Screening Of A Novel Telomerase Regulatory Factor And Its Function And Mechanism In Pancreatic Cancer

Background: The mortality rate of pancreatic cancer is the seventh among all malignant tumors.Due to late diagnosis and lack of effective treatment strategy,the overall5-year survival rate is less than 10%.At present,there are many clinical treatments for pancreatic cancer,including tumor surgery,chemotherapy,radiotherapy,immunosuppressive therapy,targeted drug therapy,etc,but the effect is limited.How to effectively improve the average survival rate of pancreatic cancer is very important.Telomerase is closely related to immortalization and tumor.Immortalization is one of the ten characteristics of tumor,which is mainly due to the high expression of telomerase in 85% of tumor cells.Therefore,the importance of telomerase in the diagnosis and treatment of tumor is gradually recognized.This study aims to screen out novel telomerase activity regulators in pancreatic cancer cells,and explore their mechanisms and functions,so as to provide new methods and strategies for early diagnosis and treatment of pancreatic cancer.Telomerase is a reverse transcriptase in the eukaryotic nucleus,which can use its own RNA as the template to serve as the base for adding repetitive nucleotide sequences(TTAGGG)onto the ends of the telomere,so that telomere shortening can be avoided and cell aging and death can be avoided.Telomerase activity is negative in most normal somatic cells,while the activity of telomerase is activated with high expression in tumor cells.Therefore,telomerase activity can be used as one of the specific biological indicators of tumor cells.Its detection is of great significance for the study of tumor cells.At present,there are many methods for detecting telomerase activity,and the most traditional method is TRAP,which has very high sensitivity.However,there are also disadvantages such as too high false positives,inability to accurately quantify,isotope contamination and PCR product saturation,etc.Although some other methods have the advantages of high sensitivity and time-saving,they need nanomaterials and professional detection equipment,which are not conducive to popularization in general laboratories.Therefore,this study wanted to establish a Biotin-labeled Direct Telomerase Assay(Biotin-DTA),which can accurately quantify telomerase activity,and does not need the use of PCR and isotope,so as to provide a new method for the laboratory study of telomerase.In addition,the regulation mechanism of telomerase activity is complex,involving the regulation of telomerase expression level,telomerase assembly,telomerase localization and other links,and new telomerase regulatory molecules need to be found urgently.Methods: 1.To establish a biotin-labeled direct telomerase activity detection method by the 293 T cells with high telomerase expression.293 T cells were transfected with Flag-h TERT and h TR plasmids(called 293 T super telomerase,or 293 T ST),and the expression of Flag-h TERT was detected by Western blot.Immunoprecipitation was performed on the lysate extract(Input)of 293 T ST cells,and the obtained fluid was IP Eluate.The efficiency of immunoprecipitation was evaluated by using Western blot to detect Flag-h TERT in Input and IP Eluate.IP Eluate was used for telomerase extension reaction.2.The reaction system of this method was optimized,including culture temperature,reaction time,KCl concentration,etc.Firstly,the difference of telomerase activity was compared under 32 ℃ and 37 ℃ culture conditions.Secondly,different reaction times were set to clarify the reaction conditions with the strongest signal.Finally,different KCl concentrations were set to obtain the optimal concentration of KCl.3.The telomerase activity of five same samples was detected to confirm the repeatability of the proposed method.In addition,telomerase activity was detected by 1μL,2μL,5μL,10μL,15μL,and 20μL IP Eluate.The results showed that the proposed method could be used to analyze telomerase activity accurately and quantitately.The reliability of the proposed method was further verified by detecting the changes of telomerase activity by transient transfection of si RNA of telomerase regulator PES1.The telomerase activity was detected by Biotin-DTA method through the experiment of gradient dilution sample,and the stability of the method was confirmed by comparing with TRAP method.Finally,the new method was used to detect the endogenous telomerase activity in Hep G2 cells to verify the sensitivity of the method.4.The proteins that may bind to h TERT or h TR were screened by immunoprecipitation and mass spectrometry.Then the candidate genes were selected,and the interaction between the candidate genes and h TERT and the regulation of telomerase activity were further identified through immunoprecipitation and telomerase activity detection.Through immunoprecipitation,WB and q RT-PCR,we explored whether the selected telomerase binding factor LDHB could regulate the telomerase activity of Panc-1cells by affecting the expression of h TERT or h TR;The RIP method was used to determine whether telomerase activity of PANC-1 cells was regulated by affecting the assembly of h TERT and h TR.6.Through telomere length measurement,cell senescence,cell growth curve and other experiments,we further explored whether LDHB affects the proliferation and senescence of PANC-1 cells.7.Through xenograft tumor of pancreatic cancer cells in nude mice,the function of LDHB in vivo was verified.Results: The non-radioactive direct telomerase detection method labeled with biotin can accurately detect telomerase activity.It is a reliable method for telomerase activity detection,with better resolution and sensitivity.Compared with the traditional TRAP,the quantitative method is more accurate without PCR.It is worth promoting because the required materials and conditions are relatively simple.Temperature plays an important role in the expression of exogenous telomerase.When cells were cultured at 32 ℃,the expression of Flaghtert was significantly higher than that of conventional cells cultured at37℃.After immunoprecipitation,more telomerase could be obtained and the activity of telomerase could be improved.When detecting telomerase activity,the highest activity was achieved when KCl concentration was 200 m M and 250 m M,which was recommended.LDHB interacts with h TERT,and they are directly bound,independent of h TR.When LDHB was knocked down in pancreatic cancer cells,the expression level of h TR was increased,the m RNA expression level of h TERT was not significantly changed,but the protein expression level of h TERT was slightly decreased.LDHB can inhibit the assembly of h TERT and h TR.PANC-1 cells cultured in different concentrations of sodium pyruvate and sodium lactate showed no significant change in telomerase activity.After knockdown of LDHB in PANC-1 cells,its telomerase activity decreased,telomere length shortened,cell senescence increased,and growth was inhibited.Knockdown of LDHB can inhibit the tumorigenicity of pancreatic cancer cells in nude mice.Conclusion: Biotin-labeled non-radioactive telomerase assay is a stable,reliable and feasible method for telomerase assay,which can be used to screen telomerase regulatory factors and popularized in the laboratory.LDHB plays a role in regulating telomerase activity in pancreatic cancer cells.After knockdown of of LDHB,the expression level of h TR is increased,and the protein expression level of h TERT is slightly decreased.LDHB regulate telomerase activity not through metabolic pathway,but through influencing the protein expression of h TERT and the assembly of h TERT and h TR.After long-term knockdown of LDHB,telomerase activity decreased,telomere length shortened,cell senescence increased and growth was inhibited in pancreatic cancer cells.After knockdown of LDHB,the tumorigenicity of pancreatic cancer cells decreased and tumor growth was inhibited.