| Objective: Gastrointestinal stromal tumor(GIST)is the most common mesenchymal tumor in the gastrointestinal tract,Imatinib mesylate(IM)is a first-line targeted therapy for patients with medium-high risk GIST,which can significantly prolong the median survival period and the disease-free survival period of GIST patients.However,in the course of IM treatment,the incidence of drug resistance is as high as 70%-80%,which may eventually lead to the recurrence and progression of GIST and poor prognosis of patients.Radiotherapy(RT)has long been considered insensitive to GIST,mainly due to the limitations of previous techniques and the slow clinical response indicated by early clinical trials.With the continuous improvement of radiation technology and concept(radiosensitizer),as well as the continuous understanding of the molecular heterogeneity within tumors,in recent years,the study of IM as radiosensitizer combined with RT has emerged in a variety of tumors.In the clinical practice of GIST,medical records and small-sample clinical trials of IM combined with RT have been reported continuously after the late IM resistance,but the basic research has not been reported.By cell in vitro experiments,this study separate role in IM,IR and under the joint action,to observe different groups of GIST-882 cell line of the influence of the morphology,cell proliferation and apoptosis,cell clone formation,migration and invasion,and the cell cycle,etc.The influence of the biological characteristics,study whether IM combined IR have a synergistic inhibition effect on the GIST-882 cells.Then,the possible molecular mechanism of synergistic effect of IM combined with IR was elucidation by comparing the changes of transcriptional levels and protein levels of radiation-related DNA damage repair genes between different action groups and the classical signal pathway of IM inhibition.Finally,at the histological level,the correlation between RAD51,a key DNA repair protein,and the clinicopathological characteristics of GIST patients was further analyzed by comparing the expression of radiation-related DNA damage repair protein in GIST tissuespecimens and its adjacent tissues.It has laid a molecular foundation for IM combined radiotherapy for GIST,thus providing a new intervention for the current treatment model of GIST and promoting the multidisciplinary comprehensive treatment approach for GIST.Methods: 1.In vitro study on the biological characteristics of GIST-882 cell lines in control group,IM group,IR group and IM+IR group at the cell level:(1)observe the effect of different treatment groups on cell morphology by microscope;(2)the effects of different treatment groups on cell viability and proliferation were detected by cell counting method and CCK8 method;(3)plate cloning formation experiment to detect the effects of different treatment groups on the cloning formation ability of GIST-882 cells;(4)scratch experiment and Transwell chamber experiment to compare the effects of different treatment groups on cell migration and invasion;(5)flow cytometry annexinv-fitc /PI apoptosis double staining method and flow cytometry PI method were used to detect the effects of different treatment groups on early apoptosis,cell cycle and late apoptosis of GIST-882 cells.2.To explore the possible molecular mechanism of IM combined with IR at the molecular level: QPCR and Western Blot were used to study the expression changes of radiation-related DNA damage repair genes in different treatment groups as well as the signaling pathway related genes acting on IM,so as to investigate the possible molecular mechanism of synergistic inhibitory effect of IM combined with IR on GIST-882 cells.3.At the histological level,immunohistochemical staining was used to detect the difference in the positive expression rate of radiation-related DNA damage repair protein in GIST and its adjacent tissues,and to analyze its correlation with the clinicopathological data of patients.Results: 1.(1)compared with the single action group,ionizing radiation combined with IM can significantly change the morphology of normal GIST-882 cell line,resulting in cytoplasmic atrophy,fusiform degeneration and a large number of dead cells floating;(2)in terms of cell biological behavior,a certain dose of ionizing radiation combined with a certain concentration of IM can significantly inhibit the survival rate,cell proliferation,cell colony formation ability and migration and invasion ability of GIST-882 cells compared with thesingle effect.The difference is statistically significant;In terms of the inhibitory effect on cell proliferation,the factorial analysis and chou-talalay analysis showed that IR combined with IM had a synergistic inhibitory effect;(3)in terms of cell cycle and apoptosis,flow cytometry PI method and annexinv-fitc /PI double staining method were used to detect apoptosis of cells to compare the effects of different intervention factors on cell cycle and apoptosis of GIST-882 cells,It was found that IM mainly led to direct apoptosis,while IR mainly led to cell cycle arrest,and the combined effect of the two was statistically significant compared with the single effect on promoting apoptosis.2.In the discussion of the molecular mechanism,it was found that IM may indirectly inhibit the ir-activated P53 signaling pathway by inhibiting its classical PI3K/Akt and ras-mapk signaling pathways,leading to the inhibition of the downstream DNA damage repair protein expression,difficulty in collecting the damaged DNA nearby,and finally leading to radiosensitization.3.(1)respectively,this study tested the80 patients,50 cases,30 cases of RAD51 protein,gamma H2 AX protein and Ku70/80 protein in the expression of GIST and peritumoral tissue,including RAD51 protein in 75cases(90.00%)was higher in GIST organization expression,35 cases(43.75%)in the tissue adjacent to carcinoma has high expression of RAD51 protein expression in the GIST of the organization’s positive rate was obviously higher than tissue adjacent to carcinoma,and the difference has significant statistical significance(P < 0.01);γ-H2 AX protein was highly expressed in 40 cases(80%)of GIST tissues,and was highly expressed in 13 cases(26%)of GIST tissues.The positive expression rate of-h2 ax protein in GIST tissues was significantly higher than that in GIST tissues,and the difference was statistically significant(P < 0.01);In GIST tissues,ku70-80 protein was highly expressed in 13 cases(43.43%)and3 cases(10%).The positive expression rate of ku70-80 protein in GIST tissues and adjacent tissues was significantly higher than that in GIST tissues,and the difference was statistically significant(P < 0.01).Further analysis showed that the positive expression rate of RAD51 protein and gamma-h2 ax protein in GIST tissues was significantly higher than that of ku70-80 protein(P < 0.01).4.further analysis of the correlation between the expression of the homologous recombinant protein RAD51 in GIST tissues and the clinicopathological information of patients showed that the expression of RAD51 was correlated with theexpression of CD117 protein in GIST tissues(P < 0.05)and the risk of GIST(P < 0.05).Conclusion:1.Radiation-related DNA damage repair proteins are closely related to the risk of GIST.This may be the key molecule that GIST is insensitive to radiotherapy and chemotherapy.2.IM combined with IR can not only synergistically inhibit the biological behaviors of gastrointestinal stromal tumor cells,such as cell viability,proliferation capacity,invasion and migration,and the ability of cell cloning,but also synergistically promote the cell cycle arrest and apoptosis of gastrointestinal stromal tumor cells.3.IM on joint IR GIST-molecular mechanism of the synergistic inhibition of 882 cells,possibly through IM inhibition of PI3K/AKT,Ras-MAPK signaling pathways,leading to the IR cause DNA damage repair protein expression is restrained,finally sensitization IR on the killing effect of gastrointestinal stromal tumor cells,for patients with advanced unresectable gastrointestinal stromal tumor drug resistance after theory provides a new treatment. |
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