| Objective:We detected the express of cell cycle related miRNAs(mir-122; mir-125b; mir-192; mir-195; mir-21) in the liver of rat which exposed to vinyl chloride(VC) to explore the effect of VC in the cell cycle; We also detected the express of serum miRNAs correspond to the liver in order to find evidence for the early effect biomarkers. Materials and Methods:1. Animals: Ninety-six healthy male SD(Sprague Dawley) rats were randomly divided into three exposure groups(5 mg/kg, 25 mg/kg and 125 mg/kg group) and a control group(25 mg/kg clean air), twenty-four per group. Exposure method is intraperitoneal injection, 3 times a week. After exposure VC for 6, 8 and 12 weeks, randomly sacrifice eight rats per group, collected the liver and serum.2. Liver cell cycle detection: Fresh liver 100 mg made liver monoplast suspension, the prepared cell suspension was fixed with 70 % ethanol at 4℃ for night. Then, washed with PBS, stained with 400μl PI staining solution for 15 min(shake every several minutes) in the dark. At the last, the percentage of each phase, G0/G1, S, and G2/M, in cell cycle was determined by flow cytometry.3. RNA extraction, purification, and microarray analyses: Small RNA(<200nt) in the liver and total RNA in the serum were isolation and purified. All RNA samples showed A260/280 ratios between 1.8 and 2.0 which the quality and integrity of RNA were satisfactory. Then using real-time fluorescent quantitative PCR technique to detect the expression of miRNAs(mir-122, mir-125 b, mir-192, mir-195 and mir-21). The expression levels of miRNAs were normalized to expression levels of U6(liver) and U48(serum). Use the 2-△△Ct method to calculate the mi RNAs relative expression compared with the control group.4. Statistical analysis: Using spss19.0 for data created and statistical analysis; If the variance was equal, statistical analyses were performed using ANOVA; If the variance was not equal, analyses were performed using rank sum text; Significance level was set at 0.05. Results:1. Animals: During the phase of infected, all rats were in good condition, there were no obvious poisoning symptoms.2. Liver cell cycle: Expose VC for 6 weeks, the proportion of G0/G1 phase in the 125mg/kg group were higher than control group with statistically significant differential( P<0.05). Expose VC for 8 weeks, the percentage of each phase of cell cycle were nonsignificant differential in four groups(P>0.05). Expose VC for 12 weeks, the proportion of S phase in the 125mg/kg group were higher than control group with statistically significant differential(P<0.05).3. The expression of miRNAs in the liver: The 5 miRNAs were reveal significantly time-depended and dose-depended. Exposed VC for 6 weeks, 5 miRNAs expression were significantly increased with the increasing of doses(P<0.05); Exposed VC for 8 weeks, mir-125 b and mir-21 expression were significantly decreased with the increasing of doses(P<0.05); Exposed VC for 12 weeks, 5 miRNAs expression were significantly decreased with the increasing of doses(P < 0.05). The 5 mi RNAs expression were significantly decreased with the increasing of time in 25mg/kg group and 125mg/kg group( P<0.05); the expression of mir-195 was non-significant differential in 25mg/kg group(P>0.05).4. The expression of mi RNAs in the serum: The mir-122, mir-125 b and mir-21 were non-significant time-depended and dose-depended(P>0.05). The expression of mir-192 and mir-195 were were reveal significantly time-depended.5. The correlation analysis of miRNAs expression in liver and serum: The 5 mi RNAs were non-significant correlation between liver and serum(P>0.05). Conclusions:1. The VCM induced G1 phase arrest in the early; with the extension of time, the percentage of S phase was increase.2. Exposure VC for 6 weeks, the mi RNAs(mir-122, mir-125 b, mir-192, mir-195, mir-21) in the liver were up-regulation with the increasing of doses; after the 8 weeks, the expression were down-regulation.3. The change of miRNAs(mir-122, mir-125 b, mir-192, mir-195, mir-21) in the liver may have the relationship with G1/S checkpoint.4. The low abundance miRNAs may play an important role in the liver injury processes induced by VC.5. The expression of mi RNAs(mir-122, mir-125 b, mir-192, mir-195, mir-21) in the serum were different with the expression in liver.6. The mir-192 and mir-195 in serum may have a potential as a biomarker of the vinyl chloride expose, but there need more research. |
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