| Background and ObjectiveColorectal cancer(CRC)is one of the most common tumors in the world,which remains a leading cause of cancer death.It is said that there are approximately 1.3 million new diagnoses and nearly 700 000 deaths account for CRC each year.However,the main reason that lower the living standard and cause death of the patients with CRC is the recurrence and metastasis of tumor.With the rapid development of molecular biology technology in recent years,the etiology and pathogenesis of tumor research has made important progress.However,there still lack of clear molecular biomarkers in diagnosis of colorectal cancer.So it becomes essential to identify the genes function in carcinogenesis and progression of CRC and explore the molecular mechanism of pathogenesis of CRC to develop accurate targeted therapy.The decoy receptor 3(DcR3),also known as TNFRSF6B or M68,is a soluble receptor belonging to the tumor necrosis factor receptor(TNFR)superfamily,composed of 271 amino acid residues.Specially,DcR3 is a member of cytokine of the lack of transmembrane domain in its amino acid sequence of the structure.Previous studies have shown that DcR3 was found in embryo lung,brain,liver,and in some autoimmune diseases.And abnormal DcR3 expression was also found in serials of malignancies,including lung cancer,gastric cancer.As one of the members of the family of soluble tumor necrosis factor receptor,the expression of DcR3 plays an important role in tumorigenesis,metastasis and immune suppression.However,the contribution of DcR3 on CRC and molecular mechanism between DcR3 and CRC is still not fully elucidated.Thus,the objective of our study was to trying to figure out the relationship between the expression of DcR3 and clinical significance parameters and investigated its potential role in proliferation and migration.Moreover,we aim to explore whether DcR3 involved in the process of epithelial-mesenchymal transition(EMT)induced by TGF-?/SMAD signal pathway in CRC in vitro.Methods1.The expression of DcR3 in colorectal cancer tissues and cells was detected by real time quantitative RT-PCR and western blottingThe expression of DcR3 mRNA level in 27 colorectal cancer tissues and matched intestinal mucosa tissue was detected by real time quantitative RT-PCR(qRT-PCR).Western blotting was taken out to detect the expression of DcR3 protein in 15 colorectal cancer tissues and matched intestinal mucosa tissue.At the same time,the expression of DcR3 mRNA and protein in 7 kinds of colorectal cancer cell line SW480/M5,SW480,HCT116,LS174T and SW620,RKO and HT29 were detected by qRT-PCR and western blotting.Immunohistochemical method was used to detect the DcR3 expression in 86 pairs of colorectal tissues and match normal tissues.In order to investigate the possible effect of DcR3 in colorectal cancer,we analyzed the relationship between DcR3 and clinical pathological parameters.2.The function of DcR3 on colorectal cancer cell growth and migration in vitro and in vivo(1)Lentiviral(Genechem,China)constructs expressing and control vector(LV-TNFRSF6B and LV-Control)were used to infect RKO and HT29 cells.SW480/M5 and HCT116 were transfected with LV3-shDcR3-GFP and sh-Negative control vector(GenePharma,China)separately.In order to establish cells constitutively repressing or expressing DcR3,antibiotic-resistant transfected cells were selected by selective medium applying puromycin(5ug/ml).And western blotting was used to examine the efficiency of overexpression and knockdown of DcR3.Then we established RKO and HT29 cell subline that stable overexpressed of DcR3 and SW480/M5 and HCT116 cell subline stable knockdown expression of DcR3.(2)Cell proliferation assay(CCK8)and colony-formation assay,were carried out to detect the function of DcR3 on colorectal cancer cell growth in vitro.We also subcutaneously injected RKO or HT29 cells that stably express vector(control)or DcR3 plasmid and SW480/M5 or HCT116 cells that stably express scramble(control)or DcR3 shRNA into nude mice,and then monitored the growth of the resultant primary tumors,to assess the effect of DcR3 on tumor growth in vivo.(3)Scratch wound-healing assay and migration assay were carried out to detect the function of DcR3 on colorectal cancer cell migration in vitro.To test the effect of DcR3 on the metastasis of CRC in vivo,we performed a tail vein xenograft model to investigate lung metastases of CRC cell lines.SW480/M5 cells that stably express control or DcR3 shRNA were injected into 6 mice for each group.3.Explore the possible signal pathway of DcR3 may be involved in colorectal cancer(1)Observation of morphological changes of subcell line RKO/DcR3 and HT29/DcR3 compared to control group cells.Using western blotting,immunofluorescence method to detect the expression of EMT expression related markers in E-cadherin(epithelial markers),N-cadherin and Vimentin(interstitial expression markers)between RKO/DcR3,HT29/DcR3 and control group cells repectively.At the same time,the EMT related proteins in the subcutaneous tumor tissue of nude mice was detected by IHC of E-cadherin and N-cadherin.(2)We firsrt observe the activation of SMAD pathway,EMT and the expression of DcR3 levels of RKO and HT29,which were treated with TGF-?3(5 ng/ml)for 24h,to investigate if TGF-P3 can induced the expression of DcR3.To determine if SMAD signaling pathway is required for TGF-?3-induced EMT,we used SB431542(SB)inhibitor to inhibit smad2/3 protein phosphorylation in RKO and HT29 cells treated with TGF-?3,observe the SMAD pathway,EMT process and the expression of DcR3 whether be suppressed.Moreover,TGF-?3 treatment group while adding inhibitor SB431542 after 24 h,reoccupy by plasmid expressing the expression of DcR3,and observe the expression of EMT marker protein,DcR3 protein and monitor if the SMAD pathway is activated at the same time.(3)To further analyze the correlation between DcR3 expression and TGF-?3/SMAD signaling in CRC tissues.We analyzed the association between DcR3 expression and TGF-?3/SMAD pathway in a public clinical microarray dataset of 177 CRC tissues.We performed Kaplan-Meier survival analyses to test if the altered DcR3 expression and TGF-?3/SMAD-EMT signaling related protein in CRC is related to patient's prognosis.All of these obversations were used to preliminary analysed of the relationship between DcR3 and TGF-?3/SMAD signaling pathway.4.Statistical analysisQuantitative values of all experiments are expressed as the mean SD.Differences among/between sample group were analyzed by one-way ANOVA or the independent samples t test Relationships between DcR3 expression and clinicopathologic characteristics were tested using Pearson x2 test.Survival curves were plotted by Kaplan-Meier method and compared by log-rank test by using SPSS 20.0 software.Differences were considered significant if P<0.05a*.Results1.DcR3 was up-regulated in clinical CRC tissues and correlated with human CRC development,metastasis and lower patient survival rate(1)DcR3 was up-regulated in clinical CRC tissuesThe expression levels of DcR3 mRNA and protein in human colorectal cancer tissues were detected by q-PCR and western blotting in fresh CRC tissues and paired normal tissues.We detected the protein expression of DcR3 in 15 human paired CRC tissues by western blotting.We found that DcR3 protein expression were significantly higher in tumor tissues than that in corresponding normal tissues.We also analyzed the DcR3 mRNA expression in 27 paired human CRC tissue samples by qRT-PCR and found that DcR3 mRNA expression in CRC tumor tissues was higher than the normal tissues in 19(70.37%)out of 27 matching tissue samples(t=2.024,P=0.018).As a result,we found that DcR3 was up-regulated in clinical CRC tissuesIn cellular level,we detected the protein and mRNA expression of DcR3 in seven different CRC cells(SW480/M5,SW480,HCT116,LS174T,SW620,RKO,HT29)and found that SW480/M5(with high metastatic potential),SW480 andHCT116 cells exhibited a much higher level of DcR3 expression than other cells,and RKO and HT29(with low metastatic potential)cells exhibited a much lowerlevel of DcR3 expression than other cells(F=4723.981,P<0.0001).The resultsprovided idea that if DcR3 was associated with the metastais ability of colorectal cancer cells.(2)DcR3 was correlated with human CRC development,metastasis and lower patient survival rateTo test the correlation between DcR3 with CRC development,IHC staining was performed to detect the DcR3 protein expression in 86 paired paraffin-embedded CRC tissues.We found that 62 out of these samples exhibited a much higher DcR3 level in CRC tissues than the corresponding normal tissues(?2=56.766,P<0.001).Moreover,analyses of the clinicopathologic characteristics of all the 86 matching tissue samples revealed that DcR3 level was closely associated with tumor differentiation(P=0.006),depth of tumor cell infiltration(P=0.046)and lymphatic metastasis(P=0.012).Furthermore,Kaplan-Meier survival analyses results revealed that patients with higher expression of DcR3 had a shorter survival time compared to those with lower DcR3 expression level(P=0.005).Cox regression analyses revealed that DcR3 expression(P=0.007),lymph metastasis(P=0.040)and infiltration(P<0.001)were recognized as independent prognostic factors in this study.These data suggested that theDcR3 may play an important role in CRC invasion and metastasis as well as the patient's survival.2?DcR3 promoted CRC cell growth and migration in vitro and in vivo(1)The effect of DcR3 on CRC cell growth in vitro was detected by CCK8 and colony-formation assayTo study the potential role of DcR3 in CRC tumorigenesis and progression,we performed colony formation assay and CCK8 proliferation assay to detect CRC cells proliferation.CCK8 assay result showed that overexpression of DcR3 significantly promoted both RKO and HT29 cells growth(FRKO=32.724,P<0.001;FH29=73.976,P<0.003),indicating that DcR3 plays a role in CRC growth,while knockdown of DcR3 significantly inhibited CRC growth both in SW480/M5(Fsw480/M5=75.404,P<0.001)and HCT116 cells(FHCT116=18.825,P<0.001).Colony-formation assay results showed that overexpression of DcR3 resulted in a more colony number than the control group in RKO and HT29(XRKO/control=175,XRKO/DcR3=279;tRKO=-4.221,P=0.013)(XHT29/control= 165.667,XHT29/DcR3=1 95.667,tHT29=-3.096,P=0.036).While knockdown of DcR3 resulted in a fewer colony number than the control group in SW480/M5(XSW480/M5/shControl 123,XSW480/M5/shDcR3=60.333;tSW480/M5=5.155,P=0.007)and HCT116(XHCT116/shControL=229,XHCT116/shDcR3=142.333;tHCT116=6.381,P=0.003).CCK8 and colony-formation assay results indicated that DcR3 played an important role in CRC growth in vitro.(2)The effect of DcR3 on CRC migration in vitro was detected by transwell assay and scratch wound-healing assayTranswell assay results showed that overexpression of DcR3 promoted cell migration in RKO(XRKO/control=273.4,XRKO/DcR3=459.4;tHT29 =-19.248,P<0.001)and HT29(XHT29/control=29.4,XHT29/DcR3=66.6;tHT29=-8.763,P<0.001)cell line than the control group,while knockdown of DcR3 significantly inhibited CRC migration in SW480/M5(XSW480/M5/shControl=86.4,XSW480/M5/shDcR3=39;t SW480/M5=10.583,P<0.001)and HCT116(XHCT116/shControl=84.8,XHCT116/shDcR3=33;tHCT116=8.714,P<0.001)than the control group.Wound-healing assay results showed that overexpression of DcR3 promoted the migrated ability than the control group in RKO(XRKO/control=292.306,XRKO/DcR3=371.27;tRKO=-4.221,P=0.013)and HT29(XHT29/control=244.366,XHT29/DcR3=307.32;tHT29=-4.409,P=0.001),while knockdown of DcR3 displayed opposite results in SW480/M5(XSW480/M5/shControl=393.722,XSW480/M5/shDcR3=234.216;tSW180/M5=5.397,P<0.001)and HCT116(XHCT116/shControl=386.808,XHCT116/shDcR3=245.72;tHCT116=11.471,P<0.001).Transwell assay and scratch wound-healing assay results displayed that DcR3 indeed promoted CRC cells migration in vitro.(3)DcR3 promoted colorectal tumor growth in vivoTo assess the effect of DcR3 on tumor growth in vivo,we subcutaneously injected RKO or HT29 cells that stably express vector(control)or DcR3 plasmid and SW480/M5 or HCT116 cells that stably express scramble(control)or DcR3 shRNA into nude mice,and then monitored the growth of the resultant primary tumors.The xenograft tumors developed at the injection site after 5 days.During a growing period of 25 days,primary tumors derived from DcR3 over-expressed groups grew significantly faster than that derived from control cells(FRKO=59.481,P<0.001;FHT29=55.784,P<0.001),and primary tumors derived from DcR3 deficient groups grew significantly slower than that derived from control cells(Fsw480/M5=60.529,P<0.001;FHCT116=80.71 3,P<0.001).Moreover,the tumor volumes of the DcR3 over-expressed groups were significantly bigger than those of control groups,and the tumor volumes of the DcR3 deficient groups were significantly smaller than those of control groups.IHC staining confirmed that the tumors derived from CRC cells stably expressing DcR3 plasmid displayed a higher DcR3 expression and increased cell proliferation index as shown by Ki-67 staining compared to the tumors derived from control cells,and the tumors derived from CRC cells expressing DcR3 shRNA displayed a lower DcR3 expression and reduced cell proliferation index as shown by Ki-67 staining compared to the tumors derived from control cells.These data demonstrated that DcR3 plays a crucial role in CRC growth in vivo.(4)DcR3 promoted colorectal tumor metastasis in vivoTo test the effect of DcR3 on the metastasis of CRC in vivo,we performed a tail vein xenograft model to investigate lung metastases of CRC cell lines.SW480/M5 cells that stably express control or DcR3 shRNA were injected into 6 mice for each group.We found that the number of lung metastatic foci in SW480/M5/shCtrl group was more than that in SW480/M5/shDcR3 group(XSW480/M5/shControl=11,XSW480/M5/shDcR3=3.333;t=8.056,P<0.001).Moreover,knockdown of DcR3 prolonged the survival time of nude mice(P=0.027).These results demonstrated that DcR3 plays a role in CRC metastasis in vivo.3.Explore the possible signal pathway of DcR3 may be involved in colorectal cancer(1)DcR3 facilitated epithelial-mesenchymal transition(EMT)of CRC cellsObservation of morphological changes of subcell line RKO/DcR3 and HT29/DcR3 compared to control group cells,we found that overexpression of DcR3 altered cell morphology to a spindle-like,fibroblastic cell morphology.We found that overexpression of DcR3 in RKO and HT29 cells significantly increased the expression of N-cadherin(tRKO=-5.751,P=0.0045;tHT29=-33.34,P<0.001)and vimentin(tRKO=-73.24,P<0.001;tHT29=-1138.878,P<0.001),while decreased E-cadherin(tRKO=11.87,P<0.001;tHT29=37.94,P<0.001)expression.IHC staining confirmed that the tumors derived from CRC cells stably expressing DcR3 plasmid displayed a higher N-cadherin expression(tRKO=-7.879,P<0.001;tHT29=-7.122,P<0.001)and a reduction of E-cadherin expression(tRKO=10.11,P<0.001;tHT29=7.817,P<0.001)compared to the tumors derived from control cells.These results revealed that DcR3 facilitated EMT of RKO and HT29 cells.(2)DcR3 was required for TGF-?/SMAD signaling-induced EMTThus,we tested if TGF-?3 induces EMT of RKO and HT29 cells.Indeed,TGF-p3 stimulation induced the EMT of RKO and HT29 cells as shown by the decreased E-cadherin(tRKO=16.60,P<0.001;tHT29=14.63,P<0.001)and increased N-cadherin(tRKO=-9.655,P<0.001;tHT29=-41.68,P<0.001)and vimentin(tRKO=-28.72,P<0.001;tHT29=-28.83,P<0.001)expression.Importantly,TGF-P3 also enhanced the phosphorylation of smad2/3(P-smad2(tRKO=-35.18,P<0.001;tHT29=-30.09,P<0.001);P-smad3(tRKO=-53.19,P<0.001;tHT29=-42.45,P<0.001))expression in RKO and HT29 cells.Interestingly,TGF-P3 enhanced DcR3(tRKO=-38.56,P<0.001;tHT29=-41.43,P<0.001)expression in RKO and HT29 cells.To determine if SMAD signaling pathway is required for TGF-?3-induced EMT,We used SB431542(SB)inhibitor to inhibit smad2/3 protein phosphorylation in RKO and HT29 cells treated with TGF-?3.We found that its inhibition of smad2/3 phosphorylated protein expression(P-Smad2(tRKO=28.49,P<0.001;tHT29=24.67,P<0.001);P-smad3(tRKO=40.73,P<0.001;tHT29=77.36,P<0.001))restored E-cadherin expression(tRKO=-67.18,P<0.001;tHT29=-12.83,P<0.001)while attenuated N-cadherin(tRKO=36.34,P<0.001;tHT29=66.53,P<0.001)and vimentin(tRKO=82.57,P<0.001;tHT29=67.91,P<0.001)expression in both RKO and HT29 cells.Notably,inhibition of smad2/3 phosphorylated protein expression by SB431542(SB)also blocked TGF-?3-induced DcR3 protein expression.We therefore hypothesized that DcR3 was required for TGF-?3/SMAD signaling-induced EMT,and thus DcR3 overexpression can rescue the function of TGF-?3/SMAD signalingon the EMT.Indeed,overexpression of DcR3 attenuated the E-cadherin expression but restored N-cadherin and vimentin expression was altered due to inhibition of smad2/3phosphorylated protein in TGF-?3-treated cells.These results clearly showed that DcR3 mediates TGF-?3/SMAD-induced EMT of CRC cells.To further analyze the correlation between DcR3 expression and TGF-?3/SMAD signaling in CRC tissues.We analyzed the association between DcR3 expression and TGF-?3/SMAD pathway in a public clinical microarray dataset of 177 CRC tissues.We found that DcR3 expression was positively correlated with N-cadherin expression,while negatively correlated with E-cadherin,suggesting that DcR3 was associated with the activation of CRC EMT.Importantly,DcR3 was positively correlated with both TGF-?3 and smad2,smad3 expression in CRC tissues.Furthermore,TGF-?3 was also positively correlated with both smad2 and smad3 expression,providing evidence that DcR3 indeed mediated TGF-?3/SMAD signaling pathway.Taken together,these data indicated that DcR3 was positively correlated with EMT and TGF-?3/SMAD signaling in CRC tissues.Patients with coexpression of E-cadherin(-)and N-cadherin(+),DcR3(+)and N-cadherin(+)or DcR3(+)and E-cadherin(-)had a shorter overall survival time compared to patients with coexpression of E-cadherin(+)and N-cadherin(-),DcR3(-)and N-cadherin(-)or DcR3(-)and E-cadherin(+),suggesting that cooperation between DcR3 and EMT-related protein confidently correlated with patient survival rate and length.Moreover,patients with coexpression of DcR3(+)and activated TGF-?3/SMAD signaling related protein had a poorer prognosis compared to patients with coexpression of DcR3(-)and inactivated TGF-?3/SMAD signaling related protein,and patients with the activation of TGF-?3/SMAD signaling pathway had a shorter overall survival time.More interesting,coexpression of DcR3(+)and activated TGF-?3/SMAD-EMT signaling related protein led to a poorer prognosis in CRC patients.These data indicated that cooperation between DcR3 and TGF-?3/SMAD-EMT signaling related protein correlated with patient survival rate and length,and DcR3 was critical for TGF-?3/SMAD-mediated CRC metastasis.Conclusion1.DcR3 was up-regulated in clinical CRC tissues and correlated with human CRC development,metastasis and lower patient survival rate.2.DcR3 promotes CRC cell growth and migration in vitro and in vivo.3.DcR3 was required for TGF-?3/SMAD signaling-induced EMT. |
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