Characteristic analysis of insulin secretion and sensitivity in pre-diabetes populations under new diagnostic criteria / 上海交通大学学报（医学版）

Objective: To investigate the characteristics of insulin secretion function and sensitivity and blood glucose disposition capacity in the prediabetes populations. Methods: A total of 1 317 subjects were enrolled in this study, including 382 with normal glucose tolerance (NGT) and 935 with pre-diabetes. All pre-diabetes populations were divided into seven subgroups according to the cut-off points of 2010 American Diabetes Association standards. Homeostasis model assessment (HOMA) was used to access baseline insulin secretion (HOMA-β) and insulin sensitivity (HOMA-IR). Insulin secretion and sensitivity after glucose load were evaluated by area under curve (AUC) for insulin/AUC for glucose (AUCINS120/AUCGLU120) and insulin sensitivity index (ISI) calculated from Cederholm formula, respectively. Disposition index (DI) was used to reflect blood glucose disposition capacity. Results: The most common type of pre-diabetes was impaired fasting glucose (IFG) combined with impaired glucose tolerance (IGT) and glycated hemoglobin A1c (HbA1c ) 5.7%-6.4%, followed by isolated IGT, while the proportion of isolated IFG was the lowest. The insulin sensitivity of isolated HbA1c 5.7%-6.4% group was better than that of isolated IFG group, isolated IGT group, and IFG combined with IGT group (P<0.05). And its β-cell function was similar with the other subgroups. The DI value of isolated HbA1c 5.7%-6.4% group was about 1.5 times of that of isolated IGT group and IFG combined with IGT group (P=0.000), which was similar with isolated IFG group. The function of β cell or insulin sensitivity in the pre-diabetes subjects with HbA1c 5.7%-6.4% was further damaged compared with the pre-diabetes people whose HbA1c were lower than 5.7%. Conclusion: Different types of pre-diabetes are significantly heterogeneous under new diagnostic criteria, and further prospective studies with a larger sample size are needed to clarify whether HbA1c 5.7%-6.4% is suitable as a diagnostic criteria for pre-diabetes in Chinese population.

The effect of hepatitis B virus X protein on the expression of CtIP in HepG2 Cells / 中华肝脏病杂志

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.

HBx gene down-regulates miR-192 expression and inhibits apoptosis of human hepatoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mechanism by which HBV X gene(HBx) inhibits apoptosis of human hepatoma cell line HepG2 in terms of miRNA.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>HBx could inhibit apoptosis of HepG2 cells through down-regulation of miR-192 which induces apoptosis of HepG2 cells.</p>

MiR-122 regulates the expression of PEG10 in hepatoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>miR-122 may be involved in regulation of PEG10 expression.</p>

Establishment of an I-SceI system and its application to introduce DNA double-strand break into human hepatoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair.</p><p><b>METHODS</b>The eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis.</p><p><b>RESULTS</b>Restriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.</p><p><b>CONCLUSIONS</b>Genomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.</p>

The simultaneous knock-down of Ku70 and Ku80 by a tandem Ku-shRNA-encoding plasmid expression system / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To extend the use of vector-derived siRNA by generating multiple shRNAs in the same plasmid.</p><p><b>METHODS</b>Construct a vector that expresses shRNAs targeting on Ku70 and Ku80 in tandem. The gene silencing efficiency of each shRNA was verified previously. After identification by restriction digestion and DNA sequencing, the reconstructed plasmid, named psiRNAKus, was transfected into the human hepatoma cell line HepG2. The tandem-shRNA-induced silencing of targeted genes was determined by RT-PCR at RNA level and Western blot at protein level.</p><p><b>RESULTS</b>The shRNAs encoded by psiRNAKus down-regulated both the expression of Ku70 and Ku80.</p><p><b>CONCLUSION</b>The vector-derived siRNA delivery system that allows multiple shRNA species to be expressed from the same vector may be of value in experimental and therapeutic applications.</p>