| BackgroundMost of the studies of liver regeneration focus on the initial and proliferation stages.Compared with the two stages before liver regeneration,we know little about the mechanism of liver regeneration termination stage,especially how to terminate liver regeneration in the process of liver regeneration termination,reshape liver tissue morphology,restore accurate liver size and other molecular mechanisms.When the liver regenerates to the original size,the hepatocytes will stop proliferation,restore the original metabolic function,and the liver begins to reconstruct its normal tissue structure.This series of negative regulation mechanisms in the end stage of liver regeneration,like "brake and steering wheel",controls the speed of liver regeneration and when to stop,and ensures that liver regeneration is carried out in the normal direction to prevent them from going in the wrong direction,such as tumor occurrence.Therefore,the study on the mechanism of liver regeneration related negative regulation is not only helpful to clarify the mechanism of liver regeneration termination and improve the understanding of liver regeneration,but also to provide important enlightenment for the understanding and treatment of the mechanism of acute liver failure,small donor liver transplantation and liver tumor.The positive feedback loop of P53-mir-34a-sirt1 has a strong negative regulation effect.In this study,the expression,function and mechanism of the positive feedback loop of P53-mir-34a-sirt1 in the process of liver regeneration were studied in vivo and in vitro by using the mouse liver regeneration model and primary mouse hepatocytes.Method1.In C57BL/6 mice,2/3 hepatectomy(PH)was performed to construct the liver regeneration model.The liver samples were collected and the protein and RNA were extracted on the 2nd,3rd,5th,7th and 10 th day after operation.The expression of P53-mir-34a-sirt1 positive feedback loop related genes in the process of liver regeneration was detected by Western blot analysis(WB)and PCR.P53 and miR-34 a were overexpressed by adenovirus and adeno-associated virus,WB and PCR were used to detect P53-miR-34a-sirt1 positive feedback loop related genes and its downstream genes.Serum enzymology and immunohistochemistry were used to detect liver function and regeneration level at different time points after pH,and the mechanism of P53-miR-34a-sirt1 positive feedback in LR process was discussed in vivo.2.UHPLC-MS/MS was used to detect the changes of bile acids in liver regeneration tissues at different time points.The results of mass spectrometry were analyzed by bioinformatics to identify the key bile acids in LR.3.Adenovirus was used to transfect mouse primary hepatocytes with over expression of P53.WB and PCR were used to detect 53-miR-34a-sirt1 positive feedback loop related genes and their downstream genes.The effects of T-?-MCA on FXR / SHP pathway and P53-miR-34a-sirt1 positive feedback loop on hepatocyte apoptosis were studied in vitro by adding T-?-MCA and GW4064.After gavage with T-?-MCA and PH,P53-miR-34a-sirt1 positive feedback loop was tested by WB and PCR.4.After knock-in of wild-type P53 and administration of FXR/SHP antagonist guggulsterone(GS)in liver cancer cell lines Huh-7(P53-mut)Hep3B(P53-de)and HepG2(P53 wild-type),WB and PCR were used to detect P53-miR-34a-sirt1 positive feedback loop related genes and its downstream genes.Results1.The expression of P53 increased significantly in 2d and 7d after PH,P53-miR-34a-sirt1 positive feedback loop was activated most obviously in the late stage of liver regeneration(7d).Overexpression of P53 and miR-34 a can further activate the positive feedback loop of P53-mir-34a-sirt1,increase the expression of its downstream negative regulatory protein,promote its negative regulatory effect,inhibit the proliferation of hepatocytes,and terminate the liver regeneration of mice in advance.2.kinds of bile acids were detected in the liver tissue of mice after 2,3,5,7,10 days of liver regeneration.Statistical analysis showed that there were statistical differences in 12 kinds of them.T-?-MCA increase in a time-dependent manner and peak in the late stage of liver regeneration.3.Overexpression of P53 can activate the positive feedback loop of P53-miR-34a-sirt1 in vitro and vivo.T-?-MCA can inhibit the activity of FXR / SHP pathway and promote the negative modulation of the positive feedback loop of P53-miR-34asirt1 in vitro and vivo.4.knock-in of wild-type P53 can recover P53-miR-34a-sirt1 in cell lines with P53 mutation.Administration of FXR/SHP antagonist guggulsterone(GS)can facilitate its effects.Conclusion1.The positive feedback loop of P53-miR-34a-sirt1 was activated in the late stage of liver regeneration.2.T-?-MCA can inhibit the activity of FXR / SHP pathway and promote the negative regulatory effect of P53-mir-34a-sirt1 positive feedback loop.3.HCC carcinogenesis may be caused by mutation of P53 induced absence of positive feedback loop of P53-mir-34a-sirt1... |
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