Study On The Mechanism Of BDNF In The Carcinogenesis And Development Of Ovarian Cancer

Ovarian cancer,one of the most lethal of malignant gynecological tumors with rising incidence,the lethality of which dues to difficulties in detection at an early stage and lack of effective treatments for patients.Brain derived neurotrophic factor(BDNF)is a member of the neurotrophin family.Tropomyosin receptor kinase B(Trk B)is a member of the tyrosine kinase receptor family and the specific receptor for BDNF.To data,the level of BDNF/Trk B is higher in many types of cancers.Recent researches have found that MAPK/ERK,PI3K/AKT and PLC? were downstream pathways of BDNF/Trk B,which was regulated by miR-16,miR-200 c and miR-204 at the same time.BDNF is considered as a new target for cancer therapies.However,there are few reports about the mechanism of BDNF in the development of ovarian cancer.In order to understand the mechanism of BDNF in the occurrence and development of ovarian cancer.We will study the expression of BDNF/Trk B in the ovarian cancer tissues,which influences the proliferation and invasion of ovarian cancer cells by regulating downstream pathways.And we will detect the kind of miRNA which may control the expression of BDNF by target binding to it.Part 1 BDNF influences the proliferation and invasion of ovarian cancer cellsObjective 1.We studied the expression of BDNF and Trk B protein in ovarian cancer tissues comparing with normal ovarian tissues,and further confirmed whether these two proteins had been activated in ovarian cancer.2.We studied the effect of BDNF on cell proliferation,migration and invasion of ovarian cancer cells.3.We illuminated the downstream pathway of BDNF/Trk B,which played a key function in ovarian cancer cells.Then we examined the biofuctions of ovarian cancer cells to investigate the molecular mechanism of BDNF/Trk B in ovarian cancer cells.Methods 1.Immunohistochemistry studied the expression of BDNF and Trk B protein in ovarian cancer tissues comparing with normal ovarian tissues,and further confirmed whether these two proteins had been activated.The samples were selected by Affiliated Hospital of Qingdao University and Zibo Maternal and Children Health Hospital from January 2010 to January 2012.There were 39 cases of ovarian cancer and 20 of normal ovarian tissues.2.Then we used cell proliferation,migration,invasion and ECIS assay to study the effect of BDNF in SKOV-3 cells treated with recombine human BDNF.3.By using western blot,expression and phosphorylation of Trk B under treatment of BDNF were detected.We silenced Trk B by si RNA,and then examined the biofuctions of SKOV-3 cells by cell proliferation,migration,invasion assay and ECIS assay.4.To illuminate the downstream pathway of BDNF/Trk B,we silenced AKT1 by si RNA,and then examined the biofuctions of SKOV-3 cells.Meanwhile,we used ECIS to do further exam treated with AKT inhibitor within different groups(control,AKTi,BDNF,BDNF+AKTi).5.To illuminate the downstream pathway of BDNF/Trk B,we silenced PLC?1 by si RNA,and then examined the biofuctions of SKOV-3 cells.Meanwhile,we used ECIS to do further exam treated with PLC? inhibitor within different groups(control,PLC?i,BDNF,BDNF+PLC?i).6.To explore the mechanism of the function of Trk B/PLC?1 pathway,we detected the cell apoptosis in SKOV-3 cells transfected with si RNA by TUNEL assay.Results 1.Immunohistochemistry showed BDNF and Trk B localized at the cytoplasm and cell membrane of ovarian cancer cells,Which were positive for BDNF(69.23%)and Trk B(74.36%).However BDNF and Trk B were absent in mormal ovarian tissues.2.The results showed that BDNF could promote the proliferation,migration and invasion of SKOV-3 cells(P=0.000023,P=0.0382,P=0.00047,P=0.00056).ECIS showed that BDNF dramatically enhanced the adherent,attachment,migration of SKOV-3 cells.3.Western blot showed that treatment of BDNF increased the expression and phosphorylation of Trk B significantly in SKOV-3 cells.Then we silenced the expression of Trk B by si RNA and the proliferation,migration and invasion assay were performed.The results showed that without the expression of Trk B,the number of cell proliferation reduced markedly(P=0.0178)as well as migration and invasion(P=0.000726,P=0.000878).4.To study whether this pathway regulated cell proliferation and migration in ovarian cancer,we silenced AKT1 by si RNA or AKT1 inhibitor,proliferation and invasion assay showed no difference with control group.ECIS showed the same results that AKTi had no effect on the risen resistivity caused by BDNF,comparing to control.5.We silenced PLC?1 by si RNA or PLC?1 inhibitor,results showed that without PLC?1,the cell number of proliferation and migration reduced(P=0.000634,P=0.000792,P=0.00583),and BDNF couldn't increase cell proliferation,migration and invasion.Western blot of Trk B and phosphor-Trk B showed treatment of BDNF increased the expression and phosphorylation of Trk B significantly in SKOV-3 cells.ECIS showed that PLC?i markedly reduced the risen resistivity caused by BDNF,comparing to control.6.TUNEL assay were performed and the result showed that silencing of Trk B enhanced apoptosis of SKOV-3 cells.Further,silencing of PLC?1 by si RNA was sufficient to enhance apoptosis of SKOV-3 cells(P=0.0000937,P=0.000128).Conclusion 1.Immunohistochemistry showed that the expression of BDNF and Trk B were significantly higher in ovarian cancer tissues comparing with normal ovarian tissues.It suggested that BDNF and Trk B had been activated in ovarian cancer.2.BDNF could enhance the cell proliferation,adherent,attachment,migration and invasion capacity of ovarian cancer cells.3.Silenced the expression of Trk B,BDNF couldn't enhance the proliferation,migration and invasion capacity of ovarian cancer cells.4.Trk B/AKT signaling pathway is not necessary to promote proliferation and migration of ovarian cancer cells.5.Trk B/PLC? signaling pathway is necessary to promote proliferation and migration of ovarian cancer cells.6.Trk B/PLC? signaling pathway inhibits cell apoptosis to promote proliferation and migration of ovarian cancer cells.Part 2 Mi R-101 can affect the migration and invasion of ovarian cancer cells by regulating the expression of BDNFObjective 1.To determine miRNAs which was in accordant trend both in ovarian cancer tissues and serum exosomes of ovarian cancer patients,comparing with healthy control,and to find a new methods of diagnosing early ovarian cancer.2.The mechanism of miRNA was elucidated,and the target genes regulated by miRNA were predicted by biological information method,and how to target genes in ovarian cancer cells was explored,which provided a new target for the treatment of ovarian cancer.Methods 1.We detected the expression of 6 miRNAs,(miR-101,miR-21,miR-210,miR-34 a,miR-30 a and miR-125b)in the ovarian cancer tissues from 36 patients,comparing to 36 healthy control by q RT-PCR.With the same measure,we determined the miRNA which was in accordant trend in serum exosomes in 29 ovarian cancer patients as well as 29 healthy control.2.Predicted by bioinformatics tools and confirmed by dual-luciferase assay,we confirmed that the miRNAcould target gene BDNF and interact.3.The SKOV-3 cells were transfected with the miRNA mimic and inhibitor.We detected the level of BDNF by western blot and q RT-PCR.4.Subsequently,the SKOV-3 cells were transfected with this miRNA mimic and inhibitor.We examined the function of this miRNA on migration and invasion capacity of ovarian cancer cells by transwell assay,as well as the BDNF was also suppressed or overexpressed as parallel control.Results 1.The expression of miR-101 decreased in the tumor tissue samples when compared with normal ovarian tissue(P<0.05).Meanwhile,The expression of miR-101 decreased in the serum exosomes samples from ovarian cancer patients when compared with the healthy people(P<0.01).The level of miR-101 was lower both in the ovarian tissue samples and serum exosomes of ovarian cancer patients comparing with healthy control.2.Dual luciferase assay was employed to identify the direct interaction between miR-101 and the 3'UTR of BDNF,suggested by bioinformatics tools.The relative luciferase activity was significantly reduced in the cells transfected with miR-101 mimic compared with control(P=0.00892).Meanwhile,the luciferase activity was up-regulated by miR-101 inhibitor transfection(P=0.0172).When 4 nucleotides changed,the luciferase activity was not repressed by miR-101 mimic.3.SKOV3 cells were transfected with miR-101 mimic or inhibitor.The expression of BDNF decreased in the miR-101 mimic transfected cells(P=0.00201),and increased in the miR-101 inhibitor treated cells(P=0.0323).4.The number of migrated cells reduced significantly by miR-101 mimic transfection(P=0.00932).Meanwhile,the miR-101 inhibitor could significantly up-regulate the number of migrated cells(P=0.0453).Mi R-101 mimic could significantly repress the invasion of SKOV3 cells(P=0.0178),however,only a slight increase could be found in miR-101 inhibitor treated cells.We suppressed and overexpressed BDNF in SKOV-3 cells,and western blot showed that the inhibition expression and overexpression had significant effect.Transwell assay showed that the migration and invasion could be enhanced by overexpressed BDNF(P=0.0382,P=0.0246),as same as inhibiting miR-101,and reduced by inhibiting BDNF(P=0.0198,P=0.0187).Conclusion: 1.The level of miR-101 was lower both in the ovarian cancer tissues and serum exosomes of ovarian cancer patients comparing with healthy control.2.Mi R-101 could repress the expression of BDNF by targeting 3'UTR.3.Mi R-101 negatively related to significant enhanced migration and invasion capacity of ovarian cancer cells.4.Mi R-101 controlled the migration and invasion capacity of ovarian cancer cells by negatively regulating BDNF.