The role of miRNA-122 expression during the acute liver failure in mice induced by D-GalN/LPS / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS, and to explore new biomarker(s) for early diagnosis of acute liver failure.</p><p><b>METHODS</b>BALB/C mice were randomly divided into four groups: the mice were given D-GalN (900 mg/kg body weight) and LPS (10 micog/kg body weight) intraperitoneally (i.p.) to construct the acute liver model; whereas the control groups were given D-GalN (900 mg/kg), LPS (10 microg/kg) and normal saline respectively. All biochemical and histological indexes were determined at 0, 1, 3, 5, 7 and 9 h respectively after administration. Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines, furthermore, the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot. ALT and AST levels were tested by biochemistry analyzer. Serum pro-inflammatory cytokine levels were tested by ELISA.</p><p><b>RESULTS</b>The mortality rate was about 80% at 24h after D-GalN/LPS treatment, but no mortality was observed in the other three control groups. Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice (ct is approximately equal to 14), it was up-regulated significantly (P = 0.013) at first hour after treatment then down-regulated according to the development of acute liver failure, the change was more obvious at 9 h (ct is approximately equal to 15, P = 0.002). ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply. It was found that the expression of miR-122 was faster and more durable than ALT. Pro-inflammatory cytokines related to acute liver failure including TNFa and IL-6 were all up-regulated in serum as well as liver tissue (P less than 0.05). Correlation analysis showed that miR-122 had a negative correlation with ALT (correlation coefficients -0.505) and positive correlations with TNFa and IL-6 (correlation coefficients were 0.493 and 0.674 respectively).</p><p><b>CONCLUSIONS</b>Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.</p>

Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells.</p><p><b>METHODS</b>Total RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software.</p><p><b>RESULTS</b>Microarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot.</p><p><b>CONCLUSION</b>Stem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.</p>

The individuals with rs224175 T/T genotype of the TGF-Β1 gene have an increased risk for developing IIBV infecton-associated diseases / 肿瘤

Objective: To assess the association of three new single nucleotide polymorphism (SPN) sites of the transforming growth factor β1 (TGF-β1) gene, which is prevalent in Asia, with the susceptibility to the chronic hepatitis B infection-related liver disease. Methods: The genomic DNA of 129 cases suffering from the chronic HBV peripheral blood infection (74 cases with chronic type B hepatitis, 41 cases with liver cirrhosis and 14 cases with liver cancer) as well as 41 healthy volunteers were prepared. The genotypes of the TGF-β1 gene at rs2241715, rs2241716 and rs4803455 sites were detected by an allotype-specific PCR method. Results: There was a significant difference in the genotypes for rs2241715 and allele frequencies between healthy volunteers and patients with chronic type B hepatitis, liver cirrhosis, and liver cancer (P < 0.05). The individuals with rs2241715 T/T genotype had a 2.974-fold increased risk for developing chronic type B hepatitis (95% CI = 1.209-7.314, P =0.018) and 3.228-fold increased risk for developing liver cirrhosis (95% CI = 1.201-8.675, P = 0.020), while no significant correlation was observed in both genotypes and allele frequencies at rs2241716 and rs4803455 sites. Conclusion: The TGF-β1 rs2241715 T/T genotype: a predisposing factor for the chronic HBV infection-related diseases.

Inhibitory effects of antisense oligonucleotides on VEGF gene expression by human hepatocellular carcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of antisense oligonucleotides to different sequences on VEGF gene expression by human hepatoma cells.</p><p><b>METHODS</b>SMMC7721 cells were cultured under normoxic or hypoxic conditions for 24 h, followed by being transfected with different antisense oligonucleotides (A06513 to cap structure, A06514 to translation initiation, A06515 to Exon-3 and A06516 to translation terminal). The total RNAs from the cells were extracted and the VEGF expression were examined with RT-PCR. The relative concentrations of VEGF transcripts in SMMC772 cells from different groups were determined using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA as internal standard.</p><p><b>RESULTS</b>In response to the hypoxic challenge, SMMC7721 cells upregulated VEGF mRNA; Comparative to the control (no oligonucleotides), A06513, A06514, A06515, and A06516 had obvious sequence-specific inhibitory effect on VEGF gene expression, with the ratio of VEGF over GAPDH of 0.49+/-0.08, 0.71+/-0.12, 0.72+/-0.11 and 0.86+/-0.12, respectively (F=12.21, P< 0.05). A06513 showed the strongest inhibitory effect (P<0.01).</p><p><b>CONCLUSION</b>The antisense oligonucleotides complementary to VEGF cap structure, may become a potential alternative for antisense gene therapy of HCC.</p>