Active radar guides missile to its target: receptor-based targeted treatment of hepatocellular carcinoma by nanoparticulate systems.

Patients with hepatocellular carcinoma (HCC) usually present at advanced stages and do not benefit from surgical resection, so drug therapy should deserve a prominent place in unresectable HCC treatment. But chemotherapy agents, such as doxorubicin, cisplatin, and paclitaxel, frequently encounter important problems such as low specificity and non-selective biodistribution. Recently, the development of nanotechnology led to significant breakthroughs to overcome these problems. Decorating the surfaces of nanoparticulate-based drug carriers with homing devices has demonstrated its potential in concentrating chemotherapy agents specifically to HCC cells. In this paper, we reviewed the current status of active targeting strategies for nanoparticulate systems based on various receptors such as asialoglycoprotein receptor, transferrin receptor, epidermal growth factor receptor, folate receptor, integrin, and CD44, which are abundantly expressed on the surfaces of hepatocytes or liver cancer cells. Furthermore, we pointed out their merits and defects and provided theoretical references for further research.

The emerging role of miR-375 in cancer.

MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding RNAs that are believed to play fundamental roles in various biological processes through regulation of gene expression at the level of posttranscription. MiR-375 was first identified as a pancreatic islet-specific miRNA regulating insulin secretion. However, further study revealed that miR-375 is a multifunctional miRNA participating in pancreatic islet development, glucose homeostasis, mucosal immunity, lung surfactant secretion and more importantly, tumorigenesis. Recently, miR-375 has been found significantly downregulated in multiple types of cancer, and suppresses core hallmarks of cancer by targeting several important oncogenes like AEG-1, YAP1, IGF1R and PDK1. The alteration of miR-375 in cancer is caused by a variety of mechanisms, including the dysregulation of transcription factors, aberrant promoter methylation and so on. Reduced expression of miR-375 in tissue or circulation may indicate the presence of neoplasia as well as a poor prognosis of many malignant cancers. Moreover, miR-375 stands for a promising direction for developing targeted therapies due to its capacity to inhibit tumor cell growth in vitro and in vivo. Here, we summarize the present understanding of the tumor suppressive role of miR-375 in cancer progression; the mechanisms underlying the dysregulation of miR-375; the potential use of miR-375 in prognosis and diagnosis and the therapeutic prospects of miR-375 in cancer.

Visualizing hepatitis B virus with biarsenical labelling in living cells.

BACKGROUND: Study on viruses has greatly benefited from visualization of viruses tagged with green fluorescent protein (GFP) in living cells. But GFP tag, as a large inserted fragment, is not suitable for labelling Hepatitis B virus (HBV) that is a compact virion with limited internal space. AIM: To visualize HBV in living cells, we constructed several recombinant HBV fluorescently labelled with biarsenical dye to track the behaviour of HBV in the cytoplasm of infected cells. METHODS: By mutagenesis, a smaller size tetracysteine (TC) tag (C-C-P-G-C-C) that could be bound with a biarsenical fluorescent dye was genetically inserted at different cell epitopes of HBV core protein expressed in transfected cells. RESULT: Confocal microscopy and transmission electron microscopy (TEM) observations showed that TC-tagged core proteins bound with biarsenical dye could specifically fluoresce in cells and be incorporated into nucleocapsid to form fluorescent virions. The recombinant fluorescent HBV virions retained their infectivity as wild-type ones. Moreover, tracking of fluorescent HBV particles in living cells reveals microtubule-dependent motility of the intracellular particles. CONCLUSION: To the best of our knowledge, this is the first time to generate fluorescent HBV virions with biarsenical labelling and to visualize their trafficking in living cells. The fluorescent HBV may become one highly valuable tool for further studying detailed dynamic processes of HBV life cycle and interaction of HBV with host in live-imaging approach.

[Association of HLA-DQA1 gene rs9272346 polymorphism with clinical outcome of hepatitis B virus infection in ethnic Han population from Hubei].

OBJECTIVE: To assess the association of rs9272346 polymorphism of HLA-DQA1 gene with clinical outcome of hepatitis B virus (HBV) infection in ethnic Han population from Hubei, China. METHODS: A case-control study was conducted, which have involved 1028 unrelated subjects including 238 asymptomatic HBV carriers (AHC), 173 acute liver failure (ALF), 292 liver cirrhosis (LC) and 325 hepatocellular carcinoma (HCC). Genotypes of rs9272346 were determined by real-time polymerase chain reaction with a TaqMan MGB probe. Statistical results were analyzed using Chi square test, student's t test and unconditional logistic regression. RESULTS: No significant differences were detected in the frequencies of G allele between ALF, LC, HCC and AHC groups (P= 0.312, 0.314, 0.264). Compared with the AA genotype, the GG and GA genotypes were not associated with the patients groups under the dominant model: ALF group vs. AHC group (adjusted OR= 1.08, 95%CI: 0.7-1.68), LC group vs. AHC group (adjusted OR= 1.11, 95%CI: 0.87-1.26), HCC group vs. AHC group (adjusted OR= 0.93, 95%CI: 0.65-1.33). For women, the GG and GA genotypes have conferred a protective effect for the outcome of ALF (OR= 0.30, 95%CI: 0.1-1.87). CONCLUSION: Our results suggested that rs9272346 polymorphism of HLA-DQA1 may not independently influence the outcome of HBV infection in ethnic Han Chinese in Hubei, while the GG and GA genotypes may confer a protective effect against ALF in women.

miR-375 inhibits autophagy and reduces viability of hepatocellular carcinoma cells under hypoxic conditions.

BACKGROUND & AIMS: Tumor cells survive hypoxic conditions by inducing autophagy. We investigated the roles of microRNAs (miRNAs) in regulating autophagy of hepatocellular carcinoma (HCC) cells under hypoxic conditions. METHODS: We used gain- and loss-of-function methods to evaluate the effect of miRNAs on autophagy in human HCC cell lines (Huh7 and Hep3B) under hypoxic conditions. Autophagy was quantified by immunoblot, immunofluoresence, and transmission electron microscopy analyses, and after incubation of cells with bafilomycin A1. We used a luciferase reporter assay to confirm associations between miRNAs and their targets. We analyzed growth of HCC xenograft tumors in nude mice. RESULTS: miR-375 was down-regulated in HCC cells and tissues; it inhibited autophagy under hypoxic conditions by suppressing the conversion of LC3I to LC3II and thereby autophagic flux. The ability of miR-375 to inhibit autophagy was independent of its ability to regulate 3'-phosphoinositide-dependent protein kinase-1-AKT-mammalian target of rapamycin signaling, but instead involved suppression of ATG7, an autophagy-associated gene. miR-375 bound directly to a predicted site in the 3' untranslated region of ATG7. Up-regulating miR-375 or down-regulating ATG7 inhibited mitochondrial autophagy of HCC cells, reduced the elimination of damaged mitochondria under hypoxia, increased release of mitochondrial apoptotic proteins, and reduced viability of HCC cells. In mice, xenograft tumors that expressed miR-375 had fewer autophagic cells, larger areas of necrosis, and grew more slowly than tumors from HCC cells that expressed lower levels of miR-375. CONCLUSIONS: miR-375 inhibits autophagy by reducing expression of ATG7 and impairs viability of HCC cells under hypoxic conditions in culture and in mice. miRNAs that inhibit autophagy of cancer cells might be developed as therapeutics.

Association of a single-nucleotide polymorphism within the miR-146a gene with susceptibility for acute-on-chronic hepatitis B liver failure.

Excessive activation of innate immune response contributes to the pathogenesis of acute-on-chronic hepatitis B liver failure (ACLF-HBV). miR-146a was recently found to be implicated in the regulation of innate immunity. In this study, we explored the biological significance of a single-nucleotide polymorphism (rs2910164) within the miR-146a gene in the risk of acquiring ACLF-HBV. We completed a hospital-based case-control study including 717 cases of HBV-infected patients--251 cases of ACLF-HBV and 466 cases of chronic hepatitis B. Whole blood samples were collected for isolation of DNA and peripheral blood mononuclear cells (PBMCs). The association between genotypes and risk of ACLF-HBV was analyzed by multivariate unconditional logistic regression, with adjustment for sex and age. Our results showed that the GG homozygote was a protective genotype in terms of susceptibility to ACLF-HBV, with odds ratio = 0.496, 95 % confidence interval = 0.309-0.797, P = 0.004 compared with CC+GC genotypes. The amount of mature miR-146a in PBMCs was significantly higher in the GG homozygote group than those in the CC and CG genotype groups of ACLF-HBV patients. The GG genotype group also represented lower serum level of TNF-α and higher survival rate (follow-up period = 4 months). In conclusion, The GG genotype within the pre-miR-146a is reversely associated with susceptibility of ACLF-HBV in the studied Chinese population. This may be partially explained by the relatively higher amount of mature miR-146a and the lower serum level of TNF-α in this genotype group.

Pilot validation of the Boston Bowel Preparation Scale in China.

BACKGROUND AND AIM: The Boston Bowel Preparation Scale (BBPS) is a novel bowel cleanliness rating scale that has undergone validation at Boston University Medical Center, Boston, MA, USA. Thus far, there is no standard recognized bowel preparation scale in China. The aim of the present study was to analyze the reliability and validity of the BBPS for the assessment of bowel preparation quality (BPQ) in China. METHODS: A group of 49 participants from several hospitals in Guangdong province viewed a video demonstration of BBPS provided by Boston Medical Center and participated in a continuing education seminar. Inter-observer reliability was assessed for three testing colonoscopies in the video. Three months later, 13 of the participants repeated the test, and intra-observer reliability was assessed. The BBPS was then applied prospectively in 1012 screening colonoscopies and BBPS scores were compared with polyp-detection rate. Intraclass correlation coefficients (ICC) and weighted Kappa values assessed inter- and intra-rater reliability, respectively. The association of BBPS scores with polyp-detection rates was calculated by χ(2) tests. RESULTS: The inter-observer ICC of BBPS scores was 0.987 (95% CI, 0.949-1.0). The weighted Kappa for BBPS scores was 0.671 (95%CI, 0.507-0.841). For 1012 screening colonoscopies, the mean BBPS score was 6.9 ± 1.8. BBPS scores ≥ 5 were associated with a higher polyp-detection rate (35%) than scores < 5 (18%) (P < 0.05). CONCLUSION: The BBPS is a valid and reliable measure of BPQ, and this validity and reliability was maintained for Chinese physicians taught via video.

TBX5 Variants are Associated with Susceptibility to and the Incidence of Liver Cirrhosis and Hepatocellular Carcinoma in the Chinese Population: A Multicenter and Follow-Up Study.

Purpose: Liver cirrhosis (LC) and hepatocellular carcinoma (HCC) are progressions affected by genetic predispositions, and persistent hepatitis B virus infection also demonstrates genetic susceptibility. All HBV-related outcomes have been compared in parallel to identify risk polymorphism in HBV progression. Methods: The multiple-stage association study filtered and validated the risk SNPs for HBV progression and explored their association with persistent infection, with a total of 8906 subjects in China from three sites. Cox proportional hazards models and Kaplan-Meier Log rank tests were used to determine the time to the progressive event in relation to the risk SNPs. Results: Rs3825214 in TBX5 replicated a specific association with LC and HCC in 4 progression cohorts and was not related to persistent infection, naivety to HBV infection and natural clearance in 3 persistent cohorts. In combined samples, rs3825214 was associated with an increased risk of LC (P<0.001; OR = 1.98) and HCC (P<0.001; OR = 1.68). The results of bioinformatics analysis indicated that rs3825214 genotypes change RNA structure and intron excision ratio. In the follow-up of 571 hospital-based persistent HBV infection patients, ninety-three (16.29%) developed LC, and seventy-four (12.96%) progressed to HCC at a median follow-up of 5.1 years. Rs3825214 was associated with HCC and LC events in Cox proportional hazards models (P<0.001). Conclusion: We identified and confirmed that genetic variants in TBX5 are significantly associated with susceptibility to and the incidence of LC and HCC.

Embryonic liver fodrin involved in hepatic stellate cell activation and formation of regenerative nodule in liver cirrhosis.

Transforming growth factor (TGF) ß(1) plays a critical role in liver fibrosis. Previous studies demonstrated embryonic liver fodrin (ELF), a ß-spectrin was involved in TGF-ß/Smad signalling pathway as Smad3/4 adaptor. Here we investigate the role of ELF in pathogenesis of liver cirrhosis. In carbon tetrachloride (CCl(4))-induced mice model of liver cirrhosis, ELF is up-regulated in activated hepatic stellate cells (HSCs), and down-regulated in regenerative hepatocytes of cirrhotic nodules. In activated HSCs in vitro, reduction of ELF expression mediated by siRNA leads to the inhibition of HSC activation and procollagen I expression. BrdU assay demonstrates that down-regulation of ELF expression does not inhibit proliferation of activated HSCs in vitro. Immunostaining of cytokeratin 19 and Ki67 indicates that regenerative hepatocytes in cirrhotic liver are derived from hepatic progenitor cells (HPC). Further study reveals that HPC expansion occurs as an initial phase, before the reduction of ELF expression in regenerative hepatocytes. Regenerative hepatocytes in cirrhotic liver show the change in proliferative activity and expression pattern of proteins involved in G1/S transition, which suggests the deregulation of cell cycle in regenerative hepatocytes. Finally, we find that ELF participates in TGF-ß/Smad signal in activated HSCs and hepatocytes through regulating the localization of Smad3/4. These data reveal that ELF is involved in HSC activation and the formation of regenerative nodules derived from HPC in cirrhotic liver.

ß-2 spectrin is involved in hepatocyte proliferation through the interaction of TGFß/Smad and PI3K/AKT signalling.

BACKGROUND: Transforming growth factor (TGF) ß signalling pathway plays a crucial role in liver regeneration following partial hepatectomy in mice. Evidence demonstrated that ß-2 Spectrin is involved in TGFß/Smad signalling pathway as a Smad3/4 adaptor protein. AIM: The aim of this study was to explore the role of ß-2 Spectrin in hepatocyte proliferation. METHODS: ß-2 Spectrin expression was evaluated in mice receiving partial hepatectomy. The effect of siRNA against ß-2 Spectrin on hepatocyte proliferation was determined. The interaction between TGFß/Smad and PI3K/Akt signalling was investigated. RESULTS: Hepatic ß-2 Spectrin decreased dramatically 2 days after 70% hepatectomy in mice. In AML-12 cells, hepatocyte proliferation was inhibited after the stimulation of TGF ß1 and a reduction in ß-2 Spectrin mediated by siRNA resulted in increase in proliferative response. Confocal results revealed that ß-2 Spectrin represented a key regulator in TGFß/Smad signalling through controlling Smad3/4 subcellular localization. Moreover, Alternation of Akt phosphorylation led to the change in subcellular localization of Smad2, 3, 4 and ß-2 Spectrin, A reduction in Smad2, 3 and 4 mediated by siRNA resulted in the induction of pAkt expression. CONCLUSIONS: These findings reveal that ß-2 Spectrin plays a crucial role in hepatocyte proliferation, which contributes to liver regeneration following hepatectomy in mice. In addition, PI3K/Akt is involved in TGFß/Smad signalling pathway through the interaction with Smad proteins and ß-2 Spectrin.

Downregulation of MiR-93 expression reduces cell proliferation and clonogenicity of HepG2 cells.

BACKGROUND/AIMS: MiR-93 was observed in various types of cancers. This study is to investigate a role of miR-93 in the carcinogenesis of HCC. METHODOLOGY: The expression of miR-93 in HepG2 cells and prima-ry human hepatocytes (PHHC) was measured by RT-PCR. HepG2 cells were transfected with miR-93 inhibitor or negative control. The cell proliferation was determined by using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. The migration and clonogenicity in vitro were measured by cell migration assay, colony formation analysis and anchorage-in-dependent growth assay. The apoptosis and cell cycle were detected by flow cytometry analysis. The mRNA and protein levels of transforming growth factor-beta type II receptor (TGFBR2) and integrin beta8 (ITGB8)were evaluated by RT-PCR and western blot analysis. RESULTS: MiR-93 was upregulated in HepG2 cells compared with PHHC and inhibition of miR-93 significantly suppressed HepG2 cell proliferation, migration and col-ony formation. The expressions of TGFBR2 and ITGB8 were upregulated when miR-93 was inhibited. CONCLUSIONS: Our results reveal an important contribution for miR-93 in hepatocarcinogenesis and suggest a role for TGFBR2 and ITGB8 dysregulation in this process. Thus,the use of synthetic inhibitor of miR-93 may prove to bea promising approach to liver cancer treatment.

MiR-192 inhibits nucleotide excision repair by targeting ERCC3 and ERCC4 in HepG2.2.15 cells.

Deficient DNA repair capacity is associated with genetic lesions accumulation and susceptibility to carcinogenesis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate various cellular pathways including DNA repair. Here we hypothesized that the existence of HBV products may interfere with cellular nucleotide excision repair (NER) through microRNA-mediated gene regulation. We found that NER was impaired in HepG2.2.15 cells, a stable HBV-expressing cell line, compared with its parental cell line HepG2. Altered miRNA expression profile, in particular the significant upregulation of miR-192, was observed in HepG2.2.15 cells. Additionally, ERCC3 and ERCC4, two key factors implicated in NER, were identified as targets of miR-192 and over-expressing miR-192 significantly inhibited cellular NER. These results indicated that persistent HBV infection might trigger NER impairment in part through upregulation of miR-192, which suppressed the levels of ERCC3 and ERCC4. It provides new insight into the effect of chronic HBV infection on NER and genetic instability in cancer.

Effects of variant rs346473 in ARHGAP24 gene on disease progression of HBV infection in Han Chinese population.

Host genetic, environmental and viral factors are classified as three categories that determine clinical outcomes of hepatitis B virus (HBV) infection. The objective of this study was to detect the associations between polymorphisms rs346473 and rs346482 in Rho GTPase-activating protein 24 (ARHGAP24) gene and disease progression of HBV infection in Han Chinese population. These two SNPs were found by our DNA pooling using Affymetrix Genome-Wide Human Mapping SNP6.0 Array in HBV carriers, and verified by using TaqMan 7900HT Sequence Detection System with 758 progressed HBV carriers versus 300 asymptomatic HBV carriers (AsC) in a discovery phase and 971 progressed HBV carriers versus 328 AsC in a replication phase. Multivariable logistic regression revealed that individuals with genotype TT at variant rs346473 displayed remarkable correlations with disease progression of HBV infection both in the discovery phase (OR, 2.693; 95% CI, 1.928-3.760; P=6.2×10(-9); additive model) and the replication phase (OR, 1.490; 95% CI, 1.104-2.012; P=9.0×10(-3); additive model). These two SNPs were in strong linkage disequilibrium with D'=0.99 and r (2)=0.951, and haplotype TT disclosed an increased susceptibility to HBV progression (OR, 1.980; 95% CI, 1.538-2.545; P=8.1×10(-8)). These findings suggest that polymorphism rs346473 in the ARHGAP24 gene might be a part of the genetic variants underlying the susceptibility of HBV carriers to disease progression.

[HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2].

OBJECTIVE: To explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA. METHODS: Three cell lines were prepared: HepG2 cells stably transfected with HBx (HepG2/HBx), HepG2 cells stably transfected with pcDNA3.1 (HepG2/pcDNA3.1) and HepG2 cells. Flow cytometry was adopted to measure the apoptosis of these three cells and Taqman fluorescence quantitative PCR was used to examine miR-192 expression. After HepG2 cells was transfected with miR-192, the apoptosis was analyzed by flow cytometry and the expressions of p53 and PUMA at mRNA and protein levels were evaluated by SYBR Green quantitative PCR and Western blot, respectively. RESULTS: Compared with HepG2/pcDNA3.1 cells (11.46% ± 0.69%) and HepG2 cells (12.5% ± 0.66%), the apoptosis rate of HepG2/HBx cells (2.37% ± 0.35%) was significantly reduced (F = 171.722, P < 0.01). The level of miR-192 was 49.1% ± 5.9% in HepG2 cells, which was dramatically down-regulated (F = 14.319, P = 0.019) as compared to the other two groups (HepG2/pcDNA3.1: 98.0% ± 8.9%; HepG2: 100%). Compared with HepG2 cells transfected with miR-NC (10.74% ± 1.15%), transfection of miR-192 into HepG2 cells led to increased apoptosis (15.74% ± 1.17%) (F = 18.415, P = 0.013) and higher p53 and PUMA expressions at mRNA (p53: 1.68 ± 0.12 vs 0.90 ± 0.09, F = 43.115, P = 0.003, PUMA: 1.66 ± 0.10 vs 0.98 ± 0.06, F = 22.541, P = 0.009) and protein (p53: 3.07 vs 1, PUMA: 2.13 vs 1) levels. CONCLUSION: HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.

[The effect of hepatitis B virus X protein on the expression of CtIP in HepG2 Cells].

To investigate the effect of hepatitis B virus X protein(HBx) on CtBP-interacting protein(CtIP) which is an important repair factor of DNA double strand break damage in HepG2 cells induced by bleomycin. A HBx stably expressing HepG2 cell line and a control HepG2 cell line with empty vector transfected were established. After the double strand break (DSB) damage occurred, the mRNA and protein levels of CtIP were detected by Real-time PCR and Western blot assay respectively, cell cycle profiles and apoptotic cell death were determined by a flow cytometry, and the position of CtIP in cells was observed by confocal laser scanning microscopy. It showed that HepG2 cells transfected with hepatitis B virus X gene could stably express HBx protein. After being induced by bleomycin, the percentage of apoptotic cell was 16.90%+/-0.89% in HBx stably expressing HepG2 cell line and 15.30%+/-0.86% in control cell line, respectively (q = 2.074, P is more than to 0.05). While the percentage of death cell was 8.71%+/-0.74% in HBx stably expressing HepG2 cell line and 4.90%+/-0.46% in control cell line, respectively (q = 7.126, P is less than to 0.01). The two cell lines manifested the increase of G2/M arrest and significant difference existed between the two cell lines. HBx down regulated the expression levels of CtIP and its mRNA. The CtIP level was 0.66+/-0.04 in HepG2-HBx cell and 0.73+/-0.05 in HepG2-vec cell, respectively (t = 2.314, P is less than to 0.05). The relative mRNA level was 1.00+/-0.06 in HepG2-HBx cell and 1.23+/-0.08 in HepG2-vec cell, respectively (t = 2. 732, P is less than to 0.05). We also found that CtIP was concentrated in the cell nucleus. The research suggests that HBx may affect DNA-repair pathways by disrupting the expression of CtIP.

Hepatic stellate cell-specific gene silencing induced by an artificial microRNA for antifibrosis in vitro.

BACKGROUND: We previously reported that the anti-transforming growth factor-beta1 (TGF-beta1) ribozymes directed by T7 and CMV promoters could reverse the character of activated hepatic stellate cells (HSCs) in vitro and improve fibrotic pathology in vivo. However, nonspecific elimination of the effects of TGF-beta1 without selectivity might have unfavorable consequences, such as overwhelming inflammation, tissue necrosis, etc. AIMS: To establish an activated-HSC-specific gene silencing method and validate its feasibility for antifibrosis in vitro. METHODS: An artificial intronic microRNA (miRNA) expression system was established, containing three parts: (1) a 1,074-bp SM-alpha actin promoter SMP8, which is a kind of RNA polymerase II promoter and has no activity in normal liver-derived cells but is switched on during the activation of HSCs, (2) intron1 modified by inserting an artificial pre-miRNA sequence against TGF-beta1, and (3) report gene enhanced green fluorescent proteins (EGFP). The feasibility of this system for artificial microRNA expression was validated through microRNA detection by real-time polymerase chain reaction (PCR). Alteration of biological characteristics of HSCs with the anti-TGF-beta1 miRNAs was preliminarily evaluated by measuring the expression levels of TGF-beta1 and its downstream molecules, including collagen I, matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP-1), etc. RESULTS: The microRNA expression system could successfully produce mature anti-TGF-beta1 miRNAs in an activated-HSC-specific manner. The microRNA-induced inhibition rate of TGF-beta1 reached 70% and above. Accompanied by TGF-beta1 suppression, its downstream targets such as collagen I, MMP2, TIMP-1, etc. were also significantly downregulated in vitro. CONCLUSIONS: Activated-HSC-cell-specific gene silencing could be induced well by the artificial intronic microRNA expression system to realize antifibrosis in vitro.

MCM3AP, a novel HBV integration site in hepatocellular carcinoma and its implication in hepatocarcinogenesis.

A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.

DNA double-strand breaks, potential targets for HBV integration.

Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients' serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBVxgene and the I-SceI induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.

[The imprinting status of genetic imprinted gene PEG10 in human hepatocellular carcinomas].

OBJECTIVE: To study the imprinting status of genetic imprinted gene PEG10 (perternally expressed gene 10) in human hepatocellular carcinoma (HCC) tissues and liver cancer cell lines. METHODS: Genomic DNA was extracted from 20 HCC tissues and its adjacent tissues, 15 normal liver tissues, 5 liver cancer cell lines (PLC/PRF/5, smmc-7721, HepG2, Hep3B, SK-HEP-1) and 2 normal human liver cell lines (changliver, HL7702). The DNA fragments containing single nucleotide polymorphism (SNP) site of PEG10 were amplified by polymerase chain reaction (PCR) and the genotype of samples was detected by DNA sequencing. Total RNA was extracted from heterozygous samples, the imprinting status and expression level of PEG10 were evaluated by quantitative real time reverse transcription-polymerase chain reaction (qRT-PCR) and DNA sequencing. RESULTS: It was found that 16/40 of HCC and its adjacent tissues were heterozygous, 3/15 of normal liver tissue were heterozygous. A site of heterozygous mutation was found in HepG2 cells by DNA sequencing. The other liver tissues and cell lines were all homozygous. PEG10 was biallelically expressed and showed loss of imprinting (LOI) in 82.4% (14/17) liver cancer samples (16 HCC tissues and HepG2), however it was a monoallelic expression and showed genomic imprinting in17.6% (3/17) liver cancer samples. In HCC, the expression levels of PEG10 were increased apparently, but it was negative expressed in cancer adjacent tissues and normal liver tissues. Expression levels of PEG10 were not significantly different between imprinted HCC tissues and HCC tissues with LOI (t = 1.311, P value is more than 0.05). CONCLUSION: PEG10 imprinting is lost in a majority of HCC and no correlation exists between the imprinting status of PEG10 and its expressions in HCC tissues.

[MiR-122 regulates the expression of PEG10 in hepatoma cell lines].

OBJECTIVE: Our previous work indicated that overexpression of imprinting gene PEG10 is associated with malignant phenotype of hepatocellular carcinoma. The aim of this study is to explore whether disregulation of PEG10 leads to dysregulation of microRNAs. METHODS: In silico analysis using TargetScan indicated that miR-122 could regulate the expression of PEG10. The expression of miR-122 in three hepatoma cell lines, Huh7, Hep3B and HepG2 and in primary human normal liver cell were compared using real time RT-PCR. After pre-miR-122 was transfected into HepG2 cell, the levels of PEG10 mRNA and protein were measured. RESULTS: In silico analysis revealed that miR-122 could regulate the expression of PEG10. Real time RT-PCR indicated that miR-122 was not expressed in Hep3B and HepG2 cells, and only weakly expressed in Huh7 cells, but highly expressed in primary human normal liver cells. The expression of miR-122 was negatively correlated with the expression of PEG10. After pre-miR122 was transfected into HepG2, the mRNA level of PEG10 was not increased, whereas the protein level of PEG10 was increased. CONCLUSION: miR-122 may be involved in regulation of PEG10 expression.