| BackgroundGliomas are the most common malignant tumors in the central system and seriously endanger human health.At present,although the treatment for glioma has certain effect,due to the invasive growth characteristics of glioma,the boundary with the surrounding brain tissue is not clear,and the operation is difficult to completely remove.However,due to treatment limitations such as glioma tolerance,radiotherapy and chemotherapy have limited efficacy,especially for high-grade glioma,with high recurrence rate,poor prognosis and median survival time of less than one year.In recent years,gene therapy at molecular level is a new direction in glioma treatment and research.By inhibiting abnormal activation of signal transduction pathway and further regulating cell cycle,it can effectively promote tumor cell apoptosis and inhibit tumor cell proliferation.Therefore,in-depth study of the occurrence and development mechanism of glioma and finding effective therapeutic targets for glioma are of great significance for prolonging the survival time and improving the quality of life of glioma patients.Notch receptor is a type I transmembrane receptor protein,which is highly conserved in evolution and plays an irreplaceable role in cell growth,differentiation,proliferation and apoptosis.In the structure of Notch receptor,there are altogether four kinds of Notch1-4 receptors,and their biological functions are different or even completely opposite.Evidence shows that abnormal expression of Notch receptor is closely related to the occurrence of malignant tumors.Notch can not only promote cancer but also inhibit cancer in the occurrence of central nervous system tumors,which is due to the different homologous receptors of Notch.Notch 3 gene is identified as the third gap in mammals and was initially described as expressed in proliferating neuroepithelium.Foreign studies have found that Notch3 gene plays an oncogene role in glioma cell proliferation,migration,invasion and apoptosis and has prognostic value.Notch3 gene can be used as a molecular target for high-level glioma gene therapy.However,there are few reports on the role of Notch3 receptor in glioma occurrence and development and related molecular mechanisms in China.ObjectiveIn order to explore the role of Notch3 signaling pathway in the occurrence and development of glioma,this study first detected the expression of Notch3 mRNA and protein,as well as the expression of Ki-67,Bcl-2,Caspase3 protein in glioma tissue,and analyzed the relationship between Notch3 and the occurrence and development of glioma,proliferation and apoptosis.Secondly,the relationship between Notch3 and prognosis-related factor IDH was analyzed by detecting the mutation status of IDH gene in glioma tissue.Finally,Notch3 receptor inhibitor was used to inhibit the expression of Notch3 in glioblastoma cell lines U87 and U251,and the effect of Notch3 on tumor cell proliferation,invasion and apoptosis was analyzed.In order to clarify the role of Notch3 in the occurrence and development of glioma at the molecular level and provide scientific basis for its clinical treatment.Methods(1)The expression of Notch3 mRNA in 40 human glioma samples(diffuse astrocytic or oligodendrocyte tumors)and 10 normal brain tissues was detected by real-time PCR.(2)The expression of Notch3,Ki-67,Bcl-2 and Caspase3 proteins in 80 cases of human glioma tissues(diffuse astrocytic or oligodendrocyte tumors)and 49 cases of relatively normal brain tissues were detected by immunohistochemical method.(3)The IDH gene mutation in 80 glioma tissues(diffuse astrocytic or oligodendrocyte tumors)was detected by gene sequencing,and then analyzed the relationship between Notch3 and the IDH gene mutation.(4)Different doses of Notch3 receptor inhibitor DAPT were added to glioblastoma cell lines U87 and U251,and the inhibitory effect of Notch3 was detected by real-time fluorescence quantitative PCR and western blot.The cells were divided into three groups according to inhibitor dosage and inhibitory effect: control group,Notch3 low expression group 1(Low-1),Notch3 low expression group 2(Low-2).(5)After Notch3 receptor inhibition,CCK-8 cell proliferation experiment was used to detect the changes of proliferation ability in U87 and U251 cell control groups,Low-1 group and Low-2 group,and spectrophotometer was used to detect the absorbance of cells at 450 nm wavelength.(6)After Notch3 receptor inhibition,the invasion ability of U87 and U251 cells in control group,Low-1 group and Low-2 group was detected by Transwell invasion chamber experiment,and the morphological changes of cells were observed by inverted microscope.(7)After Notch3 receptor inhibition,AnnexinV-FITC/PI method was used to detect the changes of apoptosis ability of U87 and U251 cells in control group,Low-1 group and Low-2 group,and the changes of cells were observed under fluorescence microscope.Results(1)In 40 fresh glioma tissues,the expression level of Notch3 mRNA was 2.26 ?1.60,significantly higher than that of normal brain tissues(0.95 ? 0.69,P<0.05).(2)In 80 glioma tissues,the expression of Notch3 protein(55.00%)was significantly higher than that in gliosis tissues(36.70%,P < 0.01).The expression in patients ? 60 years old(75.00%)was significantly higher than that in patients < 60 years old(48.33%,P < 0.05).There was a positive correlation with histological grading of glioma(?vs?vs?: 31.81%,55.00%,68.42%)(P < 0.05),but not correlated with the sex of the patient(P > 0.05).(3)In 80 glioma tissues,the expression of Ki-67 protein(63.75%)was significantly higher than that in gliosis tissues(14.28%,P < 0.01).There was a positive correlation with histological grading of glioma(?vs?vs?: 27.27%,60.00%,86.84%)(P < 0.05),but not correlated with the patient's age and gender(P >0.05).(4)In 80 glioma tissues,the expression of Bcl-2 protein(58.75%)was significantly higher than that in gliosis tissues(46.90%,P <0.01).There was a positive correlation with histological grading of glioma(?vs?vs?: 31.81%,60.00%,73.68%)(P < 0.05),but not correlated with the patient's age and gender(P >0.05).(5)In 80 glioma tissues,the expression of Caspase3 protein(38.75%)was significantly lower than that in gliosis tissues(57.29%,P < 0.01).It was negatively correlated with histological grading of glioma(?vs?vs?:68.18%,30.00%,26.31%)(P < 0.05),but not correlated with the patient's age and gender(P >0.05).(6)In human glioma tissue,the expression of Notch3 protein is positively correlated with Ki-67(r= 0.347,P <0.01);Notch3 protein expression was positively correlated with Bcl-2(r= 0.272,P <0.01);Notch3 protein expression was negatively correlated with Caspase3(r=-0.315,P <0.01).(7)The mutation rate of IDH gene was 36.25% in 80 glioma tissues.The mutation rate in patients < 60 years old(45.00%)was significantly higher than that in patients ? 60 years old(10.00%,P < 0.05).It was negatively correlated with histological grading of glioma(?vs?vs?: 63.63%,45.00%,15.78%)(P < 0.05),but not correlated with the sex of the patient(P > 0.05).In glioma tissues,IDH gene mutation is negatively correlated with Notch3 protein expression(P <0.01).(8)When Notch3 receptor inhibitor DAPT was added to U87 and U251 cells,compared with the control group,Notch3 mRNA(U87: 1.00 ? 0.00 VS 0.45 ? 0.07 VS 0.22 ? 0.05;U251:1.00 ? 0.00 VS 0.52 ? 0.15 VS 0.15 ? 0.08)and Notch3 protein(U87:0.83 ? 0.06 VS 0.61 ? 0.09 VS 0.36 ? 0.07;U251:0.85 ? 0.08 VS 0.57 ? 0.03 VS 0.31 ? 0.04)were significantly down-regulated(P <0.05).(9)After Notch 3 receptor inhibitor DAPT was added to U87 and U251 cells,compared with the control group,the cell proliferation ability of Low-1 and Low-2 groups(U87:1.70 ? 0.03 VS 1.37 ? 0.07 VS 0.99 ? 0.11;U251:1.51 ? 0.05 VS 1.30 ? 0.05 VS 0.89 ? 0.11)was significantly weakened(P < 0.05).(10)After Notch3 receptor inhibitor DAPT was added to U87 and U251 cells,compared with the control group,the cell invasion ability of Low-1 and Low-2 groups(U87: 75.00?11.53 VS 33.50?6.28 VS 14.67 ?2.08;U251:61.50?10.69 VS 32.33 ? 6.51 VS 12.00 ? 3.61)was significantly decreased(P <0.05).(11)After the Notch3 receptor inhibitor DAPT was added to U87 and U251 cells,compared with the control group,the apoptosis rates of Low-1 and Low-2 groups(U87: 8.80 ? 3.03 VS 22.40 ? 3.05 VS 47.00 ? 3.64;U251:6.60 ? 3.36 VS 20.20? 4.21 VS 42.40 ? 5.86)was significantly higher(P <0.05).Conclusions(1)Notch3 gene is overexpressed in glioma tissues but not/low expressed in normal tissues,suggesting that Notch3 activation expression is related to the occurrence and development of glioma.(2)In glioma tissues,the expression of Notch3 protein was positively correlated with Ki-67,Bcl-2 and negatively correlated with Caspase3,IDH,suggesting that the expression of Notch3 may be related to the proliferation,apoptosis and prognosis of glioma.(3)In glioma cells,downregulating the expression of notch3 can inhibit cell proliferation,invasion and promote apoptosis,suggesting that Notch3 is related to the proliferation,invasion and apoptosis of glioma cells. |
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