Identification Of Human PDCD4 Promoter And The Early Analysis Of Its Transcription Regulation

ObjectiveProgrammed cell death, also called apoptosis, plays an essential role during many biological processes. One gene identified as being up-regulated after initiation of programmed cell death is Pdcd4(programmed cell death 4). Recent data suggest a function for Pdcd4 as a tumour suppressor, thereby implicating this protein as a promising target for anti-neoplastic therapy.Pdcd4 acts as a transcription and translation inhibitor, thereby influencing tumour progression, cell cycle and differentiation.. A down-regulation of PDCD4 was discovered in a variety of tumours,including glia-, lung-and renal-derived tumours, tongue tumours and invasive ductal breast carcinoma. In human glioma, lung tumours,reduced levels of Pdcd4 protein and mRNA were significantly linked to tumour grade.Pdcd4 seems to be regulated by multiplemechanisms at different levels. Several reports described the regulation of pdcd4 by miR-21.The conserved potential site for miR-21 within the 3'-UTR (3'-untranslatedregion) of pdcd4 mRNA was found and the functionality of this site was demonstrated. Since miR-21 was identified as the microRNA commonly overexpressed in solid tumours, the other studies showed an inverse correlation in the expression of miR-21 and Pdcd4 protein. Another possibility for regulation of Pdcd4 at the protein level is its phosphorylation by Akt and S6K1, leading to increased proteasomal degradation. In addition, our previous study showed that pdcd45'-CpG island methylation was associated with reduced Pdcd4 mRNA levels in human glioma cell lines and tissue, and restoration of pdcd4 was observed after blocking methylation in glioma cells.Summing up,current study on Pdcd4 regulation mainly focus on the translation and protein level,however,little was known about the transcription regulation of Pdcd4.To study the transcription regulation of Pdcd4,we clone and construct an array of luciferase reporter plasmids containing human PDCD4 promoter,identify the region of core promoter region.Meanwhile,to study the mechanism of transcription regulation of PDCD4 by both software analysis and truncated promoter assay.Methods1.To clone and construct luciferase reporter plasmids containing human PDCD4 promoter.(1)Prediction of human PDCD4 promoter regionThe 5'-flanking region spanning-2500 to+500 of human PDCD4 gene was submitted and analyzed by MatInspector,FirstEF and Proscan Prediction software.(2)Clone of PDCD4 Promoter and construction and confirmation of the reporter plasmid pGL4-PDCD4-PlFragment from-548 to+350 was chosen as PDCD4 Promoter and amplified using suitable primers which contains KpnⅠ,BgⅢ,The fragment is named PDCD4-P1. Then PDCD4-P1 was cloned into the multiple cloning site of a pGL4-Basic plasmid to construct pGL4-PDCD4-P1 with the correct sequence(3)Analysis of endogenous PDCD4 expression in different ovarian cell lines by RT-PCR and Western blottingtotal cellular RNA extraction using Trizol,RT-PCR detection using suitable primers. 1 x106cells of each cell lines were isolated,then cells were lysed and total protein were extracted.Detection of PDCD4 by Western blotting.(4) Transfection and dual-Luciferase reporter assaysThe plasmids constrcted above and pGL4-Basic(negative control)were transiently co-transfected with pRL-TK into ovarian cancer cell lines OVCAR3,SKOV3.Luciferase reporter gene assay was conducted 24h after transfection.The firefly luciferase activity(M 1) was normalized with the renilla reniformis luciferase activity of pRL-TK(M2) and the ratio of M1 and M1(M1/M2) stand for the relative luciferase activity of the plasmids2.To identify the core promoter region of PDCD4(1)Construction of an array of truncated promoter plasmidsFragments of-315-+350,+10-+350,-315-+l,-164-+l,-114-+1 and-67-+1 named PDCD4-P2,P3, P4,P5,P6,were selected to construct an array of truncated promoter plasmids,using pGL4-PDCD4-P1 as template.(2)Transfection and dual-Luciferase reporter assaysThe plasmids constrcted above and pGL4-Basic(negative control)were transiently co-transfected with pRL-TK into OVCAR3 and SKOV3 cells. Luciferase reporter gene assay was conducted 24h after transfection3 To study the transcription regulation of human PDCD4(1)Prediction and analysis of TFBSs within 5'-flanking region of PDCD4The 5' -flanking region from-600-+400 of human PDCD4 gene was submitted and analyzed by MatInspector software.(2)Construction of truncated promoter plasmids and dual-Luciferase reporter assaysFragment of-548-+223 and-315-+223,named P4,P8, was amplified and cloned into pGL4-Basic to construct pGL4-PDCD4-P4,P8 with the correct sequence. pGL4-PDCD4-P4 and pGL4-PDCD4-P8 were seperately co-transfected with pRL-TK into OVCAR3 cell line.Luciferase reporter gene assay was conducted 24h after transfection.(3)Construction of RREB binding site truncated and mutation promoter plasmids and dual-Luciferase reporter assaysFragment of-315-+292, named P5, and-548-+350 fragment with RREB binding site mutation, named P1-RREB3mut was amplified and cloned into pGL4-Basic to construct pGL4-PDCD4-P5, pGL4-PDCD4-P1-RREB3mut. The plasmids constrcted above and pGL4-Basic(negative control)were transiently co-transfected with pRL-TK into OVCAR3 and SKOV3 cells. Luciferase reporter gene assay was conducted 24h after transfectionResults1.To clone and construct luciferase reporter plasmids containing human PDCD4 promoter. (1)Prediction of human PDCD4 promoter region To clone PDCD4 promoter,we predict the promoter region of PDCD4.The results was MatInspector-500-+191,FirstEF-262-+307 and Proscan+25-+275.We finally choose Fragment from-548 to+350 as PDCD4 Promoter, named PDCD-P1.(2)Clone of PDCD4 Promoter and construction and confirmation of the reporter plasmid pGL4-PDCD4-PlSuccessful construction of the reporter plasmids pGL4-PDCD4-P1.Analysis of DNA sequencing ensured the fidelity of amplification and the correct orientation of the luciferase reporter plasmids constructed(3)Detection of endogenous PDCD4 expression in different ovarian cell lines by RT-PCR and Western blottingTo find a target cell model, we detect the endogenous PDCD4 expression in different ovarian cell lines.The Results showed high-expression of endogenous PDCD4 in OVCAR3 and low-expression of endogenous PDCD4 in SKOV3,3AO and CAOV3.(4) Transfection and dual-Luciferase reporter assaysThe results of dual-luciferase reporter assays showed that pGL4-PDCD4-P1 can highly express,about 67 times of pGL4-Basic(negative control),in OVCAR3 which express high level endogenous PDCD4..In SKOV3 which express low-level endogenous PDCD4,the luciferase activity of pGL4-PDCD4-P1 is about 15 times of pGL4-Basic.The results showed that PDCD4-P1 contains the maximum promoter activity.2.To identify the core promoter region of PDCD4(1)Construction of an array of truncated promoter plasmidsTo identify the core promoter region of PDCD4,we construct an array of truncated promoter plasmids.The results showed successful construction of the reporter plasmids..Analysis of DNA sequencing ensured the fidelity of amplification and the correct orientation of the luciferase reporter plasmids constructed(2)Transfection and dual-Luciferase reporter assaysThe results of dual-luciferase reporter assays showed that pGL4-PDCD4-P2,P12,P13 can highly express,about 40 times of pGL4-Basic(negative control),in OVCAR3 which express high level endogenous PDCD4.With the truncation of PDCD45' -flanking region,,the luciferase activity of pGL4-PDCD4-P14 decreased,about 13 times of pGL4-Basic.When it was truncated to+67, pGL4-PDCD4-P7 completely lost its activity.The results showed that th core promoter of PDCD4 locates at-164-67 region.3 To study the transcription regulation of human PDCD4(1)Prediction and analysis of TFBSs within 5' -flanking region of PDCD4Sequence analysis has revealed the existence of many putative transcription regulatory elements spanning-600 to+400.For example,NF-kB(+445),RREB(-153,+239,+283),IRFF(+66),P53(+310)(2)Transcription regulation of PDCD4 by SplSequence analysis has revealed the existence of three putative Spl binding sites in core promoter region of PDCD4. Our previous results showed that P13 can highly express,about 40 times of pGL4-Basic(negative control), With the truncation of PDCD45' -flanking region, the luciferase activity of pGL4-PDCD4-P14, which lacks two Sp1 binding sites, decreased to about 13 times of pGL4-Basic.When it was truncated to+67, all Spl binding sites were cut off,pGL4-PDCD4-P7 completely lost its activity.The results indicated that Spl may be responsible for the basic transcription level of PDCD4.(3)Transcription regulation of PDCD4 by NF-kBTo study the influence of NF-kB on transcription of PDCD4, dual-Luciferase reporter assay of pGL4-PDCD4-P1 and P2,which lack the NF-kB binding site(+445),was performed.The result showed that the depletion of NF-kB binding site resulted in a slight reduction of RLU.However,the effect of NF-kB on PDCD4 need a further study.(4)Construction of truncated promoter plasmids and RREB binding site mutation promoter plasmidsSuccessful construction of truncated and RREB binding site mutation promoter plasmids, Analysis of DNA sequencing ensured the fidelity of amplification and the correct orientation of the luciferase reporter plasmids constructed.,which include pGL4-PDCD4-P4,P8 and pGL4-PDCD4-P5 pGL4-PDCD4-P1-RREB3mut.(5)Dual-Luciferase reporter assays and identification of a negative transcription regulation region of PDCD4pGL4-PDCD4-P4 and pGL4-PDCD4-P8 were seperately co-transfected with pRL-TK into OVCAR3 cell line.Luciferase reporter gene assay was conducted 24h after transfection..The results showed that depletion of the region+223～+350 leads to a significant increased activity,so there have a negative regulatory element in region+223～+350.(6)Negative transcription regulation of PDCD4 by RREBTo study the effect of RREB on PDCD4 transcription, pGL4-PDCD4-P16, pGL4-PDCD4-Pl-RREB3mut and pGL4-Basic(negative control)were transiently co-transfected with pRL-TK into OVCAR3 cells. Luciferase reporter gene assay was conducted 24h after transfection.The results showed that both the depletion and mutation of RREB3 binding site lead no significant change on the transcription activity of PDCD4 promoter.The results also identify the region of+223～+292 as a negative transcription regulation region,which still have two putative RREB binding sites.ConclusionsSuccessful construction of the human PDCD4 promoter luciferase reporter plasmids and characterization of core promoter region.Meanwhile,a potential negative transcription factor binding site was found downstream the TSS.This study provides a first step towards understanding the transcriptional regulation of human PDCD4 gene...