ABSTRACT
ObjectiveTo investigate the mechanism of action of miR-196b in regulating the growth and apoptosis of hepatoma cells by targeting nuclear apoptosis-inducing factor 1 (NAIF1). MethodsReal-time PCR was used to measure the expression of miR-196b in hepatoma HuH-7, SNU-449, HepG2, and SMCC7721 cells versus normal human HL7702 hepatocytes. The hepatoma HepG2 cells were collected and divided into Control group (blank control), Anti-NC group (transfected with inhibitor control), Anti-miR-196b group (transfected with miR-196b inhibitor), si-NC group (transfected with siRNA control), si-NAIF1 group (transfected with NAIF1 siRNA), Anti-miR-196b+si-NAIF1 group (co-transfected with miR-196b inhibitor and NAIF1 siRNA), and Anti-miR-196b+si-NC group (co-transfected with miR-196b inhibitor and siRNA control). MTT assay was used to measure the change in proliferation, plate colony formation assay was used to measure colony formation ability, flow cytometry was used to measure cell apoptosis, and Western blot was used to measure the protein expression of Bax and C-caspase-3. Target gene prediction software predicted that NAIF1 might be a target gene of miR-196b, and the luciferase reporting system was used to identify the targeting relationship. The t-test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK-q test was used for further comparison between two groups. ResultsThere was a significant difference in the expression level of miR-196b between hepatoma HuH-7, SNU-449, HepG2, and SMCC7721 cells and normal human HL7702 hepatocytes (1.85±0.16/1.63±012/2.36±0.25/1.92±0.13 vs 1.00±0.09, F=29.05, P＜0.001). Compared with the Anti-NC group, the Anti-miR-196b group had significant reductions in the expression level of miR-196b (0.42±0.03 vs 1.02±0.10, P＜0.05), cell proliferation (0.20±0.02 vs 0.30±0.05, P＜0.05), and colony formation ability (64.35±6.97 vs 119.54±11.82, P＜0.05) and significant increases in apoptosis rate (22.30%±2.09% vs 4.26%±0.35%, P＜0.05) and relative protein expression of Bax (0.69±0.08 vs 0.30±0.05, P＜0.05) and C-caspase-3 (0.63±0.05 vs 0.21±0.04, P＜0.05). Compared with the si-NC group, the si-NAIF1 group had significant increases in proliferation ability (0.46±0.05 vs 0.31±0.04, P＜0.05) and colony formation ability (138.92±9.66 vs 118.47±838, P＜0.05) and significant reductions in apoptosis rate (4.12%±0.40% vs 1.23%±0.12%, P＜0.05), NAIF1 (0.10±0.01 vs 0.17±0.02, P＜0.05), and protein expression of Bax (0.18±0.02 vs 0.29±0.03, P＜0.05) and C-caspase-3 (0.12±0.01 vs 020±0.03, P＜0.05). Compared with the Anti-miR-196b+si-NC group, the Anti-miR-196b+si-NAIF1 group had significant increases in proliferation ability (0.28±0.02 vs 0.21±0.03, P＜0.05) and colony formation ability (97.12±8.23 vs 66.35±5.20, P＜0.05) and significant reductions in apoptosis rate (9.60%±1.11% vs 21.14%±1.32%, P＜0.05), NAIF1 (0.30±0.04 vs 0.52±0.06, P＜0.05), and protein expression of Bax (0.28±0.03 vs 0.67±0.06, P＜0.05) and C-caspase-3 (0.22±0.05 vs 0.60±004, P＜0.05). ConclusionDownregulation of miR-196b can inhibit the growth and induce the apoptosis of hepatoma cells via negative regulation of NAIF1.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the clinical features and gene mutation profiles of patients with chronic hepatitis B (CHB) and Gilbert's syndrome.</p><p><b>METHODS</b>Thirty-three patients with CHB and Gilbert's syndrome were enrolled in the study. Serum markers of liver function and histological features of disease-related liver injury were assessed by standard methods. Gene mutations were detected by PCR and direct DNA sequencing.Statistical analysis was carried out with the chi-square and t tests.</p><p><b>RESULTS</b>Sequencing of the Gilbert syndrome-associated gene, UGT 1A 1, revealed mutations in the upstream promoter phenobarbital-responsive element module (PBREM) (-3279 mutation, 23 cases), in the promoter TATA box (a TA insertion mutation, 21 cases), and in the coding region of exon 1 (a GGA-AGA Gly71Arg mutation, 18 cases); there was no statistical difference found for any of the three mutations among this patient population (x2 =1.640, P more than 0.05).</p><p><b>CONCLUSION</b>The traditional methods of diagnosis for patients with CHB and Gilbert's syndrome remain a technical challenge in the clinic, and gene detection may represent a more favorable method for diagnosing this patient population.</p>
Subject(s)
Humans , Base Sequence , Exons , Gilbert Disease , Glucuronosyltransferase , Hepatitis B, Chronic , Mutagenesis, Insertional , Mutation , Polymerase Chain Reaction , Promoter Regions, Genetic , TATA BoxABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of adefovir dipivoxil (ADV) on blood phosphorus metabolism in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>Patients with hepatitis B surface antigen (HBsAg)-positive CHB were treated with ADV alone, ADV combined with interferon (IFN), or ADV combined with lamivudine (LAM). Changes in levels of calcium, phosphate, urea, and creatinine were assessed at treatment weeks 4, 12, 24, 48, 72 and 96. Statistical analysis was carried out with SPSS 16 software; influential factors were analyzed by ANOVA and non-conditional logistic regression analysis.</p><p><b>RESULTS</b>During the course of treatments, 32 (42.6%) of the patients presented with low phosphorus. The highest incidence of low phosphorus was found to have occurred at treatment week 24 (25.0%, 27.5% and 36.4% respectively, with no statistical difference between three groups, x2=0.225, P>0.225). Patients with hypophosphatemia did not show a significant difference in serum phosphorus levels from the other patients (F=1.853, P=0.169). Logistic regression showed a correlation between low phosphorus and sex (x2=7.876, P<0.05), age (t=2.479, P<0.05), and serum creatinine (t =-2.256, P<0.05), but not with blood urea nitrogen or blood calcium (P>0.05).</p><p><b>CONCLUSION</b>ADV antiviral treatment can decrease the blood phosphorous levels of CHB patients, particularly over extended time of treatment, and the occurrence of low phosphorus is more common than of mild phosphorus decrease.Male and elderly patients may be at greater risk of this complication. The incidence and severity of low phosphorus is not significantly different for the different ADV-based treatment regimens.</p>