| Purpose1.To observe the effect of mouse liver-specific Brahma-related gene 1（Brg1）deletion on liver regeneration.2.To explore the role of mi R-187-5p in the regulation of LR by Brg1.3.To explore the relationship between Brg1,mi R-187-5p and Hippo signaling pathway,and to preliminarily clarify the mechanism of Brg1regulating LR.Method:1.Mating Brg1 lox P/lox Pmice with albumin-Cre mice selectively to knocked out Brg1 in hepatocytes.Western blotting（Western Blot,WB）was used to detect the expression of Brg1 protein in liver tissue samples of liver-specific Brg1-deficient(Brg1-/-)mice（KO group）to verify the Brg1gene knockout rate;The liver/body weight ratio and liver function（ALT,AST）were detected on KO group mice and compared with those of normal C57BL/6 mice（WT group）.HE staining and Ki-67 immunohistochemistry were used to detect the changes in pathology and hepatocyte proliferation of KO mice tissue samples compared with WT mice.Two-thirds partial hepatectomy（Ph）was performed on 8-week-old male KO mice and WT mice of the same age and gender.Mouse liver tissue samples were collected at 0h,24h,36h,48h,72h,and 168h（7d）after surgery to observe the regeneration of the mouse liver.Immunohistochemistry was used to detect hepatocyte proliferation indicators（Ki-67 and Brd U positive cell rate）.2.Two online databases Target Scan and mi RWalk were used to predict the correlation of potential target mi RNAs of the key regulatory kinase LATS1 in Brg1 and Hippo signaling pathway.Quantitative real-time polymerase chain reaction（RT-q PCR）was used to analyze the target mi RNAs in the samples.The rescue tria was carried out according to the verification results:Brg1-deficient mice(Brg1-/-)mice（KO group）were injected with the corresponding exogenous mi RNA functional mimics（agomir）,and combined with C57BL/6（WT group）,2/3Ph were performed.Mouse liver tissue samples were collected at 0h,24h,36h,48h,72h and168h（7d）after surgery to observe the regeneration of mouse liver tissue（Ki-67 and Brdu-positive cell rate）.3.Western blot was used to examine the expression of cyclins（Cyclin D1,Cyclin A and Cyclin E1）,cyclin kinase（CDK）,key regulatory kinase LATS1 in Hippo signaling pathway and downstream protein YAP in liver tissue samples of KO and WT mice to explore The relationship between Brg1,mi R-187-5p and Hippo signaling pathway,and preliminarily elucidate the mechanism of Brg1 regulating LR process,and the effect of mi R-187-5p mimic on the above cyclins and LATS1 after infusion.4.All data in this experiment were analyzed by SPSS 22.0 statistical analysis software,the results were expressed as mean±standard deviation（M±SD）,and P<0.05 or P<0.01 indicated that the difference was statistically significant.Result1.Hepatocyte-specific Brg1 deletion in mouse embryos did not lead to embryonic death.The expression value of Brg1 protein in hepatocytes of KO mice was（0.22±0.04）,which was significantly lower than that of WT mice（0.81±0.04）（P<0.01）.Compared with WT mice,the liver/body weight ratio（4.4%vs 4.3%）,ALT（71.3U/L vs 76.0U/L）and AST（282.0U/L vs 290.0U/L）in preoperative KO mice were not significantly difference（P>0.05）.In the early stage（36h,48h and 72h）after Ph,the regeneration of liver tissue in KO mice（liver/body weight ratio:2.48%,2.78%and 2.77%）was significantly lower than that in WT group（2.96%,3.26%and 3.27%）（P<0.01）;36h-48h,the proliferation of mouse hepatocytes in the KO group（the ratio of Ki-67 positive cells:17.67%and16.34%）was also significantly lower than that in the WT group（69.23%and 57.07%）（P<0.01）.The above results indicated that liver-specific Brg1 knockout did not lead to death of mice,but delayed early liver regeneration after Ph in mice.2.Target Scan and mi RWalk online databases carried out predictive analysis on the correlation of mi RNAs,the results suggest that mi R-187-5p has a potential regulatory effect on the key kinase LATS1 in the Hippo signaling pathway.Compared with the WT group.（1.33）,the expression of mi R-187-5p was significantly decreased in the KO group（0.34）（P<0.01）.Through luciferase reporter experiments,we demonstrated that mi R-187-5p can directly regulate LATS1.Compared with the WT group（liver body weight ratio:2.87%,3.50%and 3.43%）,the supplementation of exogenous mi R-187-5p in the KO group partially restored the effect of Brg1 knockout in the early postoperative period（36h-72h）after Ph.Liver tissue regeneration was delayed（2.53%,3.18%and 3.41%）from 36h to 48h,the proliferation of hepatocytes in KO mice（the ratio of Ki-67 positive cells:71.33%,65.50%）was no statistical difference（P>0.05）from that in the WT group（73.33%,67.53%）at the same time.The rate of Brdu positive cells was also similar to that of Ki-67.The above results indicated that Brg1 could positively regulate the expression of mi R-187-5p and promote the regeneration of early hepatocytes after Ph.3.At 36h and 48h,the expression of cyclin kinase CDK1 in hepatocytes of KO mice（0.20 and 0.13）was significantly lower than that of WT mice（0.69 and 0.74）（P<0.05）.At 0h,24h,36h and 48h after Ph,the expression of cyclin D1（0.01,0.23,0.20 and 0.26）was lower than that in WT mice（0.17,0.31,0.83 and 0.71）（P<0.05）.On 36h and 48h after Ph,the expression of LATS1 in the liver of mice in the KO group（0.83 and0.70）was significantly higher than that in the WT group（0.60 and 0.36）,while the expression of dephosphorylated YAP at the same time point was significantly lower（0.30 and 0.35 vs 0.43 and 0.64）（P<0.05）.After infusion of exogenous mi R-187-5p agomir,the statistical differences in the expression levels of cyclin D1,CDK1,LATS1 and dephosphorylated YAP between KO mice and WT mice at 0h-72h were almost eliminated（P>0.05）.It indicated that the mi R-187-5p mimic could restore the expression of the above-mentioned cyclins caused by the deletion of Brg1 gene after infusion.Therefore,Brg1 may restore mouse liver regeneration by inhibiting the Hippo signaling pathway through mi R-187-5p.Conclusion1.Liver-specific Brg1 knockout does not cause mouse embryonic death or other pathological changes,but delays early liver regeneration after Ph in mice.2.Brg1 can positively regulate the expression of mi R-187-5p.Supplementing exogenous mi R-187-5p can restore the delay of early liver regeneration after Ph in mice due to liver-specific Brg1 knockout.3.Brg1 may positively regulate mouse liver regeneration by inhibiting Hippo signaling pathway through mi R-187-5p. |
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