| Hepatocarcinoma is a kind of malignant tumor with high morbidity and mortality worldwidely.The 5-year survival rate is very low because of its rapid progress, high recurrence and metastatic rate.If the mechanism of metastasis can be clarified and some early intervention can be put up,it will greatly improve the treatment of hepatocarcinoma.To be able to arrival a second site to form a metastatic carcinoma,tumor cells have to complete a series of selective and orderly steps,including department from the carcinoma in situ,invasion into the vascular system,survival in vascular system,invasion of the matrix into the ectopic organ,spreading out of vascular system and proliferating.They have to overcome pressures before they finally forming metastases,including the immune surveillance capability,the hypoxic environment,a variety of attacks,such as antineoplastic agents.In addition,a variety of effector molecules involved in the process of tumor metastasis,many of them are upregulated at mRNA and protein expression levels under hypoxia. Hypoxia is considered a factor that tumor cells have to overcome.At the same time,there is increasing evidence indicating that hypoxia can promote tumor development.We have successfully constructed the metastatic hepatoma cell model and we further validate its validity and metastatic capacity,as well as discussing the mechnism of enhanced metastatic capacity by forcing the metastatic hepatoma cells to various stimuli(including the exogenous TRAIL as well as the AKT/ERK pathway inhibitors),and further filter the key moleculars which can resist anoikis and hypoxia at the same time. Objectives:1.To construct the metastatic hepatoma cell model and explore its aggregated rate and metastatic capacity.2.To demonstrate molecular mechanism of anoikis-resistance by study response of metastatic hepatoma cells to TRAIL-induced apoptosis to AKT/ERK pathway inhibitors.3.To explore the response of metastatic hepatoma cells to hypoxic stimuli.4.To screen the key molecules for anoikis-resistance and hypoxia-resistance in metastatic hepatoma cells.Methods:1.Metastatic hepatoma cell model and its aggregated rate and metastatic ability1.1.Metastatic model of hepatocellular carcinoma cells BEL7402 and SMMC7721 cell lines were cultured in RPMI1640 with 10%FCS at 37℃5%CO2 conditions to cultivate logarithmic phase,then digested by 0.25% trypsin into single cell suspension,adjusted the concentration,and cultured in Poly-HEMA culture plates for 24 h,that is the metastatic hepatoma cell model which analog metastatic hepatoma cells in vascular system.1.2.Conditions of metastatic hepatoma cell modelCells were cultured to logarithmic phase and prepared into single cell suspension, then seeded into the 96-well plates in different cells numbers as 0.5×104,1×104,1.5×104,3×104,3×104.The Poly-HEMA plates of the first four groups were washed 3 times using sterile PBS before seeding cells,the fifth group was not washed,each tin duplicated and cultured for another 24 h.Cell activity and mortality were detected by using CCK-8 and trypan blue staining.1.3.Aggregated rate of metastatic hepatoma cells Hepatoma cells were seeded into plates without or with poly-HEMA coating as attached or detached experimental groups and cultured for 24h.Single, non-aggregated cells were counted.The percentage of aggregated cells was calculated as(1-Ne/Nc)×100%,where Ne is the number of single cells after incubation for 24 h and Nc is the number of single cells before incubation.1.4.Transwell detection of metastatic hepatoma cells' invasion and migration About 1.5×105 separating detached or attached hepatoma cells were seeded to the top chamber(diluted with 200ul RPMI 1640 in the presence of 1%FBS) of the Polycarbonate Membrane Transwell Inserts pre-coated with diluted MatrigelTM Basement Membrane Matrix.The bottom chamber was added 0.5ml RPMI 1640 with 10%FBS in advance.After 20 h of incubation at 37℃,cells on the lower part were fixed,stained and scored in 10 randomly chosen microscopic fields to decide the invasion of anoikis-resistant cells.For migration studies,cells were seeded to the upper compartment of the uncoated inserts and cultured for 6h.2.Response of metastatic hepatoma cells to TRAIL-induced apoptosis and its mechanisms2.1.Response of metastatic hepatoma cells to TRAIL-induced apoptosis1).TRAIL induced apoptosis detected by CCK-8 and Trypan blue assayAttached and suspended groups were divided into 7 groups respectively,add 0ng/mL,5ng/mL,10ng/mL,20ng/mL,50ng/mL,100ng/mL,200 ng/mL of TRAIL protein.After 24h cell culture,CCK-8 and trypan blue staining were used to detect TRAIL-induced apoptosis.Attached and suspended groups were divided into 7 groups respectively,add 20ng/mL of TRAIL protein and cultured for 0 h,2 h,5 h,8 h,12 h,24 h and 48 h.Then,CCK-8 and trypan blue staining were used to detect TRAIL-induced apoptosis.2).TRAIL induced apoptosis detected by TUNEL and Caspase3 activation assayHepatoma cells were seeded into 24-well plates as attached or detached groups for 20h,and treated with 20ng/ml recombinant sTRAIL for another 24h.Apoptotic cells were labeled with TUNEL by using an In Situ Cell Death Detection Kit.The degree of apoptosis was determined by flow cytometry.Caspase-3 activities of hepatoma cells in 6-well plates treated with sTRAIL for 3 h,6 h,9 h,12 h and 24 h were detected by Caspase-3/CPP32 Colorimetric Assay Kit.2.2.Mechanism of metastatic hepatoma cells resist TRAIL-induced apoptosis1).Expression of TRAIL and its receptors in metastatic hepatoma cellsTotal RNA was extracted by trizol one-step extraction from attached and detached cells.The mRNA expression of TRAIL and its receptors were analysesed by profiling microarray.RT-PCR was used to detect the mRNA expression of TRAIL and its receptors of metastatic hepatoma cells.ELISA assay was used to indicate the secretion of sTRAIL protein in culture supernatant,FCM was used to detect the surface-bound TRAIL and its receptors on metastatic hepatoma cells.2).Caspase-8,-9 and -3 activation of metastatic hepatoma cells determined by Western blotWe further detected the Caspase-8,-9 and -3 activation after 20ng/ml of sTRAIL treatment by using Western blot.3.Responses of metastatic hepatoma cells to PI-3K/AKT or ERK pathway inhibitorsCells in either adherent or suspended culture conditions were treated with 1μM of Wortmannin or 50μM of PD98059 for 24 h.cell viability was measured by CCK-8 kit as well as trypan blue exclusion assay.Phosphorylation level of protein was detected by western blot.4.Response of metastatic hepatoma cells to hypoxie stressAttached and detached cells in 96-well plates were cultured at 37℃with 2%O2 for 24 h as hypoxia treated group or cultured at 37℃with 20%O2 as nomoxia control.Another two groups of conventional culture and then placed under hypoxic conditions for another 24 hours.Cell viability and proliferation status were measured by using the CCK-8 assay or trypan blue staining.Inhibition rate calculated by the following formula InhibitRate=Anomoxia-Ahypoxia/Anomoxia.Mortality rates in accordance with the following formula:DeadRate=DeadCells/DeadCells+LiveCells.5.Screening and demonstrating associated genes in anoikis-resistance and hypoxia-resistance5.1.Analysesing the microarray expression profiling of attached and detached hepatoma cellsExpression profiling of attached and detached hepatoma cells were analysesed by gene chip.Using cluster software to define the functional annotation clustering of different expressed genes and find genes participated in anoikis-resistance and hypoxia-resistance.5.2.Analyses the different microRNAs in attached,detached and hypoxia hepatoma cellsMicroRNA chip was used to find different microRNAs in attached,detached and hypoxia hepatoma cells.The results were analyzed at http://www.targetscan.org/ website,target genes of up-regulated and down-regulated microRNA were screened and compared with the results of gene chip.Genes participated in anoikis-resistance and hypoxic stimulus were selected.5.3.Block efficiency of different antisense protocolWe used four protocol to block aggregated cells by antisense oligonucleotide.①Transfecting attached and detached hepatoma cells directly by antisense oligonucleotide.②Transfecting attached and detached hepatoma cells by liposome-mediated antisense oligonucleotide.③Transfecting attached hepatoma cells by liposome-mediated antisense oligonucleotide without FBS,and then divided the attached cells into attached group and detached group.④Transfecting attached hepatoma cells by liposome-mediated antisense oligonucleotide with FBS,and then divided the attached cells into attached group and detached group.The efficiency of different programs and experimental stability was compared.5.4.Biological activity of cells blocked by antisense oligonucleotides of associated genesWe designed antisense oligonucleotide sequence of ANGPTL4,CA9,NDRG1, TRAIL and TrkB,and blocked the attached and detached hepatoma cells for 24h, then detected block efficiency by Real-Time PCR and RT-PCR as well as determined the biological activity by using CCK-8 or trypan blue staining assay.6.Statistical analysisResults were expressed as mean±SD.The statistical significance of differences between the groups was determined by Student's paired T tests or one-way ANOVA using SPSS10 statistical software.P＜0.05 was considered statistically significant.。Results1.Aggregated rate and metastatic ability of metastatic hepatoma cell model1.1.Metastatic hepatoma cell modelConventional cultured BEL7402 and SMMC7721 cells spread out as monolayers, while the suspension cells became round and self-aggregated into big cell clusters after cultured for 24 h.The aggregation rate stability was very well.It was always more than 99.7%.Poly-HEMA coated plates must be washed before using.The best cell density of 96 well plates is 3×104 per well.1.2.metastatic hepatoma cells' invasion and migrationMobility analysis revealed no significant difference between the attached and detached cells.However,the amount of invasive cells in the anoikis-resistant detached cell group was much greater than that of attached cells(19473±1104 vs. 9110±706 cells,p＜0.05). 2.Response of metastatic hepatoma cells to TRAIL-induced apoptosis and its mechanisms2.1.Metastatic hepatoma cells resist TRAIL-induced apoptosis in a dose-dependent and time-dependent mannerMetastatic hepatoma cells resist TRAIL-induced apoptosis in a dose-dependent and time-dependent manner.Specifically,the apoptosis rate in the attached hepatoma cells after treatment with 20ng/ml of sTRAIL for 24 h was 20%while that in the detached cells was 5%,a value similar to that of control hepatoma cells without sTRAIL treatment.We used the dose of 20ng/mL and processing time for 24 h in the following experiments according to time-dependent and dose-dependent curve.2.2.Metastatic hepatoma cells were more resistant to TRAIL-induced apoptosisAfter treated with TRAIL,metastatic hepatoma cells' apoptotic rate was significantly lower than the control group cells detected by TUNEL method. Caspase-3 activated level arrived the peak at 9 h after treated with TRAIL. Caspase-3 activated level of metastatic hepatoma cells were lower than control group cells at different time points and the difference at 9 h was most obviously.2.3.Expression of TRAIL mRNA was increased significantly in metastatic hepatoma cellsAll assays of detecting TRAIL mRNA,including microarray analysis and semi-quantitative RT-PCR indicated that expression of TRAIL mRNA was increased significantly in Synoikis-like suspended BEL7402 cells compared with that of attached counterpart.2.4.A discrepancy of TRAIL protein expression in metastatic hepatoma cells There is a discrepancy at the protein level of TRAIL expression.sTRAIL secreted in the supernatant was increased significantly in the detached hepatoma cells,while membrane binding TRAIL(mTRAIL) was down-regulated in the detached cells compared with that of attached cells. 2.5.TRAIL receptors of metastatic hepatoma cells were decreased significantly on mRNA level and protein levelGene chip and semi-quantitative RT-PCR indicated that expression of DR4,DR5, DcR1,and DcR2 mRNA of detached synoikis-like hepatoma cells was decreased significantly compared with that of attached control groups.There is also a substantial decreased expression of TRAIL membrane binding receptors at the protein level on the detached cells as compared with that of attached cells detected by FCM.2.6.Loss of anchorage blocked TRAIL-induced caspase cascade activation TRAIL induced significant activation of procaspase-8,procaspase-9 and procaspase-3.However,this activation of caspase cascade was effectively blocked upon loss of cell anchorage.3.Response of metastatic hepatoma cells to AKT/ERK pathway inhibitors3.1 Metastatic hepatoma cells were less compromised to AKT or ERK pathway inhibitionGrowth of attached hepatoma cells were greatly inhibited under the effect of AKT or ERK inhibition,however,the detached hepatoma cells were much more resistant to protein kinase inhibition when the inhibitor was used separately. Surprisingly,when both of the AKT and ERK pathways were blocked together, the viability of detached cells decreased dramatically to a degree even greater than that of attached groups.3.2 A strong compensated activation mechanism exit in the metastatic hepatoma cellsIt showed that the ERK phosphorylation level was dramatically increased when AKT pathway inhibitor was used,which indicated a strong compensated activation mechanism in the metastatic hepatoma cells.4.Response of metastatic hepatoma cells to hypoxiaThere was no difference of cell viability between detached and attached hepatoma cells when placed under hypoxic conditions before they obtained the ability of anoikis-resistance detected by CCK-8 and trypan blue assay.They were all able to tolerant the hypoxia stimuli to some extent.If expand the anoikis-resistant hepatoma cells to hypoxia,its ability to resist hypoxic environment is much higher than the attached hepatoma cells detected by CCK-8 and trypan blue assay.5.Screening and demonstrating associated genes in anoikis-resistance and hypoxia-resistance5.1.Genes involved in resistance to anoikis and hypoxia in gene chipThere were a large number of genes that involved in resistance to anoikis and regulated by hypoxia in gene chip.Such as IER3(up-regulated for 7.95 times), CA9(up-regulated for 10.31 times),BNIP3(up-regulated for 7.88 times), NDRG1(up-regulated for 18.4 times),ANGPTL4(up-regulated for 16.29 times), IGFBP3(up-regulated for 31.07 times).5.2.Crosstalk between microRNAs regulate anoikis and hypoxiaThere was a large proportion of crosstalk between microRNAs which regulate anoikis and hypoxia.For example,the crosstalking proportion of upregulated microRNA is more than 44.4%,and that of downregulated is about 18.7%.5.3.There was a well consistent between the gene chip and microRNA chip There was a well consistent between the differentially expressed genes of gene chip and target genes of different microRNAs in microRNA chip.For example, NDRG1,CA9 and BNIP3 were increased in metastatic hepatoma cells,and their possible regulate microRNAs hsa-miR-620,hsa-miR-516a-3p/516b*, hsa-miR-342,hsa-miR-558 were down-regulated significantly.5.4.Methods of PS-asODNs/ANGPTL4 blockThe results of PS-asODNs/ANGPTL4 direct transfection were unstable. Transfection efficiency of liposome-mediated transfection of detached cells was lower than that of attached cells.We cultured attached cells to logarithmic phase and transfected them with liposome-mediation for 6 h,then divided them into attached and suspension groups to continue to culture for another 24 h,the efficiency of antisense had no difference.5.5.Biology activity of cells whose ANGPTL4 had been blockedReal-Time PCR and RT-PCR results showed that,PS-asODNs/ANGPTL4 can significantly reduce the mRNA synthesis of ANGPTL4(p＜0.05).When ANGPTL4 was blocked,the viability of metastatic hepatoma cells was much more inhibited than that of the attached hepatoma cells.The mortality rate was also higher than the normal cultured hepatoma cells.Conclusions:1.Cell aggregated rate of metastatic hepatoma cell model at 24 hours reached 99.7%steadily,which further verify the validity of the metastatic hepatoma cell model.2.Metastatic hepatoma cells had significantly higher invasiveness than the adherent cells.These results suggest that anoikis-resistant metastatic hepatoma cells are more invasive to go through the third microenvironment and arrive the second microenvironment,that is to say they are more aggressive.3.Metastatic hepatoma cells secret more sTRAIL on the one hand and it can resist TRAIL-induced apoptosis on the other hand,which can induce apoptosis of immune effector cells in order to escape from immune system surveillance,the process is called "tumor counterattack".Metastatic hepatoma cells membrane-binding DR4,DR5 were significantly reduced. Maybe it is one of the mechanisms of metastatic hepatoma cells resist TRAIL-induced apoptosis.4.When AKT or ERK pathway was inhibited separately,it did not take much effect on the viability of the anoikis-resistant cells.However,when both of these two protein kinase pathway were blocked,it caused a dramatically cell death.This indicates that there is a strong interplay between these two pro-survival pathways.When one of these ways was inhibited,the signal of the other pathway will be over-activated and compensate for loss of this survival signal.Interestingly,our western blot data showed that inhibition of AKT pathway dramatically increased the phosphorylation level of ERK pathway.5.Metastatic hepatoma cells may be resist anoikis by resisting TRAIL-induced apoptosis and interplay of AKT and ERK pathway.6.There was no difference of cell viability between detached and attached hepatoma cells when placed under hypoxic conditions before they obtained the ability of anoikis-resistance.They were all able to tolerant the hypoxia stimuli to some extent.But,if expand the anoikis-resistant hepatoma cells to hypoxia,its ability to resist hypoxic environment is much higher than the attached hepatoma cells.That is to say,acquisition of anoikis-resistance contributes to hypoxia tolerance of metastatic hepatoma cells.7.There was a well consistent between the differentially expressed genes of gene chip and target genes of different microRNAs in microRNA chip.For example,NDRG1,CA9 and BNIP3 were increased in metastatic hepatoma cells,and their possible regulate microRNAs hsa-miR-620, hsa-miR-516a-3p/516b*,hsa-miR-342,hsa-miR-558 were down-regulated significantly.8.It is difficult to block genes of metastatic hepatoma cells by transfecting oligonucleotide directly.It is better to transfect antisense oligonucleotide into attached hepatoma cells and block for 6 h,followed by dividing them into attached and detached cell groups.9.When ANGPTL4 was blocked,the viability of metastatic hepatoma cells was much more inhibited than that of the attached hepatoma cells.The mortality rate was also higher than the normal cultured hepatoma cells.These indicate that ANGPTL4 may play an important role in the growth of metastatic hepatoma cells.Innovations and significances:1.This is the first report that metastatic hepatoma cells can escape from immunological surveillance by "tumor counterattack" and have increased invasiveness.2.It is the first time to report this compensated activation in detached hepatoma cells when one of the major survival pathway was blocked.This vigorous compensated activation may offer anchorage-deprived hepatoma cells more survival opportunities and metastatic potential when using relative therapeutic agents.Thus,combination of kinase inhibitors is likely to yield a substantial advance in successfully producing downstream phenotypic response in anoikis-resistant hepatoma cells.3.In this study,we first put forward that the ability of resistance to hypoxia of metastatic hepatoma cells was significantly higher than that of normal adherent cells.4.In this study,we combined the gene chip and microRNA chip to screen key molculars that may be involved in regulation of metastatic hepatocellular carcinoma cells.It will have higher clinical application value.5.This study explored firstly the methods of PS-asODNs transfecting aggregated cells,which will provide a useful reference for PS-asODNs as well as other small molecules to transfer into the aggregated cells.
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