Trefoil Factor 3 Contributes To The Malignancy Of Glioma

ObjectiveTrefoil factor 3(TFF3)is a member of the TFF-domain peptide family and essential in regulating cell migration and maintaining mucosal integrity in gastrointestinal tract.It contains 6 cysteine residues which are banded by disulphide bonds and form a trefoil domain.Recently,TFF3 was reported playing an important role in epithelia-remodeling in various kinds of tissues.Furthermore,analysis for serum level of TFF3 in patients was considered to be a novel biomarker to predict the process of the GI cancer.However,the roles of TFF3 and its downstream regulating mechanisms of cancer progression remain unclear.In this study,we started from detecting TFF3 expression in glioma cell lines as well as protein form glioma patients.We confirmed that TFF3 is overexpressed in human glioma samples and cell lines.We determined function of TFF3 by knocking down its expression in glioma cell lines U87 and U251.We found that TFF3 promotes the proliferation of glioma cells and facilitates their migration and invasion.Further more,TFF3 inhibits glioma cells from apoptosis.Later,by co-staining TFF3 and HIF-1αin glioma patients’samples,we found that the expression of TFF3 and HIF-1αare positive correlated.The interaction between TFF3 and HIF-1αwas detected by confocal microscope and co-ip assay although the details of mechanism needs further exploration.Last,we involve xenograft mouse model to study the function of TFF3 in vivo.The results showed that the mice who were bearing TFF3 knockdown glioma cells had longer survival time,compared with control group.The goal of this study is to discover the expression and function of TFF3 in glioma,thus offering a novel therapeutic strategy of clinical practice.Methods1.Construction of TFF3 and HIF-1αknockdown plasmid,transfected glioma cell lines U87 and U251,puromycin was used to screening stable knockdown clones.2.Western blot assay was preformed to detect the expression of TFF3 in glioma cell lines and glioma samples.3.Transwell migration assay was preformed to observe the ability of glioma cells migration after knocked down TFF3.4.Transwell invasion assay with matrix gel coated membrane was to see the ability of glioma cells breaking through the gel and invasiveness.5.Immunohistochemistry assay was preformed to estimate the expression of TFF3 and HIF-1αin glioma samples.6.C~2 analysis was to find correlation between TFF3 and HIF-1αin glioma samples.7.Immunofluorescence assay of TFF3 and HIF-1αwas made to find their co-localization.8.Dual-luciferase assay was preformed to detect the change of HIF-1αtranscriptional activity after TFF3 knocked down.9.Co-ip was to find the interaction of TFF3 and HIF-1α.10.Rt-qPCR was to detect mRNA level of TFF3 in glioma cell lines and glioma samples.11.BrdU incooperation assay was to test the rate of glioma cells proliferation.12.Flow cell analysis was preformed to detect the apoptosis of glioma cells.13.Xenograft mouse model was preformed to confirm the function of TFF3 in vivo.14.Kaplan-Meier curve was to find the survival time of glioma-bearing mice and glioma patients.Results1.Expression of TFF3 was elevated in glioma cell lines and glioma patient samples.2.mRNA level of TFF3 was elevated in glioma cell lines and glioma patient samples.3.High expression of TFF3 in glioma patients indicates poor clinical outcome.4.TFF3 promotes glioma cell lines proliferation,migration and invasion,and prohibits its apoptosis.5.Expression of HIF-1αwas elevated in glioma cell lines and glioma patient samples.6.Expression of TFF3 was positive correlated with HIF-1α.7.TFF3 was co-localized with HIF-1αin glioma cell lines and glioma patient samples.8.There is interaction between TFF3 protein and HIF-1αprotein.9.TFF3 elevated expression of HIF-1αand promoted its transcriptional activity.10.Knocking down TFF3 prolonged survival time of glioma-bearing mice.ConclusionTFF3 promotes glioma cells proliferation,migration and invasion,and inhibits apoptosis.TFF3 positively regulates HIF-1αexpression and its transcriptional activity under normoxic condition.In vivo study shows that knockdown TFF3 prolongs the survival time of glioma-bearing mice.In conclusion,TFF3 is a promising target of glioma therapy.