臺灣博碩士論文加值系統

肝臟細胞的增生反應主要發生於發育、細胞修護(肝再生) 及肝癌中。一般研究肝臟再生最理想的模式為小鼠的部分切肝實驗，在肝臟進行細胞分裂的過程中，基因的活化表現會受到細胞週期精密的調控。因此，藉由肝再生的模式來瞭解基因調控的重要機制，將有助於未來控制或抑制肝癌細胞的增生。
利用微陣列分析的方法，我們發現了一群在肝癌組織中會大量表現，但在小鼠肝再生中則是特殊表現於G2/M時期的基因，如Hurp、mFLJ10468、mFLJ10540、mRHAMM等基因。為了探討這些co-regulated genes在轉錄層次上的調控，我們結合生物資訊分析系統RgSMINER (http://rgsminer.csie.ncu.edu.tw/)，來預測基因群上起動子所有可能的轉錄因子。結果發現，在G2/M基因群的起動子有轉錄因子Sp1及CREB的結合序列高度表現；相較於另一PHx96h表現基因群的起動子上，則有轉錄因子GATA-1及GATA-4的結合序列。然而，這些轉錄因子是否真的調控了肝再生不同時間點表現的基因群，尚待實驗進一步的驗證。
另一方面，本論文以Luciferase-Reporter-Assay的方法，來研究G2/M基因起動子及順式作用因子的活性。在HEK293T細胞株及小鼠CL.2 細胞株中，證實我所選殖的Hurp, mFLJ10468, mFLJ10540 及 mRHAMM的起動子具有活性。然而在細胞週期同步化的情況下，起動子的活性似乎不受細胞週期的調控。但在G2/M時期的HeLa 229細胞株中Hurp及Birc5的起動子活性有高度活化的表現。此外利用Electorphoretic Mobility Shift Assay (EMSA)、Enhancerless-Luciferase-Reporter-Assay及site-specific mutagenesis的實驗，我們進一步的定義Hurp基因在intron 1的順式作用因子，而且這些順式作用因子對於基因的調控可能扮演重要的角色。同時，將NF-Y (ATTGG)的中心結合序列及幾個over-represented repeats, Y-2 (CGCTG) 及Y-4 (CAGCA) 刪除後，會造成順式作用因子的活性下降。未來將針對這些順式作用因子測試對於細胞週期調控的關係。

Cell proliferation in liver occurs during development, compensatory repairing (regeneration) and in hepatocellular carcinoma (HCC). Liver regeneration is faithfully reproduced within the experimental partial heratectomy (PHx) system of rodents, in which a rapid and tightly controlled cascade of gene activation followed by one or two rounds of cell division occurred. Understanding the critical steps required for the transitions from regulated to regenerative mode, furthermore, to constitutive mode of proliferation in hepatocytes may help control and/or halt the process of hepatocarcinogenesis.
Using a comparative microarray analysis approach, we have identified a group of genes commonly overexpressed in HCC but are specifically induced during the G2/M phase of regenerating mouse livers. This group of genes consists of Hurp, mFLJ10468, mFLJ10540, mRHAMM and many others. In order to study the transcription regulation of these potentially co-regulated genes, we combined bioinformatics tool system, RgSMINER (http://rgsminer.csie.ncu.edu.tw/), to predict the frequently occurred transcription factor binding sites in the promoter regions of those genes. Binding sites for transcription factors, Sp1 and CREB were often found in the promoters of genes highly induced during G2/M in contrast to binding sites for GATA-1 and GATA-4 predicted for the promoters of genes expressed at tissue remodeling phase (PHx96h). Whether these transcription factors form modules to regulate the transcription activity of these co-expressed genes at different time during liver regeneration await to be demonstrated empirically.
Promoter activity of selected G2/M genes was studied using Promoterless-Luciferase-Reporter-Assay. Promoters of Hurp, mFLJ10468, mFLJ10540 and mRHAMM were identified but they failed to demonstrate a cell cycle-dependent regulation in synchronized HEK293T cells and in mouse liver CL.2 cells. But the promoter activity of Hurp and Birc5, however, was highly activated in G2/M phase of HeLa 229 cell line. The exact reason is currently unknown. Combining Enhancerless-Luciferase-Reporter-Assay, Electorphoretic Mobility Shift Assay (EMSA) and the site-specific mutagenesis, we further identified in the first intron of Hurp gene, several cis-elements that may play important roles in regulating transcription of Hurp gene. Deletion of the core sequence of NF-Y (ATTGG), and several over-represented repeats Y-2 (CGCTG) and Y-4 (CAGCA) resulted in a severe reduction of the promoter activity in the Luciferase-Reporter-Assay. Whether these cis-elements exert a cell cycle-dependent regulation is currently being tested.

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