High Expression Of Fibronectin 1 Suppresses Apoptosis Through The NF-κB Pathway And Is Associated With Migration In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC),a type of head and neck cancer,has a low prevalence throughout the world but a high incidence in southern China,especially in Guangdong,Hong Kong and Taiwan[1].In southern China,NPC has a high incidence rate,ranging from 20 to 30 cases per 100,000 people and constituting approximately 18%of all cancers in this area[9].Although NPC can be cured by radiotherapy at early stages,many patients experience local recurrence and distant metastasis,key contributors to NPC mortality.Fibronectin 1(FN1)is a member of the ligand glycoprotein family,which is widely expressed in various cell types and is involved in cell adhesion and migration[2].FN1 overexpression is an indicator of poor prognosis in head and neck cancer.It has been reported that FN1 is a potential biomarker for radiotherapy resistance in head and neck squamous cell carcinoma[3,4],and FN1 is differentially expressed in ovarian cancer with platinum resistance[4,5]molecular mechanism of cancer progression has been determined to involve FN-induced changes in gene expression.For example,FN1 can enhance the expression of matrix metalloproteinases(MMPs),which are key factors in promoting cancer invasion and metastasis[6].In colorectal cancer,FN1 can downregulate P53 and inhibit apoptosis[10].FN1 is also regarded as a prognosticator in NPC.FN1 overexpression in NPC is reportedly associated with advanced disease stage and short survival[11].However,the exact mechanism by which FN1 evokes these poor outcomes has not been investigated.In our study,we found that silencing FN1 in NPC cell lines simultaneously increased apoptosis by downregulating BCL2 via the NF-κB/P65 pathway and inhibited migration and invasion by downregulating MMP9 and MMP2;therefore,FN1 is a potential treatment target in NPC.Materials and methodsCell cultureThe human NPC cell lines 5-8F(EBV-)and C666-1(EBV+),provided by Southern Medical University,were cultured in RPMI 1640(Life Technologies,CA,USA)supplemented with 10%fetal bovine serum(FBS;Life Technologies)and maintained at 37 in an incubator containing 5%CO2.shRNA transfection and qPCRCells were transfected with FN1-specific shRNA(Ribobio,Guangzhou,China).A nonsense shRNA was provided as a negative control(NC).After 36 h of transfection,the efficacy of the FN1 shRNA was ascertained by real-time qPCR and Western blot.Total RNA was extracted using TRIzol(Life Technologies),and cDNA was generated using the PrimeScriptTM RT Reagent Kit with gDNA Eraser(Takara Bio Inc,Japan).Real-time qPCR was performed using SYBR Premix Ex Taq(Takara Bio Inc.,Japan).The qPCR primer sequences are as follows:GAPDH forward5’-GGCTCATGACCACAGTCCATG-3,and reverse5’-TCAGCTCTGGGATGACCTTG-3’;FN1 forward5’-GTTCGGGAGGAGGTTGTTACC-3’ and reverse5’-GAGTCATCTGTAGGCTGGTTTAGG-3’.qPCR assays were performed using a LightCycler 480(Roche,Basel,Switzerland).Scratch,invasion and migration assays In the scratch assays,cells were seeded in a 6-well plate at a density of 3.5 x 105/ml.After reaching confluence,the cells were starved for 12 h in FBS-free medium.Then,a wound was produced by scraping a 200-μl micropipette tip across the confluent cell layer.The migration area was observed at different times,and photos were taken using a microscope with a 20 × objective lens(Olympus,BX53;Melville,NY,USA).The wound area was evaluated with ImageJ software.Cell invasion was evaluated with transwell cell culture chambers(24-well plate,8-μm pore size)that were coated with matrigel(BD,Milan,Italy).Cells(5 × 104 in 100 μl)were transfected or not with FN1 shRNA and placed in the upper chamber without FBS,and 500 μl of culture medium with FBS was added to the lower chamber.After a 12-h incubation at 37℃,the cells in the upper chambers were washed with PBS,fixed in ice-cold methanol for 30 min and stained with crystal violet(Sigma,USA).The cells in five fields on the lower side of the membrane were counted with an upright metallurgical microscope with a 20 × objective lens(Olympus,BX53;Melville,NY,USA).Migration assays were performed similarly to the invasion assays,except the transwell chamber did not contain matrigel.Analysis of cell viability and colony formationAdhesion assay was performed as follows:after FN1 silencing,2×104 cells were plated into a 96 well plate coated with matrigal(BD,milan,Italy),incubated for 2h;then washed with PBS to remove the unattached cells.Counting the adhesion cells by CCK8 assay.After incubated for 3h with CCK8,cell attachment was counted at OD 450nm with a microplate reader(x Mark,Biorad,Milan,Italy)Cell viability was evaluated with the Cell Counting Kit-8(CCK8).C666-1 and 5-8F cells transfected with FN1 shRNA and or NC were seeded in 96-well plates at a density of 2 × 103 cells/ml.Cell viability at days 1,2,3,4,and 5 was evaluated with the CCK8 and a microplate reader at 450 nm.In the colony formation assays,cells(1000 in 2 ml)were transfected or not with FN1 shRNA,placed in 6-well plates with 20%FBS,and incubated(5%CO2,37℃)for 14 days.Every 5 days,the old medium was replaced with new medium.On the 14th day,the medium was removed,and the cells were washed 3 times with PBS,fixed in ice-cold methanol for 30 min and stained with crystal violet(Sigma,USA).The number of colonies was calculated.Apoptosis assayTUNEL assays were performed using an in situ cell death detection kit from Roche according to the manufacturer’s instructions,and the cells were fixed on microslides with DAPI-Fluoromount-G(Southern Biotech,USA).Fluorescence images of five random fields of view were obtained using an upright metallurgical microscope with a 20× objective lens(Olympus,BX53;Melville,NY,USA).Annexin V/propidium iodide(PI)(Annexin V-APC Apoptosis Detection Kit,KeyGEN BioTECH,Jiangsu,China)staining was performed as follows:the treated cells were collected and washed as instructed by the manufacturer,followed by staining with Annexin V/PI for 20 min at room temperature.A flow cytometer was used to analyze the percentage of apoptotic cells.Western blot assayProteins were extracted using lysis buffer containing PMSF and a phosphatase inhibitor(both from Beyotime,Shanghai,China).The proteins were separated by SDS-PAGE,transferred to PVDF membranes(Merck Millipore,MA,USA)and incubated in 5%BSA for 1 h.Membranes were incubated overnight at 4℃ with primary antibody(Cell Signal Technology,USA),washed 3 times with TBST and incubated with goat anti-rabbit secondary antibody(Proteintech,Wuhan,China)for 1 h.Proteins were visualized by electrochemiluminescence using Western blotting detection reagents(Thermo Scientific,UK).Subcutaneous transplantation tumor model studyTwo groups of 3 male nude mice(4 to 6 weeks old weighing 20 to 25 g)were bred under SPF conditions.Animal experiments were performed according to protocols approved by the National Institutes of Heath Guide for the Care and Use of Laboratory animals(NIH Publication No.8023,revised 1978).Cells(2 × 106 in 100μl)were subcutaneously injected into the mice.The tumor was measured every 7 days with calipers,and the tumors were harvested after 6 weeks.Statistical analysisAll the measurements were analyzed using Student’s t-test.The data are presented as the mean±SD calculated using GraphPad Prism 5.0(GraphPad Software,Inc.).The statistical significance was set at P<0.05.The statistical results are shown as*P<0.05 and**P<0.01.All the experiments were performed at least 3 times.ResultsFN1 was upregulated in NPCWe downloaded data from GEO using the accession number GSE12452;this dataset was generated using an Affymetrix Human Genome U133 Plus 2.0 Array(GPL570).To make the data easier to analyze and to obtain high quality genes,we used the median method for data standardization.The gene filters were set as follows:(1)the expression of a probe was no less than 3-fold the median,and changes were more than 20%of the that for the total sample;(2)there were fewer than 50%missing gene expression values;and(3)if a gene corresponded to several probes,only the highest IQR was retained.Finally,we found that FN1 was obviously upregulated,with a fold change of 9.12.Knocking down FN1 inhibited migration and invasion by regulating MMP9 and MMP2To confirm that downregulating FN1 inhibits the proliferation,migration and invasion of NPC cells,we silenced FN1 with shRNA.qPCR and Western blot confirmed that FN1 was significantly knocked down.Compared with the NC group,cells in the shFNl group were less aggressive,with reduced migration and invasion.In addition,the wound healed more slowly.MMP9 and MMP2 have been regarded as invasion biomarkers in many cancer cells.Therefore,we evaluated MMP9 and MMP2 expression in shFNl and control cells.MMP9 and MMP2 were downregulated in shFNl cells compared with control cells,as shown by Western blot.MMPs are secreted proteins and key factors in degrading collagen type IV.Altogether,these results suggest that FN1 can increase NPC cell migration and invasion by upregulating MMP9 and MMP2.Knocking down FN1 inhibited adhension,proliferation and induced NPC cell apoptosis via NF-κB P65The proliferative ability of C666-1 and 5-8F cells was evaluated.Cells transfected with shFN1 proliferated more slowly.TUNEL assays showed that FN1 knockdown resulted in large apoptotic cells with high-intensity red fluorescence.Western blot assays suggested that caspase3 was upregulated and that levels of the anti-apoptotic protein BCL2 were significantly decreased.Therefore,we hypothesized that FN1 inhibits NPC cell death.An accumulating body of evidence suggests that the NF-κB pathway plays a crucial role in cancer development.In addition,prominent crosstalk nodes are mediated by other factors,such as STAT3 and P65.To determine whether the NF-κB pathway is associated with the regulation of apoptosis in shFN1 cells,Western blot analysis and immunocytochemistry were employed.Western blots showed that FN1 increased IKKB,P65,and p-P65 expression and decreased IκB-α expression.To determine whether NF-κB/P65 activation is related to FN1,we suppressed NF-κB by treating NC cells for 12 h with caffeic acid phenethyl ester(CAPE,20 μg/ml)and activated NF-κB by treating shFNl cells with lipopolysaccharide(LPS,1000 ng/ml).CAPE and shFNl had the same effect on the NF-κB pathway;both inhibited NF-κB pathway activation.In CAPE-treated cells,BCL2 expression was reduced,but FN1 expression did not change.Immunocytochemistry showed that in NC cells,FN1 increased the nuclear localization of P65 by inducing cytosolic accumulation and increasing nuclear binding.shFNl cells treated with LPS,the NF-κB pathway activator,showed increased P65 and BCL2 expression over time.However,the apoptosis rate did not change considerably compared with that of NC cells;perhaps FN1 inhibits apoptosis by activating P65,and the same effect was achieved by the NF-κB pathway activator in the shFNl group.Tumor growth in the subcutaneous transplantation tumor modelThe in vitro experiments showed the effect of FN1 on tumor apoptosis and progression.Next,in vivo experiments were performed to determine whether these effects could be observed in an animal model.The subcutaneous transplantation tumor model assay showed that tumor volume was smaller in the shFNl group(0.73±0.07 cm3)compared with the control group(1.2κ0.147 cm3).In the shFNl group,the tumor growth rate was significantly reduced(p<0.05).Conclusion1.FN1 is over-expressed in nasopharyngeal carcinoma.2.Down-regulation of FN1 expression can inhibit the migration and invasion of nasopharyngeal carcinoma cells and reduce the expression of MMP2 and MMP9,3.Down-regulated FN1 expression can inhibit the growth rate and volume of subcutaneous tumor in nude mice,and can inhibit NPC cell apoptosis by up-regulating BCL2 and down-regulating Caspase3;4.NF-kB/P65 pathway involved in the regulation of FN1 on malignant biological behavior of nasopharyngeal carcinoma cells,inhibition of FN1 expression on the basis of activation of NF/kB expression can inhibit cell apoptosis,probably NF-kB/P65 Pathway in FN1 plays an important role in the regulation of apoptosis of nasopharyngeal carcinoma cells.