The Effect And Mechanism Of Antisense Tankyrase Oligonucleotide Combined Antisense Human Telomerase Reverse Transcriptase Oligonucleotide On Telomere In Human Lung Adenocarcinoma A549 Cells

Human diseases is related to gene directly or indirectly.Gene treatment,based on changing gene structure,can be used to mono-gene diseases,but nearly all multi-gene diseases,such as tumor result from regulation imbalance of multiple functional genes,namely that of genome and gene network of every layer.So "regulating gene function,not changing gene structure is main strategy to disease no matter now and future."Traditional Chinese Medicine,which core is equilibrium and integration concept(Yin-Yang theory and Zang-Fu theory),believe that disease result from the balance of integration of function.Functional regulation as a whole is just priority of traditional Chinese medicine.It has been proved by experiences of TCM of thousands years.There are many the same features between TCM and modem medicine,with the equilibrium concept and the concept of viewing situation as a whole corresponding to system biology,harmonization between human and nature corresponding to Environment Genomic Project,treat principle based on system and individualization(treat aiming at the pathogenesis ascertained by differentiating and analyzing the clinical presentation)corresponding to pharmaco-genetics,regulate treatment(strengthening vital Qi,eliminating pathogen,monarch,minister,assistant and guide)corresponding to multi-aim and medicine effect in coordination.TCM and Western medicine will reached integration gradually,and the priority of functional regulation as a whole will get more development.At present,there is an examples for telomere to regulate gene function as a whole.BACKGROUND:Telomeres,which contain repeated TTAGGG sequences,are large nucleoprotein complexes that protect the ends of chromosomes against degradation and fusion.The end-replication problem results in progressive shortening of telomeres leading to genome instability,and telomerase provides a means to replace telomere repeats which are lost during replication as a result of the inability of DNA polymerase to replicate to the end of a linear chromosome.Therefore,telomerase activity not only maintains the telomeres of proliferating cells but is implicated in the process of cellular immortalization and oncogenesis.In recent years,inhibition of telomerase used to be discussed as a promising approach for treating a variety of malignant tumors,but failed in clinic. The key is,in addition to telomerase,many other factors play a role in telomere maintenance including tankyrase.Tankyrase,which poly(ADP-ribosyl)ate telomere repeated-binding factor1 and releases it from telomere,allows access of telomerase to telomere and enhances telomere elongated.Conversely,it was possible that combination of both enzyme inhibitors can increase the risk of critically shortened telomere and promote the following crisis and death of cancer cells.OBJECTIVE:This study is to determine the effect and mechanism of antisense tankyrase oligonucleotide (asTANKS)combined antisense human telomerase reverse transcriptase oligonucleotide (ashTERT)on telomere in human lung adenocarcinoma A549 cells:1.To investigate the role of hTERT mRNA expression,telomerase activity,tankyrase activity and telomere length in t A549 cells teeated by asTANKS and/or ashTERT.2.To determine the association between expression of mcl-1,Bcl-2,Bax and action with asTANKS and/or ashTERT and obtain the mechanism.3.To observe the alteration in morphous and function for A549 cells and explore potential target of telomere-based molecular cancer therapeutics.METHODS AND RESULTS:1.The expression of hTERT mRNA by RT-PCR was markedly repressed by ashTERT and alteration was observed for neither asTANKS nor sTANKS.2.PCR-ELISA showed that telomerase activities in A549 cells were strongly suppressed by ashTERT,but not by asTANKS.3.By Western blot,asTANKS significantly inhibited tankyrase activity,while ashTERT not as well.4.Telomere length measured by Q-FISH,became significantly decreased with the treatment of ashTERT or asTANKS and the efficacy was more remarkable with the combination of them.5.Compared with the circumstance that neither asTANKS nor ashTERT was correlated with the regulation in Bcl-2 or Box,mcl-1 mRNA levels increased dramatically in the presence of asTANKS and no alteration with ashTERT.6.Both total Mcl-1 protein levels and the content of Mcl-1s were upregulated by asTANKS, while the content of Mcl-1s was downgraded by ashTERT,although total Mcl-1 protein levels had no change.However,addition of asTANKS reversed downregulation of Mcl-1s induced by ashTERT and result in elevation of them.Consistent with expression of mRNA in mcl-1,Bcl-2,either asTANKS or ashTERT was unassociateds with Bcl-2 or Bax levels.7.Cells was prone to senescence in morphous with asTANKS or ashTERT as passage time was delayed well and the trend combinated between asTANKS and ashTERT was more significant.8.Cells came more rapidly to an end and population double times was shortened more quickly with the combination of asTANKS and ashTERT,although the same effect was observed with single factor.9.Uptake rate in[~3H]-TdR trend to suppression under continuous treatment with ashTERT or asTANKS and combinated effect was more markedly.10.Transfection efficiency in X-Gal was enhanced gradually with ashTERT or asTANKS and increase combinated with them was more significant.CONCLUSION:1.Distinguished from telomerase,tankyrase is an unique negative-regulated route for telomere maintenance,in which antisense oligos diminishs poly(ADP-ribosyl)ation of TRF1,prevents it from release in telomeres,and trun down access of telomerase to telomeres and unconcerned with hTERT mRNA and protein levels as well.2.The combination between ashTERT and asTANKS can enhance the efficacy of telomere shortening induced by single factor,facilitate cancer cell senescence and hasten earlier tumor cellular crisis.3.The synergistic mechanism of them was concerned with ranscriptic and post-ranscriptic regulation in mcl-1.Concomitant with tankyrase activity downragulation,asTANKS might reverse potential resistance induced by ashTERT. In a word,pharmacological targeting of tankyrase enhances telomere shortening by means of a telomerase inhibitor and results in earlier cellular crisis.This study provides insight into strategies for telomere-based molecular cancer therapeutics by means of the iatreusis of integration concept in TCM.