Mechanism Research Of Apoptosis Signal-regulating Kinase 2 Boosting The Progression Of Human Glioma

BackgroundGlioma,with high case fatality rate and poor prognosis,is a common malignant tumor which seriously threaten the health of human beings.Since there are few breakthroughs of glioma therapy,such as surgery,chemotherapy and radiotherapy,the survival time of glioma patients is still very short.The features of glioma growth are high proliferation,robust migration and invasion,and lower apoptosis.To date,there is no report about apoptosis signal-regulating kinase(ASK)family molecules in glioma.This project mainly researched the gene and protein expression,and prognostic value of ASK2 in glioma patients;and the mechanism of how ASK2 enhanced the proliferation,migration,and invasion of glioma cells in cell lines,in vitro,and in mouse model,in vivo.Part 1.ASK2 expression characteristics and clinical parameters analysis in glioma patientsObjectiveTo explore the gene expression of ASK family genes in glioma and non-tumor brain tissue,as well as in different WHO grades.To research the gene and protein expression features,and prognostic-value of ASK2 expression in glioma patients.Methods(1)Analyze the gene expression of ASK family genes in glioma tissue and non-tumor tissue in GEO4290 dataset,and glioma tissue and non-tumor tissue from brain trauma patients from our own hospital;(2)Analyze the distribution of ASK2 gene expression in different WHO grades in GEO4290,CGGA and TCGA datasets,and the patients from our own hospital;analyze the prognostic value of ASK2 gene expression in CGGA and TCGA datasets,and in the patients from our own hospital;(3)Identify the protein expression of ASK2 in patients,from our own hospital,in different WHO grades through western blot and immunohistochemical staining.The survival difference of patients with different immunohistochemical levels of ASK2 was analyzed.Results(1)ASK2 gene expression was much higher in glioma tissue than in non-tumor tissue in GEO4290 dataset,and patients from our own hospital;and there is no difference of ASK1 or ASK3 gene expression in glioma tissue and non-tumor tissue;(2)ASK2 was highly expressed in glioblastoma(GBM,WHO grade ?)comparing with WHO ?-? gliomas in GEO4290,CGGA and TCGA datasets,as well as in patients from our own hospital;(3)Patients with ASK2 gene high expression hold shorter survival time than those with lower ASK2 expression in CGGA and TCGA datasets,and patients from our own hospital;(4)We also found that patients with higher expression of ASK2 protein level by immunohistochemical staining in our hospital had shorter survival time than those with lower protein level of ASK2.ConclusionASK2 was highly expressed in glioma tissue than non-tumor tissue;the gene and protein expression of ASK2 in GBM patients were much higher than WHO ?-?glioma patients;the high expression of ASK2 indicated worse prognosis.Part 2.The effect of ASK2 on the malignant biological behavior of gliomaObjectiveTo research the function of ASK2 on the proliferation,migration,invasion,and apoptosis of glioma cell lines U87 and U251,in vitro.Methods(1)Identify the effect on proliferation,migration,invasion,and apoptosis of glioma cell lines U87 and U251 after ASK2 gene knockdown by siRNA or shRNA;(2)Identify the effect on proliferation,migration,invasion,and apoptosis of glioma cell lines U87 and U251 after ASK2 gene knockdown by shRNA;(3)Identify the alteration of proliferation,migration,invasion,and apoptosis of glioma cell lines U87 and U251 after transfection with lentivirus which carried the full CDS sequence of human ASK2 gene(ASK2 gene over-expression);(4)After knockdown or overexpression of ASK2,CCK8 assay and clone formation assay were used to search the proliferation of glioma cell lines;the cell cycle distribution of glioma cells was tested by flow cytometry using PI staining method;(5)Wound healing assay and transwell chamber assay were used to test the migration and invasion of glioma cells with ASK2 knockdown or overexpression;(6)Cell apoptosis was tested by flow cytometry using annexin-V and PI double staining method.Results(1)ASK2 gene knockdown in glioma cell line U87 and U251 by siRNA or shRNA obviously decreased the OD value on 450 nm of CCK8 assay,the clone formation number,and the proportion of S phase in cell cycle;(2)ASK2 gene over-expression of glioma cell line U87 and U251 evidently increased the OD value on 450 nn in CCK8 assay,the clone formation number,and the S phase proportion in cell cycle;(3)Knockdown ASK2 gene in glioma cell lines by siRNA or shRNA obviously inhibit the speed of wound healing,and decreased the number of migrated and invaded cells;(4)ASK2 gene over-expression in U87 and U251 cell lines elevated the speed of wound healing,and the number of migrated and invaded cells;(5)The apoptosis of glioma U87 and U251 cell lines had no difference under the circumstances of ASK2 gene knockdown or over-expression.ConclusionASK2 gene knockdown inhibits and ASK2 gene over-expression enhances the proliferation,migration,and invasion of the both glioma cell lines U87 and U251,respectively;the alteration of ASK2 gene expression do not change the apoptosis of U87 and U251 cell lines.Part 3.ASK2 enhances the proliferation,migration,and invasion of glioma cells via regulating ERK-CDK1/MMP2/MMP9 pathwayObjectiveTo investigate the molecular mechanism that how ASK2 enhanced proliferation,migration,and invasion of glioma cell lines U87 and U251.Methods(1)Western blot was used to test protein levels of ERK,p38 and JNK,in both total and phosphorylated protein,which are the potential downstream molecules of ASK2;(2)To test whether ERK inhibitor could decrease the proliferation,migration,and invasion of glioma cell lines U87 and U251 through CCK8 assay,and transwell chamber assay;(3)To seek the CDK family molecules which were significantly positively correlated with ASK2 in CGGA and TCGA datasets;and the changes of these genes' expression were tested by qPCR in U251 cell line with ASK2 gene knockdown or over-expression comparing with the corresponding control cell lines;(4)To seek the MMP family molecules which were significantly positively correlated with ASK2 in CGGA and TCGA datasets;and the changes of these genes'expression were tested by qPCR in U251 cell line with ASK2 gene knockdown or over-expression comparing with the corresponding control cell lines;(5)Western blot was used to test the changes of CDK1 and MMP2/MMP9 in U87 and U251 cell lines with ASK2 gene knockdown or over-expression,comparing with the corresponding control cell lines;(6)To search whether ERK inhibitor could down-regulate the protein levels of CDK1 and MMP2/MMP9 in U87 and U251 cell lines with ASK2 gene over-expression;(7)To test whether CDK1 inhibitor could attenuate the proliferation of U87 and U251 cell lines with ASK2 over-expression;to test whether MMP2/MMP9 inhibiter could attenuate the migration and invasion of U87 and U251 cell lines with ASK2 over-expression.Results(1)ASK2 gene knockdown decreased the protein level of phospho-ERK,and ASK2 over-expression boosted the protein level of phospho-ERK;and there are no changes in total protein level of ERK,p38 and JNK or phosphorylated protein level of p38 and JNK;(2)ERK inhibitor obviously decreased the proliferation,migration,and invasion of ASK2 over-expression glioma cell lines U87 and U251;(3)Correlation analysis showed that CDK1,CDK2,CDK6 and CDK7 were significantly positively correlated with ASK2 in CGGA and TCGA datasets;and CDK1 was the most remarkably decreased or increased gene in ASK2 gene knockdown or over-expression U251 cell lines,respectively;CDK1 inhibitor evidently decreased the proliferation of U87 and U251 cell lines with ASK2 gene over-expression;(4)Correlation analysis indicated that MMP1,MMP2,MMP7,MMP9,MMP10,MMP11,MMP13,and MMP14 were significantly positively correlated with ASK2 in CGGA and TCGA database;and MMP2 and MMP9 were the most remarkably decreased or increased in ASK2 knockdown or ASK2 over-expression U251 cell lines,respectively,respectively;MMP2/MMP9 inhibitor obviously attenuated the migration and invasion of U87 and U251 cell lines with ASK2 gene over-expression;ConclusionERK play an important role in the downstream signaling transduction of ASK2 in glioma cell lines;the activation of ASK2-ERK-CDK1 pathway boosted the proliferation of glioma cells;and the activation of ASK2-ERK-MMP2/MMP9 pathway boosted the migration and invasion of glioma cells.Part 4.ASK2 promotes glioma progression in animal studiesObjectiveTo test how ASK2 gene knockdown or over-expression effect the xenograft glioma of human cell lines U87 and U251,and test the protein levels of phospho-ERK,CDK1,Ki67,and MMP2/MMP9 in xenograft tumor.Methods(1)Subcutaneously implant the U87 and U251 cell lines with ASK2 gene knockdown or over-expression,and the corresponding control cell lines into immunodeficient mice;and measure the tumor size and body weight of each mouse at indicated time points;(2)To record the survival time of mice which were implanted by ASK2 knockdown or over-expression U87 and U251 cell lines,and the corresponding control cell lines;(3)Resect the above-mentioned xenograft glioma and test the prote:in levels of phospho-ERK,CDK1,Ki67,and MMP2/MMP9 by IHC assay.Results(1)ASK2 gene knockdown decreased the tumor size of xenograft glioma of U87 and U251 cell lines,and lengthened the survival time of tumor-bearing mice;(2)ASK2 gene over-expression increased the tumor size of xenograft glioma of U87 and U251 cell lines,and shortened the survival time of tumor-bearing mice;(3)ASK2 gene knockdown in U87 and U251 cell lines attenuated the protein level of phospho-ERK,CDK1,Ki67,and MMP2/MMP9,in vivo;(4)ASK2 gene over-expression in U87 and U251 cell lines boosted the protein level of phospho-ERK,CDK1,Ki67,and MMP2/MMP9,in vivo.ConclusionASK2 gene knockdown inhibited the progression of xenograft glioma and lengthened the survival time of mice.ASK2 gene over-expression boosted the progression of xenograft glioma and decreased the survival time of mice.The activation of ASK2-ERK-CDK1 and ASK2-ERK-MMP2/MMP9 pathways enhanced the progression of xenograft glioma,in vivo.