TIM-3基因多态性与肝细胞癌的关联性研究

目的:分析TIM-3 rs4704853、rs1036199和rs10515746基因多态性是否与肝细胞癌（HCC）易感性存在关联。方法:纳入HCC患者342例,健康对照组350例。采用血液基因组提取试剂盒（离心柱法）提取全基因组DNA。SNP分型采用多重扩增及高通量测序技术,采用多元Logistic回归方法分析TIM-3基因多态性与HCC易感性的关联性;采用多个独立样本秩和检验分析TIM-3基因多态性与HCC临床指标的关联。结果:TIM-3基因rs4704853、rs1036199和rs10515746存在完全连锁不平衡,以上3个SNP位点杂合子（OR=6.378,95%CI=1.414~28.776,P=0.016）或突变等位基因（OR=6.270,95%CI=1.396~28.165,P=0.017）患肝癌风险增加,且杂合子组血清TBA（总胆汁酸）水平明显高于野生纯合子组（P＜0.05）。结论:TIM-3 rs4704853、rs1036199和rs10515746基因多态性可能与HCC的发生和TBA水平具有关联性。     

microRNA-622 acts as a tumor suppressor in hepatocellular carcinoma

microRNAs (miRNAs) are important regulators of tumor development and progression. In this study, we aimed to explore the expression and role of miR-622 in hepatocellular carcinoma (HCC). We found that miR-622 was significantly downregulated in human HCC specimens compared to adjacent noncancerous liver tissues. miR-622 downregulation was significantly associated with aggressive parameters and poor prognosis in HCC. Enforced expression of miR-622 significantly decreased the proliferation and colony formation and induced apoptosis of HCC cells. In vivo studies demonstrated that miR-622 overexpression retarded the growth of HCC xenograft tumors. Bioinformatic analysis and luciferase reporter assays revealed that miR-622 directly targeted the 3′-untranslated region (UTR) of mitogen-activated protein 4 kinase 4 (MAP4K4) mRNA. Ectopic expression of miR-622 led to a significant reduction of MAP4K4 expression in HCC cells and xenograft tumors. Overexpression of MAP4K4 partially restored cell proliferation and colony formation and reversed the induction of apoptosis in miR-622-overexpressing HCC cells. Inhibition of JNK and NF-κB signaling phenocopied the anticancer effects of miR-622 on HCC cells. Taken together, miR-622 acts as a tumor suppressor in HCC and restoration of miR-622 may provide therapeutic benefits in the treatment of HCC.     

Expression profiling of serum microRNA-101 in HBV-associated chronic hepatitis,liver cirrhosis,and hepatocellular carcinoma

MicroRNAs (miRNAs) represent a class of evolutionarily conserved, non-coding small RNAs (18–25 nt) that have emerged as master regulators of several biological processes. Recently, circulating miRNAs have also been reported to be promising biomarkers for various pathological conditions. In the present study, we report the comparative expression profiling of microRNA-101 (miR-101) in serum and tissue samples from chronic hepatitis B (CHB), HBV-associated liver cirrhosis (HBV-LC), and HBV-associated hepatocellular carcinoma (HBV-HCC) patients and healthy controls. The serum miR-101 levels were found to be significantly downregulated in the HBV-HCC patients compared with the HBV-LC patients (P < 0.001), CHB patients (P < 0.001) and healthy controls but were upregulated in the HBV-LC patients compared with the CHB patients (P < 0.001) and healthy controls (P < 0.001). Consistent with the serum data, the expression of miR-101 was also upregulated and downregulated in the HBV-LC and HBV-HCC tissue samples, respectively. A receiver operating characteristic (ROC) analysis of serum miR-101 yielded an area under the ROC curve (AUC) of 0.976 with 95.5% sensitivity and 90.2% specificity when differentiating between HBV-HCC and HBV-LC. Our results suggest that the serum miR-101 level can serve as a potential non-invasive biomarker to differentiate HBV-HCC from HBV-LC.     

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.     

肝细胞癌组织microRNA-187-3p表达预后意义

目的 微小RNA (microRNA,miRNA)差异表达与多种肿瘤的发生发展密切相关,但微小RNA-187-3p(miR-187-3p)在肝细胞癌(hepatocellular carcinoma,HCC)中的表达及预后意义研究较少.本研究检测HCC组织中microRNA的表达谱,分析miR-187-3p在HCC组织中的表达及其与临床病理特征之间的关系,探讨miR-187-3p表达在其发生、发展及预后中的作用.方法 利用microRNA芯片杂交技术筛选5例HCC组织及相应癌旁组织中差异表达的microRNA,然后通过实时荧光定量PCR(real-time fuorescence quantitative-PCR,RFQ-PCR)法验证86例HCC组织及相应癌旁组织中miR-187-3p表达水平,并统计临床病理资料,随访生存期.结果 148个microRNAs在HCC组织中差异表达,其中102个microRNAs在HCC组织较相应癌旁组织表达下调,46个microRNAs在HCC组织较相应癌旁组织表达上调.RFQ-PCR结果显示,miR-187-3p在HCC组织中表达水平明显低于相应癌旁组织,差异有统计学意义,Z=-2.244,P=0.025.HCC中miR-187-3p表达状态与肿瘤大小(x2=5.031,P=0.025)相关,而与性别(x2=3.648,P=0.056)、年龄(x2=0.003,P=0.956)、是否转移(x2=0.005,P=0.943)、TNM分期(x2=0.129,P=0.719)、肿瘤数目(x2 =0.126,P=0.722)、乙肝表面抗原(x2=0.019,P=0.890)、AFP水平(x2=0.187,P=0.665)和Child分级(x2=1.665,P=0.197)无关.miR-187-3p低表达的HCC患者总生存期较高表达者明显缩短,x2=7.684,P=0.006.且多因素分析发现,miR-187-3p表达水平和TNM分期是影响HCC患者生存预后的独立危险因素.结论 miR-187-3p在一定程度上参与了HCC的发生发展,其有望成为HCC新的潜在的治疗靶点以及诊断、判断病情和预测预后的分子标志物.     

Plasma and tumoral glypican‐3 levels are correlated in patients with hepatitis C virus‐related hepatocellular carcinoma

Glypican‐3 (GPC3) is a cancer antigen expressed in approximately 80% of hepatocellular carcinomas (HCC) and is secreted into the blood. To confirm the effectiveness of GPC3 as a biomarker in HCC, we analyzed the relationship between GPC3 expression levels in cancer cells and in blood in 56 patients with HCC. Preoperative plasma GPC3 levels were determined with an immunoassay, and expression of GPC3 in resected tumors was analyzed by immunohistochemical staining. Median plasma GPC3 level in all HCC cases was 4.6 pg/mL, and tended to be higher in patients with hepatitis C virus (HCV)‐related HCC (HCV group) (9.9 pg/mL) than in patients with hepatitis B virus (HBV)‐related HCC (HBV group) (2.6 pg/mL) or in those without virus infection (None group) (3.0 pg/mL), suggesting that the virus type most likely influences GPC3 secretion. Median percentage of GPC3⁺ cells in tumors was also higher in the HCV (26.2%) and HBV (11.1%) groups than in the None group (4.2%). In the HCV group, there was a positive correlation between the two parameters (r = 0.66, P < .01). Moreover, receiver operating characteristic analysis predicted >10% GPC3⁺ cells in a tumor if the cut‐off value was 6.8 pg/mL (sensitivity 80%, specificity 100%; area under the curve 0.875, 95% confidence interval 0.726‐1) in the HCV group. Plasma concentration of GPC3 could be a predictive marker of tumoral GPC3 expression in patients with HCV‐related HCC, suggesting a useful biomarker for immunotherapies targeting GPC3, although larger‐scale validations are needed.     

Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma

MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment.     

血清磷脂酰肌醇蛋白聚糖3在肝细胞癌中的表达及其与临床病理因素的相关性

目的 初步探讨肝细胞癌（HCC）患者血清和组织中磷脂酰肌醇蛋白聚糖3（GPC3）在HCC诊断中的临床应用价值.方法 采用酶联免疫吸附试验（ELISA）法和免疫组织化学染色法检测79例HCC患者血清和组织中GPC3表达水平,并与35例肝炎后肝硬化患者和30例健康体检者GPC3含量检测结果进行比较,采用Logistic回归模型对HCC患者GPC3表达水平与影响因素进行分析.结果 HCC组患者血清GPC3水平[（143.02±40.26） μg/L]明显高于肝硬化患者组[（6.15±4.31） μg/L]和健康对照组[（4.47±3.22）μg/L],三组间比较差异均有统计学意义（均P＜0.01）;GPC3阳性表达水平在HCC组织高于癌旁和远癌肝组织;GPC3蛋白表达与患者年龄、性别、肿瘤大小及甲胎蛋白（AFP）水平无明显相关性;与临床分期（x2 =4.250,P＜0.05）及肿瘤有无远处转移（x2=13.182,P＜ 0.001）关系密切.结论 GPC3检测对于HCC的诊断具有较好的特异性,联合血清AFP更有利于提高HCC诊断的敏感性,可作为HCC患者诊断及预后评估的标志物.     

microRNA-506 regulates proliferation,migration and invasion in hepatocellular carcinoma by targeting F-spondin 1 (SPON1)

Our previous study indicates microRNA-506 (miR-506) is downregulated in hepatocellular carcinoma (HCC). In the current study, we investigate the effects of miR-506 on proliferation, migration and invasion in HCC. We report that enforced expression of miR-506 inhibits proliferation, migration and invasion in vitro, and suppresses tumor growth in vivo. Conversely, suppression of miR-506 exhibits promoting effects on proliferation, migration and invasion in vitro, and on tumor growth in vivo. In addition, miR-506 binds to the 3’UTR of F-spondin 1(SPON1), and enforced expression of miR-506 decreases accumulation of SPON1. Moreover, enforced expression of SPON1 and suppression of SPON1 alleviates effects of miR-506 mimics and inhibitors on proliferation, migration and invasion in vitro, respectively. In conclusion, microRNA-506 regulates proliferation, migration and invasion in HCC by targeting SPON1.     

血清AFP、GP73、GPC3单独及联合检测对肝细胞癌的诊断价值

目的探讨血清中AFP、GP73、GPC3三种肿瘤标志物在肝细胞癌(hepatocellular carcinoma,HCC)中的诊断价值及联合检测的意义。方法检测了45例HCC患者、32例乙肝携带者和30例正常体检者血清中AFP、GP73、GPC3的含量并进行相关分析。结果 AFP、GP73、GPC3用于诊断HCC时的敏感度和特异度分别是57.8%和90.6%、80.0%和98.3%、31.1%和92.3%,ROC曲线下面积分别为0.874、0.963、0.507;AFP与GP73联合,AFP与GPC3联合,GP73与GPC3联合以及三种标志物联合时敏感度和特异度分别是93.9%和88.9%、73.3%和83.8%、84.4%和91.5%、95.6%和82.1%,ROC曲线下面积分别为0.976、0.821、0.963、0.976。结论联合检测血清中AFP和GP73对HCC的诊断具有重要价值,血清GPC3检测对HCC的诊断意义较小。     

Comprehensive profiling of novel microRNA-9 targets and a tumor suppressor role of microRNA-9 via targeting IGF2BP1 in hepatocellular carcinoma

MicroRNA-9 (miR-9) dysregulation is implicated in a variety of human malignancies including hepatocellular carcinoma (HCC), but its role remains contradictory. In this study, we explored the expression and methylation status of miR-9 in HCC samples, as well as the tumor-related functions of miR-9 in vitro. Bioinformatics analysis, array-based RNA expression profile, and literature retrieval were used to identify miR-9 targets in HCC. The potential downstream candidates were then validated by luciferase reporter assay, real-time quantitative PCR, and western blot or enzyme linked immunosorbent assay (ELISA). The expression status and clinicopathologic significances of miR-9 target genes in clinical samples were further explored. The results showed that miR-9 was frequently downregulated in primary HCC. Its silencing was largely contributed by a high frequency (42.5%) of mir-9-1 hypermethylation, which was correlated with bigger tumor size (P = 0.0234). In vitro functional studies revealed that miR-9 restoration retarded HCC cell proliferation and migration. IL-6, AP3B1, TC10, ONECUT2, IGF2BP1, MYO1D, and ANXA2 were confirmed to be miR-9 targets in HCC. Among them, ONECUT2, IGF2BP1, and ANXA2 were confirmed to be aberrantly upregulated in HCC. Moreover, upregulation of ONECUT2, IGF2BP1, and IL-6 were significantly associated with poor post-surgery prognosis (P = 0.0458, P = 0.0037 and P = 0.0461, respectively). Mechanically, miR-9 plays a tumor suppressive role partially through a functional miR-9/IGF2BP1/AKT&ERK axis. Our study suggests that miR-9 functions as a tumor suppressor in HCC progression by inhibiting a series of target genes, including the newly validated miR-9/IGF2BP1/AKT&ERK axis, thus providing potential therapeutic targets and novel prognostic biomarkers for HCC patients.     

Arginase-1 is a more sensitive marker of hepatic differentiation than HepPar-1 and glypican-3 in fine-needle aspiration biopsies

BACKGROUND:

Distinguishing hepatocellular carcinoma (HCC) from adenocarcinoma in fine‐needle aspiration biopsies (FNAB) is often diagnostically challenging. Arginase‐1 was recently described as a marker of hepatic differentiation in surgical resection specimens. We compared the expression of arginase‐1, HepPar‐1, and glypican‐3 in FNAB of HCC and adenocarcinoma involving the liver.

METHODS:

Ninety‐eight FNABs including 37 primary or metastatic HCCs (30 well or moderately differentiated and 7 poorly differentiated) and 61 adenocarcinomas involving the liver were evaluated for immunohistochemical expression of arginase‐1, HepPar‐1, and glypican‐3 using formalin‐fixed paraffin‐embedded cell block material.

RESULTS:

Arginase‐1 was more sensitive (81%) than HepPar‐1 (70%) or glypican‐3 (54%) for HCC. Arginase‐1 more often demonstrated diffuse staining, defined as reactivity in >50% of the tumor, in HCC (21 of 37; 57%) compared with HepPar‐1 (15 of 37; 41%) and glypican‐3 (12 of 37; 32%). Of the 7 poorly differentiated HCCs, 3 (43%) were immunoreactive for both arginase‐1 and glypican‐3, whereas only 1 (14%) demonstrated HepPar‐1 staining. Arginase‐1 expression was identified in adenocarcinomas of pancreatic, colorectal, and breast origin, and reactivity was diffuse in 2 pancreatic adenocarcinomas (2 of 15; 13%).

CONCLUSIONS:

Arginase‐1 is a more sensitive marker of hepatic differentiation than either HepPar‐1 or glypican‐3 in FNAB. In addition, arginase‐1 exhibits more diffuse staining in HCC than either HepPar‐1 or glypican‐3, making interpretation easier in limited FNAB samples. Arginase‐1 is not entirely specific for hepatic differentiation, as immunoreactivity can be identified in adenocarcinomas, particularly of pancreatic origin. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society.     

肝细胞癌survivin和caspase-3的表达及其与预后的关系

目的研究肝细胞癌（HCC）癌组织及癌旁组织survivin、caspase-3的表达及其与患者术后生存率之间的关系。方法免疫组织化学S-P法检测PCNA、survivin、caspase-3的表达,并以PCNA阳性强度计算增殖指数（PCNA-PI）,原位末端转移酶标记（TUNEL）法检测凋亡指数（AI）。结果survivin在肝细胞肝癌中表达阳性率高于癌旁组织（P〈0.05）。其阳性表达与肿瘤大小、肿瘤细胞分化程度、门静脉癌栓有关。癌组织中survivin表达阳性组的增殖指数PCNA-PI高于阴性组（P〈0.05）,凋亡指数低于阴性组（P〈0.05）。caspase-3在肝细胞癌中表达阳性率低于癌旁组织（P〈0.05）;其表达与肿瘤细胞分化程度和HBsAg有关。癌组织中caspase-3表达阳性组的增殖指数PCNA-PI低于阴性组（P〈0.05）,凋亡指数高于阴性组（P〈0.05）。在肝癌组织中survivin与caspase-3的表达呈负相关（r=-0.242,P〈0.05）。survivin阳性组患者生存率低于阴性组（P〈0.05）。caspase-3阴性组患者生存率低于阳性组（P〈0.05）。结论肝细胞癌组织survivin和caspase-3的表达影响癌细胞的增殖凋亡,对于判断HCC预后具有一定价值。     

The role and therapeutic implications of RING‐finger E3 ubiquitin ligases in hepatocellular carcinoma

Increasing evidence indicates that deregulation of RING‐finger ubiquitin‐protein ligases (E3s) involves in the development of hepatocellular carcinoma (HCC). These RING‐finger E3s serve as oncoproteins or tumor suppressors in HCC under specific conditions. In this review, we summarize current knowledge about abnormal RING‐finger E3s and their clinical significance in the development of HCC, and discuss parts of critical substrates for these RING‐finger E3s in detail. Furthermore, in light of success of Bortezomib in treating hematological malignancies, we describe the preclinical and clinical studies of therapeutic approaches targeting aberrant RING‐finger E3s in HCC.     

GPC3作为肝癌预后指标和免疫治疗新靶点的研究进展

磷脂酰肌醇蛋白聚糖-3(glypican-3,GPC3)是硫酸乙酰肝素糖蛋白(heparan sulfate proteoglycans,HSPGs)家族中的一员,它的成员通过糖基磷脂酰肌醇锚(glycosylphosphatidylinositol,GPI)连接于细胞表面.GPC3在胎儿肝脏中含量丰富,而在成人肝脏中几乎不表达.值得注意的是GPC3在肝细胞癌(hepatocellular carcinoma,HCC)中高表达,同时一些研究结果表明GPC3过表达的HCC患者预后较差.目前,一些关于靶向GPC3免疫治疗的研究已取得一定的成果,例如人源化抗-GPC3单克隆抗体,肽疫苗和免疫毒素治疗.因此,GPC3有望成为分子标记物并用于HCC的诊断,可作为靶点用于HCC的干预治疗,也可为判断预后情况提供一项指标.本文总结当前的一些研究,说明在HCC治疗过程中检测GPC3及其靶向作用具有重要的临床意义.     

Peptide vaccine as an adjuvant therapy for glypican‐3‐positive hepatocellular carcinoma induces peptide‐specific CTLs and improves long prognosis

There is no established postoperative adjuvant therapy for hepatocellular carcinoma (HCC), and improvement of patient prognosis has been limited. We conducted long‐term monitoring of patients within a phase II trial that targeted a cancer antigen, glypican‐3 (GPC3), specifically expressed in HCC. We sought to determine if the GPC3 peptide vaccine was an effective adjuvant therapy by monitoring disease‐free survival and overall survival. We also tracked GPC3 immunohistochemical (IHC) staining, CTL induction, and postoperative plasma GPC3 for a patient group that was administered the vaccine (n = 35) and an unvaccinated patient group that underwent surgery only (n = 33). The 1‐y recurrence rate after surgery was reduced by approximately 15%, and the 5‐y and 8‐y survival rates were improved by approximately 10% and 30%, respectively, in the vaccinated group compared with the unvaccinated group. Patients who were positive for GPC3 IHC staining were more likely to have induced CTLs, and 60% survived beyond 5 y. Vaccine efficacy had a positive relationship with plasma concentration of GPC3; high concentrations increased the 5‐y survival rate to 75%. We thus expect GPC3 vaccination in patients with HCC, who are positive for GPC3 IHC staining and/or plasma GPC3 to induce CTL and have significantly improved long‐term prognosis.     

circLARP4 induces cellular senescence through regulating miR‐761/RUNX3/p53/p21 signaling in hepatocellular carcinoma

Circular RNAs (circRNAs), a novel class of non‐coding RNAs, have emerged as indispensable modulators in human malignancies. Aberrant cellular senescence is a phenotype observed in various cancers. The association of circRNAs with cellular senescence in tumors is yet to determined. Here, we investigated the role of circLARP4 in cellular senescence and cell proliferation in hepatocellular carcinoma (HCC). Downregulated circLARP4 level was observed in HCC tissues and cell lines. Low expression level of circLARP4 independently predicted poor survival outcome. Gain‐of‐function and loss‐of‐function assays demonstrated that circLARP4 suppressed HCC cell proliferation, mediated cell cycle arrest and induced senescence in vitro. Levels of p53 and p21, 2 key regulatory molecules in cellular senescence, were increased in circLARP4‐overexpressed HCC cells and decreased in circLARP4‐silenced HCC cells. In vivo experiments further confirmed the tumor‐suppressing activity of circLARP4. Further mechanistic studies showed that circLARP4 dampened HCC progression by sponging miR‐761, thereby promoting the expression level of RUNX3 and activating the downstream p53/p21 signaling. Our study revealed the role of circLARP4/miR‐761/RUNX3/p53/p21 signaling in HCC progression, providing a potential survival predictor and therapeutic candidate for HCC.     

High preoperative levels of serum glypican‐3 containing N‐terminal subunit are associated with poor prognosis in patients with hepatocellular carcinoma after partial hepatectomy

Glypican‐3 (GPC3) is a glycosylphosphatidylinositol‐anchored cell surface glycoprotein overexpressed in hepatocellular carcinoma (HCC) cells and may serve as a potential molecular target for therapeutic intervention. This study evaluated the prognostic significance of serum GPC3 in HCC patients receiving curative surgery. A novel sandwich enzyme‐linked immunosorbent assay for the quantitative and sensitive determination of serum GPC3 N‐terminal subunit antigen (sGPC3N) was developed and used to measure sGPC3N levels in 25 healthy volunteers and 115 HCC patients who underwent curative partial hepatectomy. The relationships between sGPC3N and clinicopathologic features were analyzed and the prognostic impact on overall survival (OS) or disease‐free survival (DFS) was also investigated. Mean and median levels of sGPC3N in healthy controls were 110.12 and 115.95 pg mL^?1, respectively, with 185.52 pg mL^?1 (mean + 2 SD) being set as the upper limit of the normal range. In HCC patients, sGPC3N levels were significantly increased (mean/median, 405.16/236.19 pg mL^?1) compared to healthy controls (p < 0.0001), and 60% of HCC cases (69/115) showed sGPC3N levels that were higher than the upper normal limit. High sGPC3N levels were significantly associated with serum AFP level, high Child‐Pugh score and positive HCV. Kaplan–Meier analysis indicated that elevated pre‐operative sGPC3N was associated with shorter OS and DFS after hepatectomy (p ≤ 0.01). Multivariate analysis revealed elevated sGPC3N as an independent poor prognostic marker for OS (p < 0.05) and DFS (p < 0.01). The pre‐operative sGPC3N level serves as an independent prognostic biomarker in HCC patients.