Studies Of NF1 Function And Mechanism In Inhibition The Malignant Progression In Epithelial Ovarian Carcinoma

Ovarian malignant tumors are the most common gynaecological malignancies.Epithelial ovarian tumors(EOC,epithelial ovarian carcinoma)accounted for 85% to 90% of ovarian malignant tumors.Because the limited understanding of the tumor origin,unclear initiating events and early developmental stages of ovarian cancer,lack of an effective therapy in advanced ovarian cancer cases.The 5-year relative survival rate is only 29%.Few cases(15%)are diagnosed with localized tumor(stage?).Numerous research efforts and a large number of scholars focused on the ovarian tumor.The further research of EOC may lead to understanding of the molecular mechanisms.Management and prevention,even development of novel and individual therapy in future.Neurofibromatosis type1(NF1)is a tumour predisposition syndrome as an autosomal dominant aberrations of NF1.Recently,the new generation sequencing technology have revealed somatic NF1 aberrations in various sporadic tumours.NF1 alterations appear to be associated with the occurrence of tumor and adverse outcomes in several tumour types.In addition to the nervous system tumors,there have found the aberrations or loss of somatic NF1 may related to various sporadic tumours,such as,gastrointestinal stromal tumor,lung cancer,breast cancer and ovarian tumours.Neurofibromin-deficient or mutations can lead to increased cell proliferation and migration.The NF1 gene product neurofibromin is a negative regulator of the Ras cellular proliferation pathway,and also exerts tumour suppression via other mechanisms.Recently,next generation sequencing(NGS)was revealed the NF1 genomic alterations(GA)which is one of relapsed reason in ovarian epithelial carcinoma.This suggests that NF1 gene and its protein product,as tumour suppressor,may play critical role in the ovarian cancer.Thus,to identify the specific molecular between NF1 and EOC is a new field.A better understanding of its physiologic and pathophysiologic mechanisms in ovarian carcinoma may open new opportunities for effective therapeutic intervention and better outcomes.This test was detected the expression of NF1 in the benign,borderline ovarian tumors and malignant ovarian tumor.According to the clinical information,the correlation of between the expression NF1 and prognosis of EOC patients had been retrospectively analyzed.Plasmids system which high express NF1 gene and lentivirus vector NF1-Ri had been constructed.We had tested biological characteristics after the RNA interference and overexpression,which could help us understand the pathogenesis of ovarian cancer.Finally,we had establishded gene chip mode,which could assess downstream gene expression differential display after overexpression the NF1.To analysis the mechanisms of the proliferation and invasion of ovarian carcinoma.OBJECTIVES(1)In tissue level,to detect the expression of NF1 in the benign,borderline ovarian tumors and malignant ovarian tumor,and to analyze the correlation of NF1 expression with the occurrence and development of ovarian cancer,to survey the clinical pathology characteristics and prognosis of EOC patients.(2)At a microcellular level,to examine the protein level of NF1 in different ovarian cancer cell lines,and to construct NF1-overexpress plasmids system and NF1-Ri lentiviral vector,to discuss the mechanism of NF1 on EOC.(3)In vivo and in vitro,we aimed to test biological characteristics after the RNA interference and overexpression,and to discover the pathogenesis of ovarian cancer(4)Establishment of gene chip mode,to assess downstream gene expression after change the NF1 gene express and to analysis the mechanisms of ovarian carcinoma.METHODS(1)Using real-time polymerase chain reaction and Western Blot examine the expression of NF1 in 48 ovarian fresh excision biopsy(16 benign and 32 EOC).(2)Using immunohistochemistry examine the expression of NF1 in 124 ovarian tumor tissues(17 benign,14 borderline ovarian tumors and 93 malignant ovarian tumor).That data were analyzed by Chi-square test.(3)Application of Kruskal–Wallis test,Mann–Whitney test,multivariate Cox regression analysis showed the correlation between NF1 expression and survival rate.(4)Using RT-PCR and Western Blot examine the expressions of NF1 in ovarian carcinoma cell lines A2780,HO8910,HO8910 PM,SKOV3.(5)It was studied by immunofluorescence to sub-cellular located the expression of NF1 in ovarian cancer cell lines SKOV3 and HO8910.(6)We had constructed NF1-Ri plasmids system of lentiviral vector and a negative control.The overexpression plasmids system and its negative control.The plasmid had be verified by restriction enzyme digestion and nucleotide sequencing.We had transfected by lentivirus that had been packaged and purified in ovarian cancer cell lines.Using real-time polymerase chain reaction and Western Blot examine,the best transfection of NF1 in the ovarian carcinoma cell lines were screened out(7)Ed U and Brd U test were used to evaluate the proliferation of SKON3 and HO8910 before and NF1 interference.(8)Scratch test and Transwell were used to evaluate the cell invasion of SKON3 and HO8910 before and NF1 interference.(9)The tumor growth in vivo was evaluated by subcutaneous transplantation of the tumor after silencing NF1 in HO8910 cells.(10)Establishment of gene chip mode,to assess gene expression in SKOV3 before and NF1 overexpression.(11)Using RT-PCR and Western Blot examine to verify the chip result.(12)Verified and analyzed the microarray data by clustering algorithm,GO,pathway.RESULTS(1)The levels of m RNA and protein were measured by RT-PCR and Western Blot.16 cases were with benign tumors and 32 cases with EOC.In the 16 benign tumors and 32 EOC,the relative expression of NF1 m RNA were 85.50±20.69 and 15.77±3.983(P <0.01).The relative expression of NF1 protein were 0.9279±0.36 and 0.3813±0.32(P <0.01),respectively.(2)In 124 cases,the totle positive rate of expression of NF1 was 70.97%(88/124).The expression of NF1 was 100% in benign(17/17),borderline ovarian tumor was 85.71%(12/14)and EOC was 63.44%(59/93).The difference on the three groups had statistical significance(P<0.01).(3)The expression of NF1 in ovarian cancers was correlated with lymphatic metastasis and tumor size(P<0.05),not with age,FIGO stage,ascites and pathology subtypes etc(P>0.05).By log-rank test,Patients with high NF1 expression had a longer overall survival rate and than the patients with low NF1 expression(P=0.043).Multivariable COX regression analysis revealed that FIGO stage,lymphatic metastasis,the expression of NF1(HR=0.483,P=-0.723,95%CI 0.261-0.901)were independent prognostic factors of EOC(P<0.05).(4)Using Western Blot examine the relative expressions of NF1 in ovarian carcinoma cell lines A2780,HO8910,HO8910 PM,SKOV3 were 1.000± 0.777,3.371±0.344,1.897±0.316,0.883±0.328.The expression level in HO8910 and SKOV3 cells is most significant(P<0.01).(5)The localization of NF1 in SKOV3 and HO8910 cells with immunofluorescence.in cytoplasm and nuclear membrane(6)We had transfected by lentivirus that had been packaged and purified in ovarian cancer cell lines.Stably transfected ovarian cell lines were finished.MOI of HO8910 was 20.By RT-PCR and Western Blot examine,the m RNA and protein expression level of NF1 were be best inhibited in the NF1-Ri-2 were screened out.By transfection plasmid which carries NF1 gene,Constructs the ovarian cancer cell line SKOV3 which overexpression express NF1 gene.Observe the cell form after transfection 24 h.To screened out the condition of gene transfection in SKOV3(7)Ed U and Brd U test were used to evaluate the proliferation of SKON3 and HO8910 before and NF1 interference.(8)Scratche test and transwell test were used to evaluate the cell invasion and migration.ovarian cancer cell HO8910-Ri compared with the control group significantly enhance its capability of invasion,migration.Ovarian cancer cell PCMV-NF1-SKOV3 which overexpression NF1 compared with the control group reduce the migration ability.The differences between the two groups have statistical significance(P < 0.01,P < 0.01).(9)The average weight of tumors was significantly higher in LV-NF1-RNAi infected group than that in NF-NC infected group(P<0.01).The average volume of tumors in LV-NF1-RNAi infected group was significantly heavier than that of the NF1-NC infected group(P <0.05).(10)Establishment of gene chip mode to assess gene expression in SKOV3 before and NF1 overexpression.Microarray-based gene expression profiles.55 genes were up-regulated(ratio,2.0),and 58 genes were down-regulated(ratio,0.5)between HO8910 and HO8910-NF1-Ri;155 genes were up-regulated(ratio,1.5)and 250 genes were down-regulated(ratio,0.6)between SKOV3 and PCMV-NF1-SKOV3.(11)The result of chip is verified by PCR and Western blot method.(12)The microarray data were analyzed by clustering algorithm,GO,pathway.YWHAZ,CCND were relation to PI3 kinase pathway,Wnt pathway,STAT1,YWHAZ were relation to EGF Receptor signaling pathways,RAS,PI3 K and ERK signaling pathways.CCNG1,CCND2 were relation to p53 signaling pathways.IFI27,IRF7,ADA were relation to immunodeficiency,purine metabolism.At the same time,gene-gene interactions.Regulation of the Ras pathway via the NF1 gene.CONCLUSIONS:(1)The immunohistochemical staining revealed the NF1 localization in cytoplasm and nuclear membrane.The low expression of NF1 is associated with poor prognosis in EOC.(2)The deletion of NF1 gene may play a role in the development of ovarian cancer.In this Application of multivariate Cox regression analysis showed NF1 expression to be an independent prognostic factor for overall survival.NF1 may play a protect role in EOC.(3)Our present show that it had expression of the NF1 in the four ovarian cancer cell lines.Three specific NF1-Ri plasmids system of lentiviral vector were successfully constructed.NF1-Ri.The efficient had been verified in cell lines.(4)When silencing the expression of NF1,the ability of proliferation,migration,invasion were improved in vitro ovarian cancer cells and in vivo.On the contrary,when overexpression the NF1 in ovarian cell,the ability of proliferation,migration,invasion were decline.(5)The microarray data demonstrated the effect of NF1 in EOC.NF1 may via NF1-ras pathway influence the down-stream gene to change the biological of proliferation,invasion and metastasis of ovarian epithelial cancer ovarian cancer cells.