| Liver is one of the few organs which have the capacity to regenerate rapidly after injury. 7-10 days after 70% partial hepatectomy, liver recovers to its original mass and volume. LR after PHx can be divided into three distinct phases: an initiation step, a proliferation step and a cessation step. Factors such as growth factor, cytokine, insulin and hormone play important roles in initiation of LR(liver regeneration). So far the majority research of LR is focused on the positive signals,while the research to negative signal have rarely be paid attentions. The process of LR have the very big similarity with liver cancer, both of them have got a series of fierce multiplication. While a very important difference between them is he liver regeneration may terminate promptly, but not the tumour cell. Therefore, research to the negative mechanism would not only help us gain more insights into the finely orchestrated process, but also help us to understanding the mechanism and treatment target of liver cancer.MicroRNA is a kind of small non-coding RNA which are usually 22-25nt length. It can regulate gene expression in post-transcription level, which plays a vatal role in all the field of of animals and plants development, cell multiplication, cell differentation, tumorigenesis etc. MicroRNA could observed downregulation of these target mRNAs by binding to the 3'-UTR region of the target mRNAs. Present studys always concentrates to the relationship between miRNA and liver disease. Referencing the conclusion of those researches, we pridict that microRNA may play a important role in liver regeneration. The gene chip technology is one kind of parsing technique with high flux and high sensitivity. Through this method, some researches have examined and confirmed the different molecules in the process of liver regenerative. In order to prove this prediction, we use the gene chips technology. Rat microRNA microarray was used to identify different expression microRNA during LR(5d), and realtime-PCR was used to verify the result. Several microRNA were found to be discrepantly expressed, the most remarkable one is miR-34a. realtime-PCR verified the result of micro array.Bioinformatic tools such as Targetscan and MAS have been used for target genes and pathway classification analysis of candidate target genes .Results: 1. Result of gene chipWe identified 8 up-regulated and 2 down-regulated miRNAs, in which miR-34a has at least 5-fold difference in expression between PHx and SH, We then studied how miR-34a expression changes during LR on different time points. As revealed by qRT-PCR analysis, regeneration of remnant livers after PHx caused a transient increase of miR-34a expression with a ~2-fold and ~6-fold at 3 and 5 d when compared with SH control on each time point.2. miR-34a and its candidate target genes are inversely expressed in regenerating liver tissuesTo determine whether miR-34a regulate the expression of INHBB and MET during LR, we tested the expression of INHBB and MET in regenerating liver tissues at 5 d after PHx, when miR-34a was remarkably increased. By qRT-PCR and westernblot analysis, we observed a highly significant down-regulation of INHBB and MET on both mRNA and protein levels in PHx rats, indicating that miR-34a and both candidate target genes are inversely expressed. Also, an immunohistochemical evaluation of MET in regenerating livers at 5 d after resection revealed the significant down-regulation in PHx rats.3. miR-34a inhibits BRL-3A cell proliferationTo evaluate the effect of miR-34a in regulating rat liver cell proliferation, a MTT cell proliferation assay and a cell cycle analysis were used. Briefly, BRL-3A immortalized rat liver cells were transfected with miR-34a or NC. MTT assays were performed at 2-day intervals, while cell cycle analysis was conducted 48 h after infection. As shown in Fig. 2A, miR-34a markedly reduced BRL-3A cell growth at 4 d and more remarkably at 6 d. The growth inhibitory effect of miR-34a can also be sustained by the data of cell cycle analysis, in which the percentages of G2 phase cells in miR-34a and NC groups were (23.14±4.26)% and (8.48±2.93)% respectively, indicating a subpopulation of cells arrested in G2 phase by miR-34a. Cells treated with miR-34a or NC were analyzed by flow cytometry for cell apoptosis distribution analysis.4. The binding site of INHBB to miR-34aTo exam whether the regulation was direct, we fused the 3'-UTR region of INHBB to a luciferase system, in which, we validated the effect of miR-34a mimics on original and mutated plasmids in Fig 4A. Mutations in seed complementary sites fully rescued repression for INHBB5. Examinations in vivo To identify the function miR-34a plays in liver regeneration, we down regulate miR-34a in vivo with slow virus. The result confirmed that compares with the control group, down regulate the level of miR-34a in the process of liver regeneration could obstruct the termination of regeneration.ConclusionmiR-34a-mediated modulation of activin or HGF pathways would collectively contribute to the termination phase of LR by regulate the expression of INHBB and MET.
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