The Study Of Biological Effects Of Gelsolin On NK/T-cell Lymphoma And Molecular Mechanism

NK/T cell lymphoma(NKTCL)is a kind of Non-Hodgkin's lymphoma(NHL),and the most common subtype of T cell lymphoma.The characteristics of NKTCL are high invasion and poor prognosis.Epidemically,the incidence of NKTCL is significantly higher in Latin America and East Asia than that in European and American states.However,the specific cause of the disease still remains unclear.Therefore,further study of the etiology and molecular mechanisms of malignant tumor helps to evaluate the early diagnosis,treatment and prognosis of NKTCL.Moreover,further study of the molecular mechanism of tumorigenesis and development will provide important theoretical basis for exploring new therapeutic targets.Gelsolin is an 82-kD protein with six homologous subdomains,referred to as S1-S6.And it is an actin binding protein and one of the important regulatory factors playing a vital role in fiber assembly and decomposition.Widely existed in the cytoplasm and mitochondria of cells and extracellular(plasma),GSN has been reported to be a multifunctional regulator of physiological and pathological cellular process and regulate cell migration,cell morphology,proliferation and apoptosis.Playing roles as both effecter and inhibitor of apoptosis,which results in its association in a wide variety of cancer types,GSN has become a hot topic in oncology research,but less research in NK/T cell lymphoma.The molecular mechanism of the biological effect of GSN and its involving in pathways in NK/T cell lymphoma remain to be a further research.So far,there is no clinical study of the relationship between GSN and the tumorigenesis and development of NK/T cell lymphoma.In this study,the expression of GSN in tumor tissue of patients with type NK/T cell lymphoma and chronic rhinitis was detected by immunohistochemical method,and we analysed the relationship among the level of GSN,clinical pathological parameters and the curative effect.In vitro,we constructed recombinant lentivirus carried GSN cDNA,following with infection in NKTCL cell line YTS to establish stable YTS-GSN cell line.The cellular and molecular biology method were used to research the effects of endogenous overexpression of GSN on proliferation,survival,cell cycle,apoptosis and cell invasive ability of NK/T cell lymphoma cell line YTS.Finally,in vivo,we further studied the effects of the overexpression of GSN on the growth of NK/T cell lymphoma and molecular mechanism through the animal experiment.This study was to explore the impact of GSN on the biological function of NK/T cell lymphoma and its potential molecular mechanisms,which provides potential molecular markers for clinical diagnosis and treatment with NK/T cell lymphoma and provides experimental and theoretical reference to the research on target genes.This topic includes the following three parts:1.The study of expression of GSN in NK/T cell lymphoma tissue and its significance;2.The study of molecular mechanism of the biological effects of GSN overexpression on YTS cells;3.The study of the effect of GSN overexpression on the growth of NK/T cell lymphoma in vivo.Main Content:The first chapter:The study of expression of GSN in NK/T celllymphoma tissue and its significance Methods1.HE staining(hematoxylin-eosin staining):HE staining was used to observe and analyze the cell morphology and histological features of the NK/T cell lymphoma tumor.2.Immunohistochemistry was used to detect the levels of GSN and Ki-67 in NK/T cell lymphoma tissue.3.We analyzed the relationship between the expression of GSN in tumor tissues of patients with NK/T-cell lymphoma and clinical factors.Results1.HE staining revealed that the tumor of patients with NK/T-cell lymphoma developed with the diffuse and infiltrative characteristics,tissue necrosis,vascular invasion,and surface of evident mucosal ulcerate.The NK/T-cell lymphoma cells form a single,medium-sized and the cytoplasm stained from lightly to brightly.The destruction of the vessel wall was observed,pathologically.2.Immunohistological study exhibited that the level of GSN in NK/T cell lymphoma tumor tissues was significantly decreased compared with the control group.3.The results exhibited that the level change of GSN in tumor tissues of patients with NK/T-cell lymphoma has no obvious relationship with patients' age,gender,location,clinical stage and the levels of lactate dehydrogenase(LDH).However,we found that the level of GSN in tumor tissues of patients with Ki-67>80%was significantly lower than that in tumor tissues of patients with Ki-67<80%and the positive rate of the GSN in the effective treatment(CR+PR)group was significantly increased,compared with the ineffective treatment group(SD+PD).The second chapter:The study of molecular mechanism of thebiological effects of GSN overexpression on YTS cellsMethods1.The construction of GSN overexpression lentiviral vector:The pCDH-CMV-MCS-EF1-copGFP(DCE)and GSN plasmids were both digested with EcoRl and NotI,and then they were ligated and we got GSN overexpression lentiviral vector(DCE-GSN).Then Lent-GSN plasmid was transformed.Colony PCR and gene sequence in identifying the recombinant were performed.2.Recombinant lentivirus packaged:The lentiviral vectors were packaged adopting the three-plasmid packaging system package recombinant lentiviruses Lenti-con/Lenti-GSN,pCMV-A8.2 and pCMV-VSV-G plasmids were transfected into 293T cells.The GFP expression was observed by fluorescent microscopy after 48h,and the packaged recombinant lentiviruses were harvested from the supernatant of cell cultures at 72h post transfection by centrifugating.3.Cell transduction in vitro and detection of GFP expression:After,the transduction ratio was determined under a fluorescence microscope and was measured by flow cytometry 48 and 72 h post transduction,respectively.4.GSN expression in cells was identified by qRT-PCR analysis.5.CCK-8 assay and colony formation assay were used to detect the effects of GSN on cell proliferation and clonogenic survival of YTS cells.6.Flow cytomerty was used to detect the effects of GSN on cell cycle and apoptosis of YTS cells.7.Transwell assay was performed to measure the effects of GSN on the invasion of YTS cells.8.Western blot was used to detect the levels of GSN,PI3K,AKT,p-AKT,c-Myc,p53 and Caspase-3.Results1.The results of colony PCR,sequencing and comparison showed that DCE-GSN plasmid was constructed successfully.2.After the DCE-GSN vector was transfected into YTS cells,green fluorescence was obvious in the infected YTS cells,as observed under a fluorescence microscope,and the result indicated a successful transfection.3.Flow cytometry analysis showed that the transfection ratio in YTS cells was 70-80%,which can satisfy the requirement of the subsequent experiments.4.qRT-PCR analysis' demonstrated that the mRNA level of GSN was significantly increased in the YTS-GSN cells,when compared with the YTS-Con cells.5.CCK-8 assay and colony formation assay revealed that cell proliferation and colony formation of YTS-GSN cells were significantly suppressed and GSN overexpression resulted in a decrease in the cell proliferation and clonogenic survival of YTS-GSN cells,compared with YTS-Con cells.6.Flow cytomerty analysis showed that the GSN overexpression significantly promoted apoptosis in YTS-GSN cells,whereas had no obvious effect on the cell cycle.7.Transwell assay showed.that invasion of YTS-GSN cells was significantly inhibited,compared with YTS-Con cells.8.Western Blot analysis revealed that compared with control group,the levels of PI3K,p-AKT and c-Myc in YTS-GSN cells were significantly decreased,whereas the levels of p53 and Caspase-3 were obviously increased.The third chapter:The study of the effect of GSN overexpression onthe growth of NK/T cell lymphoma in vivoMethods1.The mice models bearing solid tumors were established by injecting YTS-GSN cells subcutaneously in the right side of the armpit of mice.2.When the tumor volume reached to 100 mm3 as measured using calipers,tumor volumes were assessed every 3 days.At day 32,the tumor xenografts werecollected and weighed.3.Immunohistochemistry was used to detect the GSN expression in NK/T cell lymphoma tissues of mice.4.Western blot was used to detect the levels of PI3K,AKT,p-AKT,c-Myc,p53 and Caspase-3 in NK/T cell lymphoma tissues of mice.Results1.GSN overexpression significantly inhibited growth of tumor.2.Immunohistological study showed that the GSN expression in NK/T cell lymphoma tumor tissues of mice with injecting YTS-GSN cells was significantlyincreased,compared with the control group.3.Western Blot analysis revealed that compared with control group,the levels of PI3K,p-AKT and c-Myc in YTS-GSN tumor tissue were significantly decreased,whereas the levels of p53 and Caspase-3 were obviously increased.Conclusion1.The change in level of GSN has a close relationship with Ki-67 expression and treatment efficacy in patients with NK/T cell lymphoma.These results suggested GSN may play an important role in tumorigenesis and development.2.The GSN upregulation significantly inhibited cell proliferation,colony formation and invasion,promoted cell apoptosis.Moreover,GSN upregulation may inhibit through the inhibition of the PI3K-AKT signaling pathway.3.Animal experiments further suggested that GSN overexpression may suppresse the growth of NK/T cell lymphoma through the inhibition of the PI3K-AKT signaling pathway.