Cloning Of Cell Division Autoantigen-1 Promoter And Regulation With TGFβ1 And Smad2/3siRNA

Specific promoter binding with functional protein is necessary fortranscription of deoxyribonucleic acid (DNA). Therefore, as a first steptowards the better understanding of molecular mechanise and regulation ofa gene, it is necessary to know the function and characterization of itsspecific promoter. In addition, an effective way of target gene therapy is tofind its specific promoter. According to this, it is important to study on thecloning and characterization of the promoter region of cell divisionauntoangiten-1 (CDA1). RNA interference is a process utilized by eukaryotes to modulate geneexpressio at pre-and post-transcriptional levels. Its mechanism is thatresearchers transfect cells with 12-23 bp siRNA to induce RNAi in thesesystem without eliciting the antiviral response. Since the discovery 7 yearsago that introduction of dsRNA (double-strand RNA) into cells initiatedsequence-specific gene silencing, the use of RNAi as a laboratory tool hasrevolutionized the study of eukaryotic gene function. Moreover, it holdsgreat promise as a molecular therapeutic technique for the treatment ofcancers and infectious diseases. CDA1 is a novel gene and has just been found for 5 years. It maybethe target gene of TGFβ, however, its function and characterization isunclear. Smads protein are key mediater of TGFβ and its target gene,whose binding results in the formation of a ligand-receptor complex andactivation of the type I receptor. The activated type I receptor thenphosphorylates Smad2 and Smad3. Smad4 forms complexes withphosphorylated Smad2 and Smad3, and together they accumulate in thenucleus. Smads can recruit coactivators to stimulate transcription or recruitcorepressors to inhibit transcription. The role of TGFβ and Smad in theprocess of regulating CDA1 are the key to understand the role of CDA1 inTGFβ signaling pathway. The aim of the study is to make clear thefunction and characterization of CDA1 promoter region and the regulationof TGFβ and Smad2/3. Human and the mouse CDA1 having highlyhomology has been identified, so that the study at first is to cloning mouseCDA1 promoter region and to observing the role of TGFβ and Smad inthe process of regulating CDA1. To better observing the function of theCDA1 promoter, the study selects the pGL3-Basic luciferase vector asvector of CDA1 promoter because it will better express the nomal protein invivo. RNA interference technology is utilized to silence Smad2 and Smad3to understand the role of Smad2 and Smad3 in regulating CDA1.Methods1. Identification and construction of luciferase reporter gene constructProscan software is utilized to predict CDA1 promoter region accordingto mCDA1 genomic DNA and Smad binding sites. Then different lengthmCDA1 promoter fragments were amplified by PCR using mouse livergenomic as template and cloned into BglⅡ-digested pGL3-basic plasmidto produce recombination plasmid-pGL3-basic-mCDA1promoter. Theirquality were checked by restriction enzyme digestion and sequencing.2. Identification luciferase activity of recombination plasmidRecombination plasmids were transfected into LLC and RAW 264.7cells and measured their luciferase activity.3. Observation the role of TGFβon pGL3-basic-mCDA1promoterpGL3-basic-mCDA1promoters were transfected into the cells andadded TGFβ,then measured their luciferase avtivity.4. Identification and construction of pAVU6+27-Samd2siRNA andpAVU6+27-Smad3siRNA constructs5. Identification the function of pAVU6+27-Samd2siRNA andSmad3siRNA plasmidLLC cells transfected with pAVU6+27-Samd2siRNA or Smad3siRNAplasmid, lysed after 48h, and using RT-PCR and Western-blotting toidentify the function of pAVU6+27-Samd2siRNA and Smad3siRNA plasmid.6. Observation regulation mCDA1 with Smad2 siRNA andSmad3siRNALLC cells were transfected with pAVU6+27-Samd2siRNA or/andSmad3siRNA plasmid as well as pGL3-basic-mCDA1promoter (0.97kb and6.1kb). then stimulted 12hr with TGFβ(5ng/ml) after transfectd 48hr.Finally lysed the cultured cells and measured their activity by luciferase andβ-gal.Results and discussions1. Identification and construction of luciferase reporter gene-pGL3-basic-mCDA1promoter construct successfully2.Luciferase activity of pGL3-basic-mCDA1promoter recombinationplasmid is effective, and the activity of recombination plasmid in LLC cellsis obviously higher than that in RAW264.7 cells. Luciferase activity ofdifferent length mCDA1 recombination plasmid in LLC cell or RAW264.7cells is significanely difference.The data suggested these promoters ofnucleotide sequences maybe contain some enhancer , insulator andsilencer.3. TGFβ positively regulated Luciferase activity of different lengthmCDA1 recombination plasmid in LLC cell or RAW264.7 cell was observedthrough luciferase assay of the cells transfected pGL3-basic-mCDA1promoter recombination plasmid stimulated with TGF β (finalconcentration: 5ng/ml). CDA1 is the target gene that was further proved bythe data.4. pAVU6+27-Samd2siRNA and Smad3siRNA were constructedsuccessfully. Then the function of pAVU6+27-Samd2siRNA andSmad3siRNA construct were identificated through RT-PCR andWestern-blottinng methods after LLC cells have been transfected for 48hr.5. Luciferase activity of pGL3-basic-mCDA1 promoter recombinationplasmid (0.97kb) is weakly higher when the LLC cells were transfected withpAVU6+27-Samd2siRNA or Smad3siRNA construct than that cellstransfected with pAVU6+27 empty vector. However, luciferase activity ofpGL3-Basic-mCDA1 promoter recombination plasmid (6.1kb) is notchanged in the same situation. In contrast, luciferase activity ofpGL3-basic-mCDA1 promoter recombination plasmid (0.97kb and 6.1kb) isdownregulated when the LLC cells transfected withpAVU6+27-Samd2siRNA and Smad3siRNA construct together. The datasuggested Smad protein indeed plays a key role in regulation of CDA1.Moreover, the regulation maybe related with Smad binding site of CDA1.Conclusions1. Luciferase activity is different when the same pGL3-basic-mCDA1promoter recombination in LLC and RAW cells and Luciferase activity ofthe same length pGL3-basic-mCDA1 promoter recombination is alsodifferent in LLC cells and RAW cells.2. Luciferase activity of the different length pGL3-basic-mCDA1promoter recombination in LLC cells or RAW cells is significantly high whenthe transfected cells were treated with TGFβ.3. Luciferase activity of mCDA1 promoter in which LLC cellstransfected with pGL3-basic-mCDA1 promoter recombination plasmid(0.97kb) and pAVU6+27-Samd2siRNA or Smad3siRNA construct is weaklyhigher than that of cells transfected with empty pAVU6+27. In contrast,luciferase activity of pGL3-basic-mCDA1 promoter recombination (6.1kb) isnot changed in the same situation. Luciferase activity of mCDA1 promoteris decreased when LLC cells transfected with pGL3-basic-mCDA1promoter recombination plasmid and pAVU6+27-Samd2siRNA andSmad3siRNA construct. The data suggested Smad upregulated the activityof mCDA1 promoter.In a word, different length mCDA1 promoter region are cloned and theresearch proved that the activity of mCDA1 could be regulated by TGFβthrough Smad2 and Smad3 siganling pathway.