大鼠肝癌细胞与胎肝细胞间抑癌微小RNAs的表达差异

微小RNAs(miRNAs)是一类短序列、非编码、具有调控功能的单链小分子RNA[1],通过与其靶mRNA分子互补结合,在翻译水平上特异性抑制基因表达,参与调控生物生长和发育等许多复杂的生命过程[2,3],异常的miRNAs表达可能与人类许多疾病乃至肿瘤的发生和发展都密切相关[3,4].已知的miRNAs参与癌症发生的机制包括细胞黏附、血管生成、蛋白质水解、细胞信号系统、细胞增殖、侵袭转移和细胞死亡[5].miRNAs在肝癌组织中异常表达,它可能参与了肝细胞癌变及肝癌转移的病理过程[6].本研究应用Exiqon miRNA基因芯片技术,建立大鼠肝癌细胞和胎肝细胞之间miRNAs的差异表达谱,探索肝癌诊断和预后的新指标.DOI:10.3760/cma.j.issn.1007-3418.2009.02.016     

细胞调亡与肝细胞肝癌

细胞凋亡是细胞自然衰老、死亡的一种形式，其退化或受抑参与包括肝细胞肝癌在的各种恶性肿瘤的发生与发展过程，许多因素调控和影响着肝癌的细胞凋亡。     

转化生长因子-β1在肝细胞性肝癌中表达增强

目的转化生长因子－β１（ＴＧＦβ１）在细胞生长的负性调节上有重要作用,是肝细胞生长和增殖的强抑制物但肝癌（ＨＣＣ）患者肝组织中ＴＧＦβ１ｍＲＮＡ表达高．现检测ＨＣＣ患者外周血中ＴＧＦβ１ｍＲＮＡ表达,血清ＴＧＦβ１水平,以及肝组织中ＴＧＦβⅡ型受体（ＴＧＦβＲⅡ）的表达．方法ＨＣＣ患者外周血分离单个核细胞（ＰＢＭＣ）,提取总ＲＮＡ,用反转录聚合酶链反应（ＲＴＰＣＲ）技术扩增ＴＧＦβ１ｍＲＮＡ．血清ＴＧＦβ１水平测定用Ｐｒｏｍｅｇａ公司产的试剂盒．肝组织ＴＧＦβＲⅡ表达的分析用原位杂交法．结果ＨＣＣ患者２０例ＰＢＭＣＴＧＦβ１ｍＲＮＡ用ＲＴＰＣＲ检测阳性率达７０％,对照组阴性．ＨＣＣ患者４０例血清ＴＧＦβ１水平（２７５８ｍｇ／Ｌ±８１０ｍｇ／Ｌ）明显高于对照组（８２７ｍｇ／Ｌ±３７２ｍｇ／Ｌ）．原位杂交表明,ＴＧＦβＲⅡ在ＨＣＣ细胞的胞质中有弱表达．结论ＨＣＣ患者ＴＧＦβ１基因在转录水平和翻译水平上均显示表达增强．ＰＢＭＣ有可能代替肝组织用以检测ＴＧＦβ１ｍＲＮＡ的表达应用于基础与临床的研究．     

CD44v6及nm23-H1表达与肝细胞癌侵袭转移

目的原位检测肝细胞癌(HCC)中CD44v6 mRNA及nm23-H1 mRNA的表达,了解CD44v6 mRNA及nm23-H1 mRNA的表达与HCC侵袭转移的关系,了解HCC中CD44v6 mRNA表达与nm23-H1 mRNA表达的相关性.方法采用PCR法合成CD44v6 cDNA探针,体外转录法合成nm23-H1 cRNA探针,采用原位杂交法检测HCC 33例 CD44v6 mRNA和nm23-H1 mRNA的表达.结果侵袭转移倾向高危组的10例HCC标本中,CD44v6 mRNA和nm23-H1 mRNA的阳性率分别为80.0%(8/10)和40.0%(4/10);侵袭转移倾向低危组的23例HCC标本中,CD44v6 mRNA和nm23-H1 mRNA的阳性率分别为21.7%(5/23)和91.3%(21/23).CD44v6 mRNA的表达与HCC的侵袭转移倾向具有正相关性(P＜0.01),nm23-H1 mRNA的表达与HCC的侵袭转移倾向具有负相关性(P＜0.01),CD44v6 mRNA及nm23-H1 mRNA的表达具有负相关性(P＜0.01).结论检测CD44v6 mRNA及nm23-H1 mRNA的表达可能成为HCC转移预后判断的指标.     

微RNA 18与B细胞易位基因2在肝癌细胞中差异表达相关性的研究

目的 研究微RNA 18(miR-18)对肝癌细胞HepG2基因表达谱的影响,预测其靶基因,初步探讨miR-18与抗增殖基因B细胞易位基因2(BTG2)两者在肝癌发生过程中差异表达的相关性.方法 利用基因表达谱芯片技术筛选HepG2的微RNA差异表达谱,应用生物信息学方法预测表达明显上调的miR-18的靶基因,初步筛选出直接调控的靶基因为抗增殖基因BTG2;应用RT-PCR和Northern blot法分析BTG2正常与肝癌组织中的表达情况.结果 生物信息学分析结果显示miR-18分子调控的下游靶基因有609个,涉及细胞增殖、分化和凋亡,转录调节等众多生理和病理过程;抗增殖基因BTG2在肝癌组织和细胞中呈明显低表达.结论 miR-18在肝癌细胞中表达明显上调,并可能负性调控抗增殖基因BTG2在肝癌细胞中的表达,两者共同在肝癌细胞增殖方面发挥着重要作用.     

microRNA在皮肤血管瘤血管组织中的表达及意义

目的探讨皮肤血管瘤组织中microRNA的表达及意义,为从基因水平上治疗该疾病提供依据。方法收集手术切除的血管瘤、血管肉瘤、静脉畸形组织,并取正常皮肤组织作为对照。分离并提取microRNA,进行实时PCR分析。免疫组化法检测MEK-1、cyclin-E1的表达。结果 PCR结果显示,皮肤血管瘤组织microRNA-96水平比其他血管异常组织低;microRNA-96目的基因MEK-1和cyclin-E1蛋白表达较正常皮肤和其他血管异常组织增加,但在mRNA水平上表达没有统计学差异。结论 microRNA-96表达的减少和MEK-1、cyclin-E1水平的增加可能是导致皮肤血管瘤发生的原因。     

microRNA与原发性肝细胞癌发生机制相关性的研究进展

MicroRNA(miRNA)是一类长约19-22个核苷酸的内源性非编码的小分子RNA,通过对靶基因转录后水平的负性调控作用,从而降低靶基因的表达水平.近年研究发现miRNA参与调节肿瘤细胞的增殖、分化和凋亡等多个生物学过程,其突变、缺失或表达水平的异常与人类肿瘤发生发展密切相关.本文就miRNA和人原发性肝细胞癌发生的相关性研究的最新进展作一综述.     

大鼠肝纤维化模型肝细胞凋亡及肝细胞坏死的动态分析

目的　观察肝细胞凋亡和肝细胞坏死在肝纤维化形成过程中的变化 ,探讨其在肝纤维化发病机制中的作用。方法　雄性Wistar大鼠 32只 ( 85～ 95克 )随机分为 4组。A ,B ,C为实验组 ,采用腹腔注射CCl4 的方法造模 ;D为对照组 ,代之以生理盐水。于 2 ,4,6周末分别处死A ,B ,C三组大鼠 ,D组同期处理。取肝脏石蜡包埋 ,连续切片做病理学检查、细胞凋亡原位检测。测定血清ALT。结果　病理学检查A ,B ,C ,D四组分别符合肝纤维化的S1,S3 ,S4 ,S0 期。各实验组 (A ,B ,C)的肝细胞坏死记分值均高于对照组 (D) ( 1 6 7± 0 82 ) ,( 2 83± 0 75 ) ,( 2 5 0± 1 0 5 )vs( 0 16± 0 4 1) ,(P <0 0 1) ,肝细胞凋亡级数也均高于对照组 (D) ( 2 5 0± 0 5 5 ) ,( 2 83± 0 4 1) ,( 2 0 0± 0 6 3)vs( 0 33± 0 5 2 ) ,(P <0 0 1)。各实验组 (A ,B ,C)的ALT均高于对照组 (D) ( 76 83± 2 1 87) ,( 117 0 0±15 94) ,( 10 3 6 7± 36 6 8)vs( 39 16± 2 32 ) ,(P <0 0 5 )。结论　肝细胞凋亡和肝细胞坏死在肝纤维化发生过程中存在动态变化 ,二者在其不同时期发挥的作用不同。     

胎肝提取物对BEL—7402肝癌细胞的影响

从４个月胎肝中制备提取物，采用３Ｈ－ＴｄＲ掺入，ＭＴＴ比色法和细胞形态学等技术观察了胎肝提取物对ＢＥＬ－７４０２肝癌细胞的影响。１％和２％ＤＭＳＯ对细胞ＤＮＡ合成的抑制率为５６％和５６％；对线粒体脱氢酶的抑制率为４３％和４８％；形态学改变主要表现为有丝分裂相减少，胞质出粗细不等的突起，相互连成网状结构。     

Demonstration of direct lineage between hepatocytes and hepatocellular carcinoma in diethylnitrosamine-treated rats

The question whether hepatocellular carcinoma (HCC) arises from dedifferentiation of mature hepatocytes or from proliferation of liver stem cells is still debated. In the present study, we used retroviral-mediated genetic labeling to investigate the fate of mature hepatocytes in rats after administration of diethylnitrosamine (DEN). Mature hepatocytes were genetically labeled by intravenous injection of retroviral vectors containing the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal (nls-LacZ) 1 day after partial hepatectomy. Liver biopsies performed after completion of hepatic regeneration showed that 18.3% of hepatocytes expressed the nls-LacZ transgene. Rats were then treated with DEN in drinking water for 12 weeks and sacrificed between 98 and 151 days after the onset of DEN administration. Clones of beta-galactosidase positive cells were observed, half of which (53%) also expressed the placental form of glutathione-S-transferase (GSTp), a marker of preneoplastic cells. HCCs of various sizes expressing GSTp were present in all animals. Careful examination of 90 HCCs revealed that 16 (17.7%) also expressed nls-LacZ. This figure precisely matched the proportion of labeled hepatocytes before DEN treatment (18.3%). In conclusion, a random clonal origin of HCC from mature hepatocytes is seen in the DEN model of hepatocarcinogenesis.     

Studied microRNA gene expression in human hepatocellular carcinoma by microRNA microarray techniques

AIM: To achieve a better understanding of the molecular mechanisms of micro RNA expression changes involved in hepatocellular carcinoma.METHODS: In this research process, patients were not treated with antivirals, immunosuppressants or immunomodulators for at least 6 mo before collecting serum. The study population was composed of 35 outpatient hepatitis B virus(HBV) cases and 12 healthy control cases from the Affiliated Hospital of Inner Mongolia Medical University(Inner Mongolia, China) from July 2013 to April 2014. The 35 HBV cases were divided into two groups: a hepatocirrhosis group with 20 cases and a liver cancer group with 15 cases. All 35 cases carried HBs Ag. The diagnostic criteria followed the European Association for the Study of the Liver 2012(EASL2012) standards. Micro RNA(mi RNA) was extracted from a control group of patients, a group with hepatocirrhosis and a group with liver cancer and its quality was analyzed using the human V2 micro RNA expression beadchip. Cluster analysis and a radar chart were then applied to the mi RNA changes.RESULTS: The mi RNA-qualified rate of human serum samples was 93%. The concentration of a single sample was 200 ng/μL and the volume was 5 μL.All mi RNA serum samples were uncontaminated by the genome. The Mann-Whitney test showed significant differences in mi RNA between each group, with a detection P-value of 0.05. Illumina software was set up with Diff Score set to ± 13, meaning that P = 0.001.There were significant changes in mi RNA expression between the three groups. mi RNA-183 was the most up-regulated, followed by mi RNA-373. mi RNA-129 and mi RNA-188 were both strongly down-regulated and mi RNA-378 was down-regulated a small amount. The liver cancer group had greater changes, which indicated that changes in mi RNA expression levels were caused by hepatocirrhosis. The liver cancer disease course then further increased these changes. In the pentagon created by these five mi RNAs, three groups showed significant deviation. The liver cancer group had a bigger deviation trend. The chart indicated that mi RNA expression changes occurred in the hepatocirrhosis group, which increased in the liver cancer disease course and were irreversible.CONCLUSION: There was a significant relationship between the irreversible up-regulation of mi RNA-183/373 and down-regulation of mi RNA-129/188/378 and incidences of hepatocirrhosis and liver cancer.     

Sequential alterations of microRNA expression in hepatocellular carcinoma development and venous metastasis

Hepatocellular carcinoma (HCC) is a prevalent cancer with an extremely high mortality rate attributed to HCC metastasis, which is the major cause of tumor recurrence and organ failure. Presence of tumor thrombi in the portal veins (venous metastases) is a clinicopathological feature of metastatic HCCs. In this study, we analyzed the microRNA (miRNA) expression profiles of nontumorous livers, primary HCCs, and venous metastases in the same livers from 20 HCC patients by way of TaqMan low-density array (TLDA) and identified the precise alterations of miRNA expression from nontumorous livers to primary HCCs and venous metastases globally. By unsupervised clustering analysis, nontumorous livers were distinctly segregated from primary HCCs and venous metastases, whereas no discernible difference in the expression pattern could be found between primary HCCs and venous metastases. However, a marked global reduction of miRNA expression levels was detected in venous metastases, as compared with primary HCCs. These data suggest that miRNA deregulation is an early event in liver carcinogenesis and the later global miRNA down-regulation aggravates the preexisting miRNA deregulation to further promote HCC metastasis. CONCLUSION: Our study has enriched the current understanding of the deregulation of miRNAs in HCC progression and highlighted the sequential and distinctive alterations of miRNA expression in primary HCC and venous metastasis formation.     

肝癌组织生长抑素受体亚型的表达及其意义

目的研究原发性肝癌患者肝癌组织中生长抑素受体亚型(SSTR2、3和5)的表达情况及其意义。方法应用免疫组化法检测50例原发性肝癌患者癌组织、癌旁组织以及24例非癌肝组织中SSTR2、3和5的表达;采用RT-PCR法检测10例肝癌组织及非癌肝组织SSTR2、3和5mRNA的表达。结果肝癌组织中SSTR2、3和5蛋白表达阳性率分别为72%、34%、14%,SSTR2的表达率明显高于SSTR3和5,差异有统计学意义(P0.05);SSTR2在肝癌组织中表达率较非癌肝组织高(x2=7.372,P0.01),差异有统计学意义;肝癌组织SSTR2、3和5mR-NA表达率分别为100%、80%和30%,均高于非癌肝组织。结论肝癌组织SSTR亚型表达增高,SSTR2为肝癌组织中主要表达的生长抑素受体亚型。     

Differential expression analysis of piwi-interacting RNAs in hepatocellular carcinoma

<正>Objective To investigates the role of piwi-interacting RNAs (piRNA) in the occurrence and development of hepatocellular carcinoma. Methods Second-generation small RNA sequencing was performed on cancer and pa-racancerous tissues,metastatic and non-metastatic liver cancer tissues of patients with liver cancer,and the sequencing data were filtered out for the common RNA sequences to be repeated. The piRNA predictor was used to forecast the possible new piRNA merged with the downloaded known piRNA to screen out distinction. A     

Alterations of epigenetics and microRNA in hepatocellular carcinoma

Studies have shown that alterations of epigenetics and microRNA (miRNA) play critical roles in the initiation and progression of hepatocellular carcinoma (HCC). Epigenetic silencing of tumor suppressor genes in HCC is generally mediated by DNA hypermethylation of CpG island promoters and histone modifications such as histone deacetylation, methylation of histone H3 lysine 9 (H3K9) and tri‐methylation of H3K27. Chromatin‐modifying drugs such as DNA methylation inhibitors and histone deacetylase inhibitors have shown clinical promise for cancer therapy. miRNA are small non‐coding RNA that regulate expression of various target genes. Specific miRNA are aberrantly expressed and play roles as tumor suppressors or oncogenes during hepatocarcinogenesis. We and other groups have demonstrated that important tumor suppressor miRNA are silenced by epigenetic alterations, resulting in activation of target oncogenes in human malignancies including HCC. Restoring the expression of tumor suppressor miRNA by inhibitors of DNA methylation and histone deacetylase may be a promising therapeutic strategy for HCC.     

HBsAg对正常肝细胞及肝癌细胞诱导产生NO的影响

采用ＨＢｓＡｇ分别刺激体外培养的正常肝细胞及肝癌细胞,观察培养液中ＮＯ＾－２／ＮＯ＾－３,以及ＮＯ对这两种细胞ＤＮＡ合成的影响,结果发现,ＨＢｓＡｇ可以诱导正常肝细胞及肝癌细胞产生ＮＯ,并抑制这两种细胞ＤＮＡ合成。但以正常肝细胞受抑较严重,提示：乙肝病毒及乙肝病毒表面抗原也引起肝硬化病人ＮＯ水平增高的重要原因,ＮＯ可能肝硬化组织易于癌变的可疑诱因。