| The first partObjectivePrevious studies showed that the the Notchl-MMP pathway and the PKCζ-MMP pathway promoted the malignant progression of human tumors,these two pathways are involved in the migration and invasion of tumor cells.However,the interaction between Notchl-MMP pathway and PKCζ-MMP pathway in human glioma has not been reported.The purpose of this study is to investigate the expression of Notchl,PKCζ,MMP2,MMP9 and the correlation among them in glioma,and to reveal the potential role of PKCζ signaling and Notchl signaling in the malignant progression of glioma.Methods1.Paraffin-embedded tissue from 103 patients were collected,patients were followed up.2.Observed the different expression of Notchl,PKCζ,MMP2 and MMP9 in adjacent noncancerous tissues and glioma tissues.3.Patients with glioma(grade I-IV)were investigated for the correlation among Notchl,PKCζ,MMP2 and MMP9 expression by immunohistochemistry.4.The correlation of Notchl,PKCζ,MMP2 and MMP9 with clinicopathological features was analysed by SPSS 17.0.5.Combined with clinicopathological parameters to investigate the role of Notchl,PKCζ,MMP2 and MMP9 in the development of glioma and patients survival.Results1.Notchl and PKCζ immunoexpression were cytoplasmic and nuclear in glioma,MMP2 and MMP9 immunoexpression were cytoplasmic,rate of positivity is(77/103;74.76%),(57/84;67.86%),(72/103;69.90%)and(49/103;47.57%),respectively.2.The expression of Notchl was positively correlated with PKCζ,MMP2 and MMP9,PKCζ was positively correlated with MMP9,but there was no correlation between MMP2 and MMP9 expression.3.Notchl and PKCζ expression levels were both significantly higher in human glioblastoma multiforme tissues than that in low-grade tumors.MMP2 and MMP9 expression levels were both significantly higher in high-grade tumors than that in low-grade tumors.4.Univariate survival analysis showed that Notchl,PKCζ,MMP9,histological grade,pathological type and family history predicted shortened overall survival,PKCζ,MMP2,MMP9,histological grade and pathological type predicted shortened progression-free survival.5.In multivariate survival analysis,Notchl,PKCζ and histological grade were significant independent prognostic factors for overall survival and progression-free survival.Conclusion1.Notchl and PKCζ immunoexpression were cytoplasmic and nuclear in glioma,MMP2 and MMP9 immunoexpression were cytoplasmic.2.This is the first study report the expression of Notchl was positively correlated with PKCζ,MMP2 and MMP9,PKCζ was positively correlated with MMP9,but there was no correlation between MMP2 and MMP9 expression.3.Multivariate survival analysis showed that Notchl,PKCζ and histological grade were significant independent prognostic factors for overall survival and progression-free survival.4.This is the first study report the positive correlation among Notchl,PKCζ and MMP9 expression and their effect on glioma prognosis,we speculate that the crosstalk between PKC(and Notchl may exist,further regulating their downstream protein MMP9 expression and affecting the malignant progression of glioma.The second partObjectiveITNS1(Intersectinl)is a highly conserved adaptor/scaffold protein with a unique multidomain structure.Our previous report indicated that the ITSN1-S expression was positively associated with the WHO grade of human glioma,and negatively associated with a shorter over survival of glioma patients.ITSN1-S mediated glioma cells invasion by regulating the expression and activity of MMP2.The SH3 domain(Src homology 3 domain)implicated in actin cytoskeleton rearrangement and signal transduction by binding to numerous proteins.In this study,we focus on the role of SH3 domains of ITSN1-S in regulating the proliferation and migration of glioma cell line LN229 and to explore the possible molecular mechanisms.Methods1.Construction of the ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group:ITSN1-S and fragmental EH1-EH2-CC domain of ITSN1-S were spliced into the linear PCDH-CMV-MCS-EF1-Puro vector which was excised by incision enzyme digestion,and used PCDH-CMV-MCS-EF1-Puro vector with no treatment as control group.2.Each of the three recombinant plasmids was transfected into the LN229 by lentivirus.3.Western blotting was applied to detect the expression of each protein,and then screened stably cell clones.4.MTT assay was performed to detect the state of cell proliferation which was persistently cultured for 6 d.5.Soft agar assay was performed to detect the colony formation ability of glioma cell which was persistently cultured for 2 weeks.6.Wound-healing assay was performed to detect the state of cell migration at 0,3,6,9,12,24 h.Results1.The recombinant plasmids ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was constructed correctly which was detected by incision enzyme digestion and plasmid DNA sequencing.2.Glioma cell LN229 was respectively infected with total-length ITSN1-S,EH1-EH2-CC fragment and vector lentiviruses to enhance related protein expression,which was identified by western blotting analysis.3.MTT assay showed that ITSN1-S total-length group proliferated rapider than EH1-EH2-CC fragment group and vector control group(P<0.05),but there was no significance difference between EH1-EH2-CC fragment group and vector control group(P>0.05).The proliferation rate at 6 day in ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was(523.60±32.12)%,(409.54 ± 31.33)%and(353.89 ± 13.98)%,respectively.4.Soft agar assay showed that ITSN1-S total-length group proliferated rapider than EH1-EH2-CC fragment group and vector control group(P<0.05),but there was no significance difference between EH1-EH2-CC fragment group and vector control group(P>0.05).The clone formation rate at 14 day in ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was(46.49 ± 2.34)%,(23.00 ± 1.66)%and(17.68 ± 3.47)%,respectively.5.Wound-healing assay found that ITSN1-S total-length group migrated faster than EH1-EH2-CC fragment group and vector control group after 12 h(P<0.05),but there was no significance difference between EH1-EH2-CC fragment group and vector control group(P>0.05).The migration distance at 24 h in ITSN1-S total-length group,EH1-EH2-CC fragment group and vector control group was(0.388 ± 0.023)mm,(0.307 ±0.021)mm and(0.287 ± 0.011)mm,respectively.ConclusionOur results showed that ITSN1-S total-length group proliferated rapider and migrated faster than EH1-EH2-CC fragment group and vector control group,which suggests that the SH3 domain may be the crucial functional domain of ITSN1-S to regulate the proliferation and migration of glioma cell. |
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