克隆形成试验和裸鼠成瘤试验检测HCCS1的生物学功能。结果　HCCS1有 1 8个外显子 ,cDNA全长约2 .0kb ,蛋白产物定位于线粒体。HCCS1在肝癌组织中有高频率的突变 ,免疫组化检测表明HCCS1在癌旁组织的表达明显高于癌组织。HCCS1转染肝癌细胞明显抑制其克隆的形成及在裸鼠体内的成瘤。结论　上述发现表明HCCS1具有肝癌抑癌基因的作用     

Integrated bioinformatics analyses of key genes involved in hepatocellular carcinoma immunosuppression

Hepatocellular carcinoma (HCC) is a typical inflammation-driven cancer. Chronically unresolved inflammation may remodel the immunosuppressive tumor microenvironment, which is rich in innate immune cells. The mechanisms via which HCC progresses through the evasion of the innate immune surveillance remain unclear. The present study thus aimed to identify key genes involved in HCC immunosuppression and to establish an innate immune risk signature, with the ultimate goal of obtaining new insight into effective immunotherapies. HCC and normal liver tissue mRNA expression and clinicopathological data were obtained from the Cancer Genome Atlas database. The immunosuppressive innate immune-related genes (IIRGs) in HCC were screened using integrated bioinformatics analyses. Gene expression was then validated using the Gene Expression Omnibus database and the Human Protein Atlas database, and tissues were obtained from patients with HCC who underwent surgery. In total, 3,676 genes were identified as differentially expressed mRNAs after comparing the HCC tissues with the normal liver tissues in TCGA. Gene Set Enrichment Analyses revealed 21 highly expressed IIRGs in HCC tissues. A survival analysis and Cox regression model were used to construct an innate immune risk signature, including three IIRGs: Collectin-12 (COLEC12), matrix metalloproteinase-12 (MMP12) and mucin-12 (MUC12) genes. Univariate and multivariate Cox analyses revealed that the signature of the three IIRGs was a robust independent risk factor in relation to the overall survival (OS) of patients with HCC. The expression of the three aforementioned IIRGs was confirmed through external validation. Moreover, COLEC12 and MMP12 expression significantly correlated with that of immune checkpoint molecules or immunosuppressive cytokines. The tumor immune dysfunction and exclusion tool predicted that the increased expression of the three IIRGs in patients with HCC was significantly associated with the efficacy of relatively poor immune checkpoint blockade therapy. Conclusively, a novel innate immune-related risk signature for patients with HCC was constructed and validated. This signature may be involved in immunosuppression, and may be used to predict a poor prognosis, functioning as a potential immunotherapeutic target for patients with HCC.     

Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection

Background

Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen and may stimulate the proliferation and invasiveness of human hepatocellular carcinoma (HCC) cells through the c-met receptor. This study evaluates the significance of serum HGF levels in patients undergoing HCC resection.

Study design

The peripheral and portal sera and HCC and non-tumorous tissues of 40 HCC patients, with tumor TNM stage I (n = 12), II (n = 17), and III (n = 11) diseases, who underwent hepatic resection were prospectively collected. Serum HGF levels were determined by enzyme-linked immunosorbent assay. The c-met protein expressions were examined by immunohistochemistry. Median follow-up time was 69 months.

Results

The prehepatectomy portal HGF levels (median, 622 pg/mL) were significantly higher than peripheral HGF levels (564 pg/mL) (P = 0.026). The posthepatectomy portal HGF levels (699 pg/mL) were significantly higher than prehepatectomy portal HGF levels (P < 0.001). C-met expression was detected in 87.5% HCC and in 85.0% non-tumorous liver tissues. By Cox multivariate analysis, posthepatectomy portal HGF level >699 pg/mL (P < 0.001), multiple tumors (P = 0.042), and TNM stages II (P = 0.019) and III (P = 0.009) were independent factors related with survival. Patients with a posthepatectomy portal HCG level >699 pg/mL and with a positive c-met expression in HCC tissue have the worst survival.

Conclusions

In HCC patients, high peripheral and portal HGF serum levels related with poor prognosis after hepatic resection. Hepatocyte growth factor and c-met receptor can be targets of future HCC postoperative treatment.