The Mechanism Of Hypoxia Inducible Factor-1α On The Regulation Of P53RFP Gene Transcription

BackgroundIn recent years, therapeutic angiogenesis has become the new direction of ischemic vascular disease treatment, which is also the most popular field of study. The researchers have been looking for an effective stimulus for angiogenesis gene. Hypoxia inducible factor1(HIF-1) is a critical gene regulating of angiogenesis. As a nuclear transcription factor, it directly or indirectly regulated more than100downstream target genes. Those genes, which mainly enable the cells to cope with oxygen stress, are involved in erythropoiesis, cell proliferation/survival,angiogenesis, and glucose metabolism, respectively. On the other hand, some studies do suggest that HIF-1also plays a detrimental role in ischemic reperfusion injury by inducing the pro apoptotic molecules, cytokines such as Nix, BNip3, and IL-20which cause mitochondrial dysfunction leading to cell death. Hence, modulation of HIF-1activity seems to provide an innovative therapeutic target to reduce the cellular damage, which arises from ischemic injury. Further study of the mechanism of HIF-1a on the transcriptional regulation of its downstream target genes would help to understand the role and mechanism of HIF-la involved in the hypoxic adaptive response.It has been clear that the structure of HIF-1and the key points of its regulation. HIF-1is a heterodimeric transcription factor composed of two subunits:the oxygen regulated HIF-la and the constitutively expressed HIF-1β subunit. Intracellular oxygen concentration regulates HIF-1activity by influencing both stability and transcriptional activity of the HIF-1α subunit. Under normoxic conditions, HIF-la protein stability is regulated by prolyl hydroxylases via the modification of conserved prolines (Pro402, Pro564) within the oxygen-dependent degradation domain of HIF-la. These are subsequently recognized by the von Hippel-Lindau tumor suppressor protein, which targets HIF-la for ubiquitination and proteosomal degradation. Likewise, transcriptional activity of HIF-la is regulated by hydroxylation at an asparagine residue(Asn803) of HIF-la that blocks the interaction between C-terminal activation domain (CAD) and the p300/CBP coactivator. Therefore, we have finished the mutation of Pro402, Pro564and Asn803in HIF-la gene and successfully constructed the expression vector of HIF-la of triple mutant (pcDNA-(3.1+)-HIF-la-Trip) in order to get high levels of gene expression under normoxic condition.P53RFP, a p53-inducible E3ubiquitin ligase, is firstly isolated as a target gene of p53. It has been reported that p53RFP can cause p21ubiquitination and degradation, which is a p53downstream protein promoting growth arrest and antagonizing apoptosis. There have been less studies on this gene. We have found that increased expression of HIF-la resulted in upregulation of p53RFP gene expression. Therefore, we hypothesized that p53RFP may play an important role in the process of regulating the proliferation and apoptosis of endothelial cells by HIF-la. On the basis of previous research, we will explore whether HIF-la regulate p53RFP gene expression and the mechanism and effects of HIF-la on the regulation of p53RFP gene transcription.ObjectiveTo further study the mechanism and effects of HIF-la on the regulation of p53RFP gene transcription, we will construct Luciferase reporter vectors with different fragments of p53RFP promoter.Method1. For hypoxic exposure, HEK293and SW480cells were placed in a hypoxic incubator in an atmosphere consisting of94%N2,5%CO2, and1%O2. At the time point of0,6,12,24h, quantitative real-time RT-PCR was performed to detect the mRNA levels of p53RFP, and the protein levels of p53RFP and HIF-la were detected by Western blot.HEK293cells, SW480cells were treated with DFO or CoCI2for24h, quantitative real-time RT-PCR was performed to detect the mRNA levels of p53RFP. HEK293cells, SW480cells were treated with different concentrations of DFO for24h, the protein levels of p53RFP and HIF-la were detected by Western blot.HEK293and SW480cells were transfected with a plasmid (pcDNA3.1(+)-HIF-1α-Trip) encoding a mutant form of HIF-la, which is resistant to degradation under normoxic condictions. Quantitative real-time RT-PCR was performed to detect the mRNA levels of p53RFP after24h of transfection, and the protein levels of p53RFP and HIF-la were detected by Western blot after48h of transfection.2. Constructed Firefly luciferase reporter vectors of p53RFP gene promoter with different fragments by gene cloning technology. HEK293cell were co-transfected together with p53RFP promoter Firefly luciferase constructs and pcDNA3.1(+)-HIF-la-Trip, then detected the effect of HIF-la overexpression on the p53RFP promoter activity, and found out the most active promoter fragment.3. Mutated the hypoxia-responsive element (HRE) CGTG into TACA by gene site-directed mutagenesis technique. HEK293cell were co-transfected together with the mutant p53RFP promoter Firefly luciferase constructs and pcDNA3.1(+)-HIF-la-Trip, then detected the effects of mutant HRE on the p53RFP promoter activity induced by HIF-1α and found out the core regulatory element within p53RFP promoter.4. Chromatin co-immunoprecipitation was performed to testify whether HIF-1α is directly connected to the core HRE region of p53RFP gene promoter, and verified whether the process of HIF-1α on the p53RFP gene transcriptional regulation required for the participation of transcription coactivator p300.5. The full coding sequences of p53RFP gene were amplified by PCR, then cloned into pcDNA4/Myc-HisA vector and constructed the p53RFP expression vector (pcDNA4-p53RFP). SW480cells were transfected with pcDNA4-p53RFP for48h, CCK-8detection to find the effect of p53RFP overexpression on SW480cell proliferation, and flow cytometry to detect the effects on SW480cell cycle and cell apoptosis. SW480cells were transfected with pcDNA4-p53RFP for24h, quantitative real-time RT-PCR was performed to detect the mRNA levels of Cyclin B1, CyclinD1, p21WAF1/Cip1and CDK1.ResultThe first part:With the growth of cells in the hypoxic exposure time, the mRNA levels of p53RFP in HEK293and SW480cells showed a growing trend. Compared to Oh group, the mRNA levels of p53RFP in HEK293cells after12h,24h exposed to hyoixa were significantly different (P=0.000,0.020); the mRNA levels of p53RFP in SW480cells after12h,24h exposed to hyoixa were significantly different compared with the group of Oh (P=0.001,0.012). With the growth of cells in the hypoxic exposure time, the protein levels of p53RFP and HIF-1α showed a growing trend in both HEK293and SW480cells. HEK293cells were treated with DFO or CoCl2, the relative mRNA levels of p53RFP of both groups were significantly higher than that of blank group (P=0.000,0.017).The relative mRNA levels of p53RFP of SW480cells treated with DFO or CoCI2were also significantly higher than that of blank group (P=0.000,0.001)HEK293and SW480cells were transfected with a plasmid encoding a mutant form of HIF-la for24h, the mRNA levels of p53RFP were significantly higher than that of empty vector group in both HEK293and SW480cells (P=0.015,0.001); and there were no significant differences in the mRNA levels of p53RFP of blank group and empty vector group in HEK293and SW480cells(P=0.362,1.000).There was almost no HIF-1α protein in blank group and empty vector group of both HEK293and SW480cells. In the group of trip mutant HIF-la, the protein level of HIF-la increased, and the p53RFP protein levels also increased compared with the groups of blank and empty vector.The second part:We have constructed luciferase reporter vectors (pGL3-pl, pGL3-p2and pGL3-p3) of different fragments (1780bp,1230bp,780bp) of p53RFP gene promoter. Sequencing results matched with GenBank sequence by NCBI blast. Over expression of HIF-la increased luciferase activity (by2-fold,5.4-fold,3.4-fold in pGL3-1l, pGL3-p2, pGL3-p3respectively) compared with empty vector group.We have constructed luciferase reporter vectors (pGL3-p2M, pGL3-p2M2, pGL3-p3M) with mutant HRE and pGL3-p2-AHRE2vector with deletion of HRE2. Mutation (pGL3-p2M) or deletion of the HRE2(pGL3-p2-AHRE2)significantly reduced the increase in reporter gene activity by HIF-la(P=0.000,0.000),while there was no significant difference between the luciferase activity of pGL3-p3M with mutation of HRE3and that of pGL3-p3(P=0.953), and there was also no significant difference between the luciferase activity of pGL3-p2M2and that of pGL3-p2M (P=0355)The third part:Chromatin co-immunoprecipitation assay revealed that HIF-la binding to the HRE2and HRE3of the p53RFP promoter, the occupancy of HIF-la was inhibited on the p53RFP promoter where both the HRE sites were mutated, while p300was recruited to the promoter binding to the HRE2region, rather than HRE3region.The fourth part:Cloned the whole encoding sequence of p53RFP and constructed the expression vector pcDNA4-p53RFP. Sequencing results matched with GenBank sequence by NCBI blast.SW480cells were transfected with pcDNA4-p53RFP, the result of CCK-8showed that p53RFP overexpression cells grew significantly slower than blank control cells and vector control cells on the second, third, and fourth day after transfectiontP<0.004); the cell cycle analyses by flow cytometry were performed,the results showed that there were more cells accumulating in G2phrase of cell cycle in p53RFP overexpression cells compared with vector control cells(.P<0.05), and the apoptosis rates for p53RFP overexpression cells significantly increased(P=0.006).We detected the transcriptional levels of cell cycle-related proteins, such as cyclinBl,cyclinD1, p21Wafl/CiP1and CDK1after SW480cells were transfected with pcDNA4-p53RFP or empty vector for48h. The result showed that the mRNA level of p21waf1/CiPi was up-regulate in PcDNA4-p53RFP transfeted cells compared with vector control cells (P=0.043). While, transfection with pcDNA4-p53RFP had no significant effect on the mRNA levels of cyclin B1,cyclinD1, and CDK1.ConlusionThe first part:p53RFP expression is up-regulated in response to hypoxia through stabilizing HIF-la.The second part:HIF-la induced activation of the p53RFP promoter, and the effect on the activity of pGL3-p2was highest.The third part:HIF-la induced activation of the p53RFP promoter mainly through HIF-la binding on the HRE2region, which subsequently caused recruitment of the transcription coactivator p300.The fourth part:p53RFP overexpression inhibited the growth of SW480cells, induced the G2phase cell cycle arrest and apoptosis of the cells may through up-regulation of p21WAF1/Cipl gene expression.