MiR-199a-3p Regulates Liver Regeneration Post Rat Reduced Size Liver Transplantation Targeting NM23

Part Ⅰ microRNA expression profile after rat reduced-size liver transplantationBackground:Liver regeneration is the key factors influencing the prognosis of living donor liver transplantation. There hasn’t been the research about special miRNA of liver regeneration after living donor liver transplantation.Objective:To analyse variation of miRNAs expression profile after rat reduced-size liver transplantation at certain time point, selecting and verying significative miRNA which can provide targeting intervention strategies in liver regeneration after rat reduced-size liver transplantation.Methods:The reduced-size liver transplantation model was established, using miRNAs microarray to detect miRNA expression.In differentially expressed microRNAs, we tried to find one certain microRNA targeting NM23 through target gene prediction software.Results:compared with control group rats liver tissue, there are 11 miRNAs up-regulated in reduced-size liver transplantation,they are let-7b-5p, let-7c-5p, miR-101-a-3p, miR-103-3p, miR-130-a-3p, miR-142-5 p,miR-186-5p, miR-199a-3p, miR-21-5p,miR-221-3p、miR-34a-5p; At the same time,miR-26 b-5 p, miR-150-5p miR-19-3p and rno-miR-146-5p were down-regulated. After 70% hepatectomy, let-7b-5p、let-7c-5p、let-7e-5p、 miR-101a-3p、miR-103-3p、 miR-130a-3p、miR-199a-3p、miR-221-3p、miR-342-3p were up-regulated while miR-23b-3p、miR-150-5p were down-regulated.In differentially expressed microRNAs after reduced-size liver transplantation, we found miR-199a-3p which targets NM23 through "targetscan.org".Then we used real-time quantitative PCR to detect miR-199a-3p expression on 6 time points, verfing the microarray results of miR-199a-3p is credible or not.Conclusion:It exists variation of miRNAs expression profile after rat reduced-size liver transplantation, picked out and verified the objective miRNAs.Part II miR-199a-3p regulates rat BRL-3 liver cells proliferation targeting NM23Background:MiR-199a and NM23 were deeply researched in the field of cancer,however they were rarely studied in liver regeneration and liver cell proliferation.Objective:To observe how miR-199a-3p mimics and miR-199a-3p inhibitors influence the proliferation of rat BRL-3a liver cells in vitro, construcing a theoretical foundation for the experiment in vivo.Methods:miR-199a-3p mimics,miR-199a-3p inhibitors and their negative controls were artificially synthesized.Rat BRL-3a liver cells,divided into four groups(Groups mimics, Groups mi-nc, Groups inhibitors, Groups in-nc), were respectively transfected by four artificial miRNAs.At 12hours,24hours,48hours,72hours after transfction, NM23,Cyclin D1 and PCNA mRNA expression changes were examined by real-time fluorogenic quantitative PCR; At 24hours,48hours after transfction,NM23,Cyclin D1 and PCNA protein changes were detected by Westemblotting; Positive cells of NM23,Cyclin D1, PCNA and ki-67 were counted in immunohistochemical analysis;Cell cycle was measured by flow cytometry;cell proliferation was assessed by CCK 8;Results:At 12hours, mRNA expression changes of NM23,Cyclin D1 and PCNA had no statistical difference in four groups (P> 0.05). At 24hours,48hours,72hours,Both mRNA and protein expression of NM23,Cyclin D1 and PCNA of Groups inhibitors were significantly increased (P< 0.05) than that of Group in-nc, on the other hand, Both mRNA and protein expression of NM23,Cyclin D1 and PCNA of Group mimics were significantly reduced (P<0.05) than that of Groups mi-nc.In immunohistochemical test,positive cells rate of NM23,Cyclin D1, PCNA and ki-67 in Groups inhibitors were significantly higher than Group in-nc(P<0.05),the reverse result happened in Group mimics.The cell cycle G0/G1 phase cells ratio of Group inhibitors was significantly lower than Groups in-nc (P<0.05), G0/G1phase cells ratio of Group mimics was significantly higher than than that of Group mi-nc (P<0.05).At the same time, the S phase cells ratio of Group inhibitors was significantly higher than Group in-nc (P<0.05), the S phase cells ratio of group Group mimics was obviously lower than Group mi-nc (P<0.05).CCK 8 test showed that cells of Group inhibitors proliferated significantly faster than that of Group in-nc (P<0.05), cells of Group mimics proliferated obviously slower than than that of Groups mi-nc (P<0.05).Conclusion:Four artificial miRNAs were successful transfected into SD rats BRL-3a liver cells. miR-199a-3p inhibitors upregulated NM23 expression,promoting rat liver cell proliferation; miR-199a-3p mimics downregulated NM23 expression.inhibiting rat liver cell proliferation.Part Ⅲ miR-199a-3p regulates liver regeneration post rat reduced size liver transplantationBackground:liver regeneration alter living donor liver transplantation ls not only the decrease of liver volume, but also cold perfusion, cold storage and ischemia-reperfusion injury, postoperative liver regeneration has its uniqueness. The second part of the experiment confirmed that miR-199a-3p mimics, miR-199a-3p inhibitors can regulate rat liver cell proliferation by changing the NM23 expression.Objective:To observe how miR-199a-3p overexpression and inhibition lentivirus vectors influence the liver regeneration after rat reduced size liver transplantation targeting NM23.Methods:miR-199a-3p overexpression and inhibition lentivirus vectors,also their negative controls were artificially synthesized.Rat donors were respectively transfected by four vectors 3days before transplantation.We set four groups(Group OE, Group OE-NC, Group IN, Group IN-NC) according to different vector. On lday,3day,5day,7day after transplantation, NM23,Cyclin D1 and PCNA mRNA expression changes were examined by real-time fluorogenic quantitative PCR; On 3day,5day after transplantation,NM23,Cyclin D1 and PCNA protein changes were detected by Westernblotting; Positive cells of NM23,Cyclin D1, PCNA and ki-67 were counted in immunohistochemical analysis;Serum aminotransferase and total bilirubin were detected, calculating liver regeneration rate, drawing survival curve.Results:On 1st day, mRNA expression changes of NM23,Cyclin D1 and PCNA had no statistical difference in four groups(P> 0.05). On 3rd day,5th day,7th day,Both mRNA and protein expression of NM23,Cyclin D1 and PCNA of Group IN were significantly increased (P<0.05) than that of Group IN-NC, on the other hand, Both mRNA and protein expression of NM23,Cyclin D1 and PCNA of Group OE were significantly reduced (P< 0.05) than that of Group OE-NC. In immunohistochemical test,positive cells rate of NM23,Cyclin D1, PCNA and ki-67 in Group IN were significantly higher than Group IN-NC (P<0.05),the reverse result happened in Group OE.The serum aminotransferase and total bilirubin test in Group IN,Group OE had no statistical difference comparing with their own negative controls. The postoperative survival rate and the median surial in Group IN,Group OE had no statistical difference comparing with their own negative controls.Conclusion:Four lentivirus vectors were successful transfected into liver tissues of rat donors. miR-199a-3p overexpression lentivirus vectors raised the expression of NM23, miR-199a-3p inhibition lentivirus vectors decreased the NM23 expression. CyclinD1, PCNA and Ki67 changed according to NM23 changes.More interestingly, rat liver regeneration rate changed at the same time points.Sum up,we draw the conclusion that:miR-199a-3p regulates liver regeneration post rat reduced size liver transplantation targeting NM23.