| Background: Our country is one of the most serious region of liver disease in the world and many patients have be infected with hepatitis B, we hope to cut more issues to treat liver diseases in clinical, but the problem is particularly acute followed with liver function failure. Although liver transplantation is an effective way to treat, but a serious shortage of donor limits the application and development of liver transplantation, however, the ideal way to stimulate liver regeneration itself effectively can solve these problems. Transmembrane ubiquitin-like protein 1(Tmub1) is a newly protein which plays a negative regulation role in liver regeneration process. Former research focused on its downstream effects, and for post-transcriptional regulation stage there is not much research to study.So, to study the effect between Tmub1 protein and its superior control factor can help us further understand the molecular mechanisms of liver regeneration. At present, to be an important upstream regulatory factor, the Micro RNA can aim the target protein on the level of post-transcriptional regulation. We found that mi R-27a/b have the potential binding sites in Tmub1 3 ’non-coding region sequence through bioinformatics, so we speculate mi R-27a/b can be combined with Tmub1 and may play an important role in the process of liver regeneration. Specific mechanisms of liver regeneration is extremely complex, to study the role of mi R-27a/b and Tmub1 protein in liver regeneration can be able to further elucidate the molecular regulation of liver regeneration network and promote the treatment of liver regeneration. Also, it has positive guidance about preventing liver failure in clinical.Objective: We detecte the expression of two subtypes of mi R-27a/b and Tmub1 after liver resection in rat at different periods of times, to clarify the process of Tmub1 which is regulated by mi R-27a/b in liver regeneration.Materials and Methods:1. 40 SD rats weighing in 160-200 g were selected and randomly divided into two groups: partial hepatectomy group(PH group, the experimental group) and laparotomy group(sham group). And the liver tissue and primary hepatocytes were sequentially extracted at the time of 0h, 12 h, 24 h, 48 h, and 72 h. 2. Using the Target Scan and micro RNA.org software to analyse the bioinformatics of Tmub1 gene, the mi R-27a/b potential binding sites is in the Tmub1 gene 3’UTR sequence. 3. To detecte the volume of mi R-27a/b and Tmub1 m RNA in postoperative liver tissue at different time in each group by using RT-PCR, also, the protein expression of Tmub1 was analyzed by western blot in same condition.Then study the time correlation between mi R-27a/b and Tmub1 expression.And we can use luciferase reporter gene technology to analyze the binding activity of mi R-27a/b and Tmub1. Results: 1. Bioinformatics testing results: Tmub1 gene 3’UTR sequence has a potential binding site with mi R-27a/b. we use reverse analysis method to analyze mi R-27a/b and find that Tmub1 is the target genes of mi R-27 a and mi R-27 b. 2. Real-time PCR results : We found that mi R-27a/b expression have different degrees of decline compared with sham group at the time of 12 h, 24 h, 48 h, 72h(P <0.05) while there is no significant difference at the time of 0h(P> 0.05). We detect the expression of PH group and the sham group at different time periods and found Tmub1 m RNA in PH was significantly higher than the sham group at the time of 12 h, 24 h, 48 h, 72h(P <0.05). 3. Western blot showed that Tmub1 was a little expression in normal liver cells. Rat hepatocytes Tmub1 expression start significantly increased in PH group after 12 h and reached the peak at 48 h, compared with the sham group, the Tmub1 effect has increased significantly in PH group at the time of 12 h, 24 h, 48 h, 72 h,(P <0.05), while there is no significant difference(P> 0.05) on Tmub1 protein expression at the time of 0h in PH group. 4. Luciferase reporter assay results suggest that mi R-27a/b mimetics Tmub1 reporter gene vectors are co-transfected luciferase activity was significantly decreased, espically in mi R-27 b, it was suggested that two subtypes of mi R-27a/b can combine with gene expression and play a regulatory role.The present research found that mi R-27 a and mi R-27 b expression have the negative regulation effect on Tmub1, and espically in mi R-27 b. So Tmub1 expression can be regulated by mi R-27a/b on the proliferation of liver cells regulation, then to regulate liver regeneration process. Conclusions:... |
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