Study Of The Underlying Mechanism Of Small Double Strand Rna To Activate Tumor Suppressor Gene P21^WAF1/CIP1 Expression

Part1Small double strand RNA molecule dsP21-322induce the expression of tumor suppressor gene p21WAF1/CIP1in human cellsOBJECTIVE:To select more human cell lines susceptible to small double strand RNA, dsP21-322that mediated tumor suppressor gene p21WAF1/CIP1activation and investigate whether this RNA activation (RNAa) process is dependent on transcriptional factor p53expression.MATERIALS AND METHODS:A dsRNA, dsP21-322, targeting the tumor suppressor gene p21promoter at position-322relative to the transcription start site was synthesized. A dsControl lacking significant homology to all known human sequences was also synthesized and used as a negative control. In order to certify the availability of dsP21-322, dsP21-322or dsControl were transfected into PC-3and T-24cancer cells and monitored p21gene or protein induction by using RT-qPCR or Western-Blot. Subsequently,four kinds of human cell lines which express different types of p53protein were chose, including human osteosarcoma cell U2-OS (p53wide type), human embryonic kidney cell line293T (p53wide type), human cervical cancer cell Hela (p53mutant type) and human lung cancer cell NCI-H1299(p53null). All above these cell lines were cultured in vitro and transfected with available dsP21-322or dsControl, RT-qPCR and Western-Blot were applied to detect the expression level of p21, p53gene transcription and p21, p53protein, respectively.RESULTS:The results of RT-qPCR and Western-Blot showed that dsP21-322caused an obvious induction in p21mRNA and protein levels compared with dsControl transfections in both PC-3and T-24cells, respectively, which confirmed the availability of dsP21-322. Further analysis demonstrated that dsP21-322also caused a significant induction in p21mRNA expression in p53wide type or p53mutant cell lines. Compared with dsControl transfections, induction of p21mRNA was3,97-,4.94-, and4.64-fold in U2-OS, Hela and293T cell lines, respectively. Western blot further analysis elevated levels of p21protein strongly correlate to the increase in p21mRNA expression in these three cell lines. The p21protein in dsP21-322transfections was significantly more than in dsControl transfections (P<0.05). Meanwhile, dsP21-322did not affect the expression of p53in three kinds of cell lines. However, dsP21-322was unable to elevate the p21mRNA and protein level in p53null NCI-H1299cell line.CONCLUSIONS:Activation of p21expression by dsP21-322in human cell lines is pervasive phenomenon. Furthermore, dsP21-322failed to elevate the p21expression in p53null cell, indicating this process might rely on the expression of p53, which needs to further study. Part2Defining Features for Small double strand RNA molecule dsP21-322induce tumor suppressor gene pQAF1/CIP1gene activationOBJECTIVE:To observe the kinetics of a small activating RNA(dsP21-322)-mediated tumor suppressor gene p21activation, as well as identify the site of action in cells for dsP21-322and determine the effect of strand modifications to manipulate dsP21-322activity, in order to define features of RNAa.MATERIALS AND METHODS:Using standard RT-PCR and RT-qPCR assess expression levels of p21gene from Oh to72h after transfected dsP21-322into PC-3cells or T-24cells, respectively. Modified dsRNAs molecules derived from dsP21-322and dsControl that were covalently linked to Fluorescence group Cy3at either the5'-end of the antisense (dsP21-322-AS-5'Cy3/dsControl-AS-5'Cy3) or sense (dsP21-322-S-5'Cy3/dsControl-S-5'Cy3) strand were synthesized and transfected into PC-3cells. The site of action for fluorescence labeled dsP21-322was detected by using converted fluorescence microscope. Further synthesized modified dsRNAs that were covalently linked to biotin at the3'-end of the antisense (dsP21-322-AS-3'Bio/dsControl-AS-3'Bio), sense (dsP21-322-S-3'Bio/dsControl-S-3'Bio),5'-end of the antisense (dsP21-322-AS-5'Bio/dsControl-AS-5'Bio) or sense (dsP21-322-S-5'Bio/dsControl-S-5'Bio). Using standard RT-PCR and RT-qPCR evaluate whether strand modifications of dsP21-322could affect its activity.RESULTS:The results of standard RT-PCR and RT-qPCR showed that induction of p21expression began to emerge following48hours of dsP21-322transfection with levels continuing to increase by72hours. Fluorescence labeled dsP21-322actively migrated into the nuclear fraction from cytoplasm in PC-3Cells after transfection for48h. Transfection of dsP21-322modified at its5'-end of the antisense strand completely blocked induction of p21, while other three kinds of modification to the sense or antisense strand retained its activity.CONCLUSIONS:RNAa is nuclear process acting on gene transcription, with the rate at which RNAa activity emerges is48hours after saRNA transfection. Moreover,5'-end of the antisense in saRNA may be critical for initiating transcriptional activation. These findings reveal functional features of saRNA-mediated gene activation that offer the theory support for exploring RNAa mechanistic studies. Part3Studying mechanism for Small double strand RNA molecule dsP21-322induce tumor suppressor gene p21WAF1/CIP1activationOBJECTIVE:To characterize the transcriptional landscape of the p21promoter in human prostate cancer cells, as well as identify the molecular target for small activating RNA dsP21-322. Further analysis whether transcriptional activation is associated with AG02, RNA Pol â…¡ and epigenetic Changes.MATERIALS AND METHODS:Detected potential non-coding transcripts originated from or overlapping the p21promoter by RT-PCR using several primer sets that cover different regions on the p21promoter. Transfected dsP21-322into PC-3and mapped the5'end of p21mRNA by5'rapid amplification of cDNA ends (5'RACE) assay before and after dsP21-322transfection. Using a well-characterized antibody that recognizes biotin protein, Chromatin immunoprecipitation (ChIP) was performed to evaluate potential binding of dsP21-322at the p21promoter after individually transfecting the three activating biotinylated dsP21-322(dsP21-322-AS-3'Bio, dsP21-322-S-3'Bio and dsP21-322-S-5'Bio) into PC-3cell. Moreover, ChIP was also performed to determine whether enrichment of RNA Pol â…¡ and AG02proteins at chromosomal DNA of p21was associated with dsP21-322induced gene expression, as well as determine the potential role of histone modification marks H3K9acetylation(AcH3K9) after transfecting dsP21-322into PC-3cell.RESULTS:The results of RT-PCR and5'RACE showed that none of the p21promoter primer sets amplified PCR products from the cDNA samples, as well as failed to observe extended or new5'end of p21mRNA in dsP21-322transfected cells compared to untransfected cells. Compared to the enrichment of similarly labeled dsControl, ChIP revealed that all three biotin labled dsP21-322had significant enrichment at its target site in a region ranging from-395bp to-127bp relative to the p21transcription start site. In addition, ChIP also suggested that activated dsP21-322 enhanced association of RNA Pol â…¡ and AGO2proteins with the dsP21-322target site. However, no significant changes for AcH3K9were detected at the dsP21-322targeted site or p21transcription start sites after transfecting dsP21-322into PC-3cell.CONCLUSIONS:These results indicate that RNAa is resulted from specific targeting of promoter for saRNA and this process is associated with recruitment of RNA Polymerase â…¡ and AGO2protein to the saRNA-target site. These findings reveal more mechanistically aspects of RNAa, accordingly provide important theoretical basis and effective means for RNAa application.