| Objective:Utilizing the luciferase reporter gene system to find out the full length promoter of p55PIK gene and the core DNA sequence within it. Using bioinformatics technique to predict the DNA sites which may influence the p55PIK transcription activity. Using site mutagenesis technique to confirm the which site and the corresponding transcription factor may influence p55PIK transcription activity.Methods:Human Genome DNA of healthy people as template,PCR assay was utilized to amamplify the 5' terminal of p55PIK promoter DNA with different length, these DNA fragments were cloned into luciferase reporter gene plasmids pGL3-Basic,obtained(-651/+45)-p55PIK, (-839/+45)-p55PIK,(-1064/+45)-p55PIK,(-1243/+45)-p55PIK and (-1633/+45)-p55PIK The relative luciferase activity of these vectors was examined in HepG2 or Hela cell line respectively find out the most active fragment of p55PIK full length promoter and the core promoter with most activity change; Utilizing the bioinformatics to analysis the core promoter sequence to predict the transcription factors might bind with and the corresponding cis-acting element; use site mutagenesis to make sure the effects of it on the transcription activity of p55PIK.Results:Human p55PIK promoter reporter plasmids with different length were cloned into pGL3-Basic plasmids,obtained (-651/+45)-p55PIK,(-839/+45)-p55PIK,(-1064/+45)-p55PIK,(-1243/+45)-p55PIK and (-1633/+45)-p55PIK plasmids, enzyme reaction and DNA sequencing show that the length and base pair were correct. The relative luciferase activity of these plasmids was examined,in HepG2 cell (-1243/+45)-p55PIK activity was 4.90±0.21, p<0.01; in Hela cell (-1243/+45)-p55PIK activity was 29.68±0.54, p<0.01; the change between (-839/+45)-p55PIK and (-1243/+45)-p55PIK were with most significance, the P value in two cell line were both<0.01; Utilizing the bioinformatics to analysis the core promoter sequence to predict that the transcription factor MZF1, YY1, RUNX1, ADR1, IRF1, Delta E or p300 might bind with correspongding DNA site to activate the p55PIK promoter; Full length p55PIK reporter plasmids (-1243/+45)-p55PIK was used as template and the binding site of transcription factor was mutated respectively.RLA of the mutant plasmids the wild type plasmids was examined,the results show that the RLA of reporter mutant MZF1-mut1 with TGGGGA mutated to CTAGTG at-900/-896 was inclined (P<0.01).Conclusions:A series of different length fragments of human p55PIK gene luciferase reporter plasmids with the same 3'terminal was successfully constructed,obtained (-651/+45)-p55PIK,(-839/+45)-p55PIK,(-1064/+45)-p55PIK,(-1243/+45)-p55PIK and (-1633/+45)-p55PIK. The full length promoter of human p55PIK gene located in (-1243/+45) and the core promoter located at (-1243/-840) of upstream of p55PIK gene. the transcription factor MZF1, YY1, RUNX1, ADR1, IRF1, Delta E or p300 might bind with correspongding DNA site to activate the p55PIK promoter. MZF1 may transcriptional activate the expression of p55PIK through direct interaction with TGGGGA site located at-900/-896 of p55PIK promoter. Objective:To determine the role of MZF1 in p55PIK transcriptional regulation and the mechanisms on it,and to observe the proliferation influence of cancer cell.Methods:At first, ChIP assay was used to to confirm the interaction between MZF1 protein and TGGGGA DNA motif located at (-900/-896) of p55PIK promoter with the aim to enclose the mechanism of p55PIK transcriptional regulated by MZF1.MZF1 protein was over expressed or down regulated in cancer cell line. Transcription activity of p55PIK promoter, mRNA or protein expression of p55PIK was examined by luciferase reporter gene assay,real time PCR or Western Blot Assay respectively, to determine whether p55PIK was transcriptional regulated by MZF1 protrein. BrdU corporation assay was performed to evaluate the DNA thynthesis percentage of cells, Ki67 expression was examined to assess the proliferation ability. Real time PCR assay was utilized to examine the expression of MZF1 and p55PIK mRNA in 10 colorectal cancer tissue sample and corresponding normal tissue,with the purpose to demonstrate the coefficient correlation between MZF1 and p55PIK.Results:ChIP assay result show that apparent bands were found in MZF1 antibody group and the input but with no band in IgG group.Relative luciferase activity (RLA) of MZF1-GFP group was higher than GFP group (P<0.01),RLA of MZF1-SiRNA group was lower than SiRNA control group (P<0.01);The p55PIK mRNA expression of MZF1-GFP group was higher than GFP group (P<0.01), the p55PIK mRNA expression of MZF1-GFP group was MZF1-SiRNA group was lower than SiRNA control group (P<0.01 in SW480, P<0.05 in LOVO). p55PIK protein was up regulated in GFP-MZF1 group (P<0.05). DNA synthesis was enhanced in GFP-MZF1 group (P<0.01)or repressed in MZF1 SiRNA group (P<0.01 in SW480, P<0.05 in LOVO); Ki67 mean density was higher in GFP-MZF1 group (P<0.01) or lower in MZF1 SiRNA group (P<0.01). Expression of MZF1 and p55PIK in 10 samples of clinical colorectal cancer tissue compared with normal tissue were examined and analyzed,the results show that MZF1 and p55PIK expression in tumor tissue were enhanced compared with normal tissue significantly (P<0.05), the correlation coefficient was 0.945.Conclusions:p55PIK gene was transcriptional activated by MZF1.MZF1 protein transcriptional regulates the p55PIK expression by interact with TGGGGA DNA motif located at-900/-896 of p55PIK gene upstream. In clinical colorectal cancer sample,MZF1 and p55PIK expression were enhanced compared with normal tissue and with significant direct correlation... |
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