The Regulation Of Autophagy By Human Telomerase Reverse Transcriptase (hTERT) In Glioblastoma

Objective:Glioblastoma(GBM)is the most common and aggressive malignant brain tumor in adults with high morbidity and mortality.Despite the improvement of medical technology and the rigorous treatment that patients recieved,it is still difficult to remove GBM tumors by surgical resection due to their invasion to the surrounding tissues.As a result,recurrence often occurs in GBM patients.Human telomerase reverse transcriptase(hTERT),the catalytic subunit of human telomerase,is overexpressed in most cancers including GBM.It is well known that hTERT can compensate telomere shortening to immortal cells.However,in addition to the canonical function,hTERT has extra-telomeric roles.Autophagy is a conserved pathway,by which cells deliver cellular organic material and impaired organells to lysosomal for degradation and recycle these cargo to produce energy under a stressful condition.It is reported that an increase of cellular reactive oxygen species(ROS)can activate the process of autophagy,which will facilitate the removal of excessive ROS.On the contrary,when autophagy is impaired under certain conditions,ROS level will be disturbed,which can affect the growth and proliferation of cells.So the paper is going to talk about the non-telomeric role of hTERT in regulating autophagy as well as the subsequent effects on cellular ROS level and survival.Our study will provide an additional basis on the telomerase-tageting therapy for future clinical anti-cancer treatment.Methods:Firstly,by digging data from the database,we compared the mRNA level of hTERT between human glioblastoma samples and normal samples,as well as the overall survival of patients based on different expression levels of hTERT.Next,to construct a stable hTERT-knockdown cell line,we infected glioblastoma U87 cell line with lentivirus vectors carrying hTERT-targeting sh RNA.The effect of hTERT on cell survival and proliferation was measured by clone formation and cell count kit.The relationship between hTERT and autophagy was investigated by immunoblotting.The regulation of hTERT on ROS level was detected by fluorescence probe.Next,we treated cells with antioxidant NAC and detected cell viability.Then,we overexpressed autophagy-related gene BECN1 to analyse the relationship among hTERT,autophagy and ROS.Finally,we investigated the effect of hTERT,autophagy and ROS on cell survival by cell viability assay.Results:1.The mRNA level is increased in glioblastoma samples compared with normal samples,and patients with a higher level of hTERT show a relatively low survival rate.Besides,hTERT knockdown impairs the growth and proliferation of glioblastoma cell line.2.hTERT deficiency impairs the developmemt of autophagy,causes an increase of ROS level,and suppresses cell survival and proliferation.3.Overexpression of BECN1 in hTERT-deficient cells can restore the level of autophagy,lower ROS level and improve cell viability.Conclusion:The results of bioinformatics and biological experiments demonstrate that hTERT might play an important role in the progression of glioblastoma.In vitro biological experiments show that in addition to the role of lengthening telomeres,hTERT could activate the process of autophagy mediated by BECN1,lower the level of cellular ROS and thereby regulate cell survival and proliferation.The research provides more molecular mechanisms for hTERT acting as a target of clinical anti-tumor therapy.