Effect And Mechanism Of Mutant Type PUMA On The Biological Function Of Hela Cells In Vitro

PUMA is a potent BH3-only protein that antagonises anti-apoptotic BCL-2proteins, has an essential role in multiple apoptotic models. PUMA expression isnormally kept very low, but can be induced by several transcription factors includingp53, p73, E2F1and FOXO3a, whereby it can induce an apoptotic response.Recentstudies show that PUMA is subject to post-translational control throughphosphorylation.Evidence that PUMA is phosphorylated at multiple sites, with themajor site of phosphorylation being serine10.Replacing serine10with alanine causesreduced PUMA Protein turnover,Prolong the half life,and enhanced the stability andcell apoptotic death. This topic is to investigate on the differences of Wild typePUMA Protein mutant PUMA Protein function and the apoptosis-inducingmechanism,which may pave the way for deeply study on tumor suppressor functionof PUMA and its Post-translational regulate on mechanism.Objective:To investigate the differences of Wild type PUMA Protein mutant PUMAProtein in physiological and biochemical function and the apoptosis-inducingmechanism,which may pave the way for deeply study on tumor suppressor functionof PUMA and its Post-translational regulate on mechanism.Methods:PUMA was amplified by PCR from plasmid of pCEP4-(HA)2-PUMA andsubsequently cloned into the eukaryotic vector pIRES2-EGFP to generatepIRES2-EGFP-(HA)2-PUMA. pIRES2-EGFP-(HA)2-PUMA-T28G was constructedthrough site-directed mutagenesis. Sequence of the inserted gene was identified byPlasmid PCR and analyzed by sequencing subsequently.Two plasmids respectivelytransfected into HeLa cells by Lipofectamine2000.The expression of PUMA wasdetected by Western blot and RT-PCR. Hela cells were into3groups:Wild typePUMA transfection group,mutant PUMA transfection group,and the empty vectorcontrol group.After transfection24h and48h the expression of PUMA was detected by Western blot and RT-PCR.The cellular proliferation inhibition were examined byMTT The cellular apoptosis rates were examined by FCM. Characteristicmorphological changes of apoptosis by PI and Hoechst33342staining.Wild typegroup and mutant group after joining protein synthesis inhibitors cycloheximide,degradation of PUMA Protein was detected by Western blot, respectively.RT-PCRwere used to detect mRNA and protein of PUMA, Bax and Bcl-2respectively.Caspase-3activity was tested by spectrophotometer.Results:DNA sequencing showed that TCC,which encoded the S10of PUMA,waschanged to GCC;there were no mutation sites in other codons of PUMA inpIRES2-EGFP-(HA)2-PUMA-T28G recombinant plasmid.High expression of EGFPwas detected by fluorescence microscope. Western blot and RT-PCR displayed thehigh expression of PUMA gene. The eukaryotic expression vectors for PUMA and itsmutant can be successfully constructed.We analysed the expression levels of Wild type PUMA and mutant PUMA bywestern blotting. This revealed that mutant PUMA was expressed at a higher steadystate level than Wild type PUMA in the same under the condition of transfectionefficiency. This difference was not due to transfection efficiency or differenttranscription rates from the two plasmids, as equal amounts of Puma mRNA could bedetected by qPCR in the WT and mutant PUMA-expressing cells. Cells expressingWild type PUMA and mutant PUMA were treated with cycloheximide,and we nexttested Wild type PUMA and mutant PUMA protein degradation. This assay revealedthat degradation of mutant PUMA protein prolong the half life and enhanced thestability, due to we changed the PUMA protein phosphorylation sites. Mutant PUMAand WT displayed certain degree proliferation inhibitory effect in a time-dependentmanner Hela cells.There was significant difference(P<0.05) to compare empty vectorgroup OD value of cells transfected with mutant PUMA and WT.Moreover the ODvalue after transfected with mutant PUMA was significantly lower than that aftertransfected with WT at the24h and48h time point, and there was significantdifference(P<0.05). The inhibition ratio on the Hela cells growth after transfectedwith mutant PUMA was higher than that after transfected with WT.we evaluated quantification of early and late dying cells nuclear morphology by PI andHoechst33342staining amongst the transfected population.Of the GFP-positivepopulation,25%of those transfected with mutant PUMA displayed apoptotic nuclei,in comparison with11.8%in cells transfected with empty vector, with a further9.2%of the transfected population undergoing death compared with WT at the24h timepoint,the difference was statistically significant. It was similarly with FCM results.We analysed the expression levels of PUMA mRNA, BCL-2mRNA and BAXmRNA by RT-PCR. The expression of PUMA mRNA increased with extension ofthe transfection time,they were all time-dependant.With the PUMA upregulation,BCL-2down-regulated and BAX has no obvious change.Caspase-3activity wasdetected after transfected into HeLa cells, and then we found that Caspase-3activitywas significantly elevated, the transfection6h group was (1.23±0.23) times ofControl group, the transfection12h group was(2.05±0.17) times of Control group,the transfection24h group was (2.75±0.22) of Control group,the transfection36hgroup was(3.32±0.24) times of Control group,the transfection48h group was(4.21±0.21) times of Control group, it has statistically significant difference.Conclusions:1. PUMA can inhibited Hela cells proliferation, promote apoptosis of tumor, andmutant PUMA in Hela cells than in wild type PUMA the inhibition of tumor cells andpromoting apoptosis function more obvious.2. PUMA is phosphorylated at multiple sites, with the major site ofphosphorylation being serine10. Replacing serine10with alanine causes reducedPuma turnover and enhanced cell death.3. PUMA effect on promoting apoptosis of Hela cells may be through the raisedPUMA expression,thereby causing BAX of oligomerization and BCL-2down-regulation, ultimately leading to the increase in Caspase-3activity mediated.