| Part 1 The effect and mechanism of Protein Kinase B inhibitor Triciribine about the radiosensitivity in ESCCObjective: Our goal is to find the Triciribine(TCN)inhibitor of Protein Kinase B(Akt),and its downstream target gene VEGF and HIF-1? in ESCC cells,and observe the mechanism of Triciribine about the radiosensitivity in ESCC.Methods: The ECA109 and KYSE450 of esophageal squamous cell carcinoma were selected,and under different concentration gradients,hypoxia state and time point,the Akt inhibitor was selected to treat esophageal squamous cell carcinoma with triciribine,and the cell proliferation-toxicity assay(cell counting Kit-8,CCK8)observed the effect of triciribine phosphate ester on different esophageal squamous cell carcinoma.The expression of Akt,P-Akt,hif-1? and VEGF protein in different concentrations of triciribine was verified by using Western blot,and the changes were given to regular oxygen,hypoxia,the radiosensitivity of triciribine in hypoxia and non-oxygen esophageal squamous cell carcinoma groups after irradiation treatment with 6MV-X of the hypoxia and the CIS-Platinum positive control group were observed by Clone Formation experiment,and the cell apoptosis rate in different treatment groups was detected by flow cytometry.The establishment of non-thymus nude mice(Nude Mouse,For short,esophageal squamous cell carcinoma cell line ECA109)were divided into four groups: control group,the simple irradiation group,the intraperitoneal injection triciribine group,and the intraperitoneal injection triciribine joint irradiation group.Radiation Group subcutaneous transplantation of a single irradiation dose of 8Gy,irradiated 14 days.Then we measured tumor volume and detected the protein expression of Akt,p-Akt,HIF-1? and VEGF in the tumor after the death of nude mice.Results: Triciribine inhibits the proliferation of ECA109 and KYSE450 in esophageal squamous cell carcinoma,and has a time and dose dependent;By analyzing the curve we find that the radiation damage of the hypoxic esophageal squamous cell carcinoma after 24 hours in the treatment of the hypoxic esophageal squamous cells by the triciribine is more obviously.After treatment of triciribine,the apoptosis rate of the cells in the hypoxia cell irradiation was increased significantly,and there was a dose dependence from triciribine treatment groups.The protein expression of p-Akt,HIF1? and VEGF were decreased when Triciribine was used in the hypoxic esophageal carcinoma cells,And there is a dose-effect correlation.Further studies revealed that triciribine significantly reduced the rate of proliferation of ECA109 cells transplanted in nude mice after radiotherapy,while the Western blot assay showed that the expression of p-Akt,HIF1? and VEGF in transplanted tumors was decreased by the detection of triciribine in the transplanted tumor model of nude mice.Conclusion: Thus,we postulate that TCN might induce radiosensitivity of ESCC.To validate the hypothesis,we identify the involvement of TCN in modulating ESCC radiosensitivity in multilevel models.The innovative proposal by our study may possibly find a new target to radiosensitivity of ESCC.Part 2 Screening and molecular biological mechanism for radiotherapy sensitization-related of Akt-dependent Lnc RNAs of esophageal squamous cell carcinomaObjective: In this paper,the clinical specimens,cell lines and nude mice animal models of esophageal squamous cell carcinoma were proposed to find the Akt-dependent Lnc RNAs of esophageal squamous cell carcinoma for radiation sensitization.By screening the results we found LINC00293 and then studied its biological phenotype,which binds to transcription factor ETS1 and increases the important anti-apoptosis genes MCL1 affect radiation sensitivity.And this study can explore the radiation sensitization molecular mechanism of Akt-inhibitor.Methods: A total of 60 patients with locally advanced esophageal squamous cell carcinoma were collected and followed up for one year.We assessed the severity of a one-year condition as a standard for patients undergoing radical chemoradiotherapy,and divided it into two groups.The Akt dependent Lnc RNAs was screened by Akt inhibitor triciribine.Then we choose LINC00293 as the key of follow-up research by combining with TCGA.Screening of downstream target genes directly combined with LINC00293 by using RNA pull-down assay combined with mass spectrometry(MS).We choose ETS1 as downstream target gene by RIP and Western Blot.The m RNA expression of LINC00293 and transcription factor ETS1 was detected by q PCR,and the expression of transcription factor ETS1 and downstream related apoptotic pathway proteins were detected by immunohistochemistry,then the correlation between the change of gene expression and prognosis was analyzed.We chose ECA109 and KYSE450 by q PCR and Western Blot.Prepare LINC00293 and ETS1 slow viral vectors.Through the cloning Experiment and CCK8 experiment for proliferation,the flow cytometry was used to measure the apoptosis,and the immunofluorescence was used to test ?-H2 AX.Furthermore,Northen Blot and Protein Atlas clarifies subcellular localization of LINC00293 and ETS1 in esophageal squamous cell carcinoma cells.Based on the successful construction of LINC00293 and ETS1-stabilized esophageal squamous cell carcinoma,a model of BALB/c nude mouse subcutaneous tumor was constructed.The tumor got 6 MVX radiotherapy after 5cm diameter,2 Gy/time,1 time/day,total irradiation for 5 days,and the total dose was 10 Gy.The changes of tumor bodies were recorded in nude mice after radiotherapy.The survival curves of the nude mice model were obtained by fixation of tumor tissue.The changes of subcellular localization,expression changes and expression of apoptosis-related proteins were tested by Northen Blot,q PCR and Western Blot in nude mice tumor tissue.Through RIP and rescue experiments,we find that LINC00293 and ETS1 directly combine with each other and participate in radiation resistance.The changes of expression of ETS1 in protein and m RNA levels were detected in Through RIP and rescue experiments,we find that LINC00293 and ETS1 directly combine with each other and participate in radiation resistance.The changes of expression of ETS1 in protein and m RNA levels were detected in LINC00293 knocking down cells.At the same time,the m RNA expression of LINC00293 was detected in the ETS1 knocking down cells.Results: After radiotherapy,the tumor has no change or progressed were in the radiotherapy resistance group,the tumor narrowed after radiotherapy and the disease stability in one year were the control group,the difference between the two lnc RNAs sequencing was screened.GO(gene Ontology)enrichment analysis gave us the main biological functions and pathways involved in the lnc RNAs of 137 groups of difference multiples ?5,and the main biological function of target gene is apoptosis,DNA damage,mitosis and DNA metabolism.We found that the change of ENSG00000253314-LINC00293 gene expression affected the survival period of esophageal cancer patients after radiotherapy by using Graph Pad Prism 7,And the higher expression,the shorter survival.Proliferation was inhibited and DNA damage increased after knockdown of LINC00293 in ECA109 cells.RNA pull-down combined with MS screening directly binds the downstream target gene of LINC00293.Transcription factor ETS1 had the highest score in MS,and the ETS1 signal was strongest by RIP combined with Western blot experiments.Using instantaneous transfection technique,LINC00293 and ETS1 decreased in m RNA and protein level in esophageal squamous cell carcinoma,and the m RNA level of LINC00293 decreased when ETS1 was knocked down.It is suggested that there is positive feedback loop between these two.Northern Blot suggests that the subcellular localization of LINC00293 in the nucleus,and the Human Protein Atlas database shows that the ETS1 subcellular localization is also the nucleus.The expression of MCL1 protein decreased when LINC00293 or ETS1 were knocked down,but the m RNA and protein expression of LINC00293 or ETS1 had no change after knockdown MCL1.The promo database chooses a fault tolerance of 0,which shows that ETS1 may be combined in the boot sub-region of LINC00293.Conclusion: The LINC00293 regulated by Akt in esophageal squamous cell carcinoma is involved in promoting the resistance of radiotherapy,and the prognosis of esophageal squamous cell carcinoma is promoted by the positive feedback loop with ETS1 to promote the MCL1 expression leading to radiation resistance.This study reveals the mechanism of increased sensitivity to radiotherapy for esophageal squamous cell carcinoma after akt small molecule inhibitor triciribine. |
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