[Efficacy of subgroup mouse bone mesenchymal stem cells on mobilizing autologous cardiac stem cells and repairing ischemic myocardial tissue].

OBJECTIVE: To search for the bone mesenchymal stem cell (MSC) subgroup which might be more effective on repairing myocardial damage. METHODS: In this experiment, four MSC subgroups were defined based on the surface differentiation antigen detection of mouse bone mesenchymal stem cells (mBMSCs): SCA-1(+)/CD45(+)/CD31(+), SCA-1(+)/CD45(+)/CD31(-), SCA-1(+)/CD45(-)/CD31(-) and SCA-1(+)/CD45(-)/CD31(+). These subgroup cells and unselected mBMSCs were injected into infarcted mouse via tail vein. Echocardiographic heart function measurement and in vivo DiR-labeled stem cells imaging were performed at 48 h after injection. In situ C-kit (a flag antigen of cardiac stem cells) and cardiac-specific differentiation antigen immunohistochemistry detection was made in the infarcted myocardium. RESULTS: The capacity of the SCA-1(+)/CD45(+)/CD31(+) cells on improving heart function was significantly higher than other cell groups (all P < 0.05). In vivo imaging showed that the mean fluorescence intensity of the SCA-1(+)/CD45(+)/CD31(+) cells was also higher than other cell groups (all P < 0.05). Number of cardiac stem cells in the infracted myocardium was significantly increased after the injection of all subgroup cells and unsorted mBMSCs cells for 48 h compared untreated infracted myocardium. The capacity of mobilizing cardiac stem cells is as follows: SCA-1(+)/CD45(+)/CD31(+) >SCA-1(+)/CD45(-)/CD31(+) >SCA-1(+)/CD45(-)/CD31(-) >SCA-1(+)/CD45(+)/CD31(-). CONCLUSION: The SCA-1(+)/CD45(+)/CD31(+) subgroups of mBMSCs exhibites the highest capacity to improve cardiac function after myocardial infarction and to mobilize autologous cardiac stem cells compared with other mBMSCs subgroups and unsorted mBMSCs cells.

E2F8 contributes to human hepatocellular carcinoma via regulating cell proliferation.

The E2F family member of transcription factors includes the atypical member E2F8, which has been little studied in cancer. We report that E2F8 is strongly upregulated in human hepatocellular carcinoma (HCC), where it was evidenced to contribute to oncogenesis and progression. Ectopic overexpression of E2F8 promoted cell proliferation, colony formation, and tumorigenicity, whereas E2F8 knockdown inhibited these phenotypes, as documented in Huh-7, Focus, Hep3B, and YY-8103 HCC cell lines. Mechanistic analyses indicated that E2F8 could bind to regulatory elements of cyclin D1, regulating its transcription and promoting accumulation of S-phase cells. Together, our findings suggest that E2F8 contributes to the oncogenic potential of HCC and may constitute a potential therapeutic target in this disease.

ARHI, as a novel suppressor of cell growth and downregulated in human hepatocellular carcinoma, could contribute to hepatocarcinogenesis.

The identification of cancer genes differentially expressed in hepatocellular carcinoma (HCC) plays an important role in understanding the molecular mechanisms of hepatocarcinogenesis. Here, ARHI gene expression was analyzed by real-time RT-PCR and it was significantly downregulated in 33 of the 42 (78.6%, more than two folds) HCC specimens compared with adjacent noncancerous livers (P < 0.01). In addition, ARHI expression was reduced in some HCC samples at protein level confirmed by immunohistochemistry. Furthermore, our data suggested that the overexpression of ARHI can significantly inhibit cell growth and colony formation of Hep3B cells (P < 0.01), whilst silencing endogenous ARHI gene by RNAi could promote cell growth of Huh-7 and Focus. LOH of microsatellite markers D1S2806 and D1S2803 was only found in 2.4% (1 of 42 HCCs) of HCC cases. The expression of ARHI was obviously re-expressed in some HCC cells, Bel-7402, Bel-7405, QGY-7703 and Hep3B, by a demethylation agent, 5-aza-2'-deoxycytidine (DAC). DNA hypermethylation within ARHI promoter was identified in 47.1% of HCC specimens without ARHI expression. Our current observations provide evidences that ARHI downregulated in HCCs could play a role in liver cancer via acting as a tumor suppressor gene, which mainly was triggered by the epigenetic events in HCC specimens.

Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A.

BACKGROUND/AIMS: The hepatitis B virus X protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanisms remain unknown. The aim of this study was to identify underlying mechanisms involved in HBx-mediated epigenetic modification. METHODS: Interactions between HBx and DNA methyltransferase (DNMT) or histone deacetylase-1 (HDAC1) were assessed by co-immunoprecipitation. DNA methylation of gene promoters was detected by bisulfite sequencing, and HBx-mediated protein binding to gene regulatory elements was evaluated by chromatin immunoprecipitation. Target gene transcriptional activity was measured by real-time polymerase chain reaction. RESULTS: HBx can interact directly with DNMT3A and HDAC1. HBx recruited DNMT3A to the regulatory promoters of interleukin-4 receptor and metallothionein-1F and subsequently silenced their transcription via de novo DNA methylation. By contrast, the transcription of CDH6 and IGFBP3 was triggered by HBx through the deprivation of DNMT3A from their promoters. Transcriptional levels of target genes in hepatocellular carcinoma (HCC) specimens were strongly correlated with the occurrence of HBx. CONCLUSIONS: The interaction of HBx and DNMT3A facilitates cellular epigenetic modification (via regional hypermethylation or hypomethylation) at distinct genomic loci, providing an alternative mechanism within HBx-mediated transcriptional regulation, and a profound understanding of hepatitis and HCC pathogenesis.

Down-regulation of SFRP1 as a putative tumor suppressor gene can contribute to human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. SFRP1 (the secreted frizzled-related protein 1), a putative tumor suppressor gene mapped onto chromosome 8p12-p11.1, the frequent loss of heterozygosity (LOH) region in human HCC, encodes a Wingless-type (Wnt) signaling antagonist and is frequently inactivated by promoter methylation in many human cancers. However, whether the down-regulation of SFRP1 can contribute to hepatocarcinogenesis still remains unclear. METHODS: We investigated the expression of SFRP1 through real time RT-PCR and immunohistochemistry staining. The cell growth and colony formation were observed as the overexpression and knockdown of SFRP1. The DNA methylation status within SFRP1 promoter was analyzed through methylation-specific PCR or bisulphate-treated DNA sequencing assays. Loss of heterozygosity was here detected with microsatellite markers. RESULTS: SFRP1 was significantly down-regulated in 76.1% (35/46) HCC specimens at mRNA level and in 30% (30/100) HCCs indicated by immunohistochemistry staining, as compared to adjacent non-cancerous livers. The overexpression of SFRP1 can significantly inhibit the cell growth and colony formation of YY-8103, SMMC7721, and Hep3B cells. The RNA interference against the constitutional SFRP1 in the offspring SMMC7721 cells, which were stably transfected by ectopic SFRP1, can markedly promote cell growth of these cells. LOH of both microsatellite markers D8S532 and D8SAC016868 flanking the gene locus was found in 13% (6 of 46 HCCs) and 6.5% (3 of 46 HCCs) of the informative cases, respectively, where 5 of 8 HCC specimens with LOH showed the down-regulation of SFRP1. DNA hypermethylation within SFRP1 promoter was identified in two of three HCC specimens without SFRP1 expression. Moreover, the DNA methylation of SFRP1 promoter was significantly reduced, along with the re-expression of the gene, in those HCC cell lines, Bel7404, QGY7701, and MHCC-H, as treated by DAC. CONCLUSION: Our data suggested that the down-regulation of SFRP1 as a candidate tumor suppressor gene, triggered by the epigenetic and/or genetic events, could contribute to the oncogenesis of HCC.

[A pig model of myocardial infarction by intracoronary embolization with gelatin sponge].

OBJECTIVE: We produced a large-animal model of myocardial infarction induced by transcatheter embolization of the left coronary artery using a gelatin sponge. METHODS: Seven pigs underwent transcatheter embolization of the left anterior descending artery (LAD) using gelatin sponge to produce anteroapical myocardial infarction (MI). 4 weeks later, Echocardiography, Coronary angiography and Pathology was performed, and the data were compared with those of the control group (n = 6). RESULTS: The procedure mortality was 1 of 7. In the MI group, the LV end-diastolic dimension increased (control versus MI: 37.0 mm +/- 3.4 mm and 50.8 mm +/- 6.1 mm, P < 0.01), the ejection fraction (EF) decreased (control versus MI: 62.3% +/- 2.9% and 36.6% +/- 2.1%, P < 0.001). Coronary angiography revealed the LAD remained occluded. The postmortem specimen showed a transmural MI scar in the anteroseptal and apical regions in the MI group and the LV volumes at 30 mm Hg were 81.4 ml +/- 4.3 ml, the scar areas were 18.4% +/- 1.6% of total left ventricular free wall and the scar thickness was 3.5 mm +/- 0.8 mm. Histologic samples in the MI group stained with Masson's trichrome showed massive fibrosis in the border zone and patchy fibrosis in the remote region in the LV free wall, whereas the control group showed no fibrosis. CONCLUSION: This pig model of myocardial infarction is reliable, reproducible, and similar to the human condition, amenable to investigate other investigation.