| Background and ObjectiveBackground:Inflammatory bowel disease(IBD)whose causes and pathogenesis are still unclear is a nonspecific chronic inflammatory bowel disease,including ulcerative colitis(UC)and Crohn's disease(CD).The important feature of IBD lies in that it has a chronic,recurrent and persistent development course.Therefore,under circumstances of long-term and recurrent IBD,more serious complications such as intestinal fibrosis and cancer may appear on patients.At present,studies have shown that a variety of inflammatory disorders such as infection,chemical and physical stimuli and immunization diseases of their own can increase the risk of cancer.The chronic inflammatory process of IBD can induce cancer.The colitis-associated cancer(CAC)with high mortality is one of the importantcomplicationsclosely related to prognosis and survival time for patients with IBD and no effective prevention and treatment schemes have been found yet so far,which receives increasing attention from the medical field.The cancerizationof inflammatory bowel disease occurs mainly on patients with ulcerative colitis(UC)whichis a form of colitis,a disease of the colon,that includes characteristic ulcers,or open sores.The main symptom of active disease is usually constant diarrhea mixed with blood,of gradual onset.At present,the molecular mechanism of CAC is still not clear,however,there are relatively complete theories for the pathogenesis of sporadic colorectal cancer and the mainstream view is that accumulated mutations lead to different stages of normal mucosa from adenoma to cancerization.However,IBD related CRC does not accord with the classical mode of adenoma-canceration and the cancerization process of inflammatory bowel disease can be basically summarized as "inflammation-nontypical hyperplasia-cancerization",and can be further divided into:inflammation-dubious atypical hyperplasia-mild atypical hyperplasia-severe atypical hyperplasia-cancerization.One or multiple stages in this process can be skipped and develop in a multi-center form(multifocal dysplasia).Epithelial Mesenchymal Transition(EMT)refers to the phenomenon that epithelial cells under certain physiological and pathological conditions transform to mesenchymal cells.At present,EMT is believed to be involved in tissue fibrosis in various inflammation and participates in a variety of tumor invasion,metastasis process.EMT is divided into three different functional types,of which type ? can participate in and promote tumor invasion and metastasis.In the type ? EMT,abnormal epithelial cells lose their original form and have high-speed mobility of interstitial cell,causing the cells receive malignant tumor invasion and metastasis phenotype,showing characteristics of early malignant lesions.The occurrence characteristics of EMT is the epithelial cell surface marker expression was decreased(E-cadherin,alpha-catenin,Zo-1,cytokeratin,mucin),and interstitial cell surface marker expression level increased(Vimentin,FN,N-cadherin,alpha-sma,matrix metalloproteinase MMP-2,MMP-9 etc.)signal pathway induced by EMT including TGF-TGF beta signaling pathway,Wnt pathway,Wnt pathway and Notch pathway and so on.Among them,the expression loss of E-cadherin the most important sign.The occurrence of EMT induced signaling pathway is very complex,and includes the positive and negative feedback of every tumor related signaling molecule.TGF-? is a key mediator in the process of EMT,in the early stages of cancer progression,TGF-? as a tumor growth inhibitor can inhibit the proliferation and differentiation of the tumor cells.Smad2/3 plays an important role in the TGF?-induced EMT.The classic TGF-? signaling pathways by stimulating epithelial cells is to bind to TGF-?? type and type ? membrane receptors on the ligand-receptor forming a complex,and then bind to and activate the phosphorylation of Smad family proteins such as Smad2/3,p-Smad2/3-Smad4 complex translocate into nuclear and afterwards the complex enters the nucleus and binds to Smad EMT-related gene promoter binding element combination to regulate?-smooth muscle actin a(?-SMA),connective tissue growth factor(CTGF),matrix metalloproteinase 2(MMP2),Snail and the interaction of Smad protein 1(SIP1)and other associated targeted gene.Syndecan-1(Sdcl)is a heparan sulfate proteoglycan(HSPG),contains heparan sulfate(HS)and chondroitin sulfate chain modified type ? transmembrane core protein,mainly distribute in the epithelial cells,a small amount of them distribute in the blood vessel endothelial cells and plasma cell surface.Sdcl and its extracellular domain combined with the extracellular matrix,activate and participate in the pathogenesis of various diseases through internal and external cell signaling.Sdcl can regulate leukocyte accumulation,proliferation and invasion,angiogenesis,adhesion and invasion of pathogenic microorganisms and host defense mechanisms.Sdcl in inflammatory diseases such as asthma and lung injury,abscess,inflammatory bowel disease,through changing leukocyte adhesion,immune activation and matrix reconstruction,plays an important regulating role.Cell surface do wnregulation of Sdcl could delay the process of epithelial repair,and might cause inflammatory reaction become more long-term and repeated.In addition,Sdc1 shedding increased process may inhibit promoting leukocyte homing and tissue repair which are necessary to human body.Syndeean-1 mediated cell adhesion failure may play a key role in the carcinogenesis of colorectal cancer.Syndecan-1 can help maintain cell epithelial morphology and increase cell adhesion,and the SDC1 expression is positively correlated with the adhesion molecule E-cadherin,the shedding of its expression might induce E-cadherin expression to decrease.Loss of Sdcl expression can destroy the cell adhesion,and is closely related with the malignant degree of colorectal cancer,invasion and metastasis and tumor staging in which process Sdc1 may be involved in the induction and the occurrence of EMT in this process,but the interaction of the relationship between the two has not been further confirmed.Low molecular weight heparin therapy can relieve the inflammatory symptoms related to protein-losing enteropathy and inflammatory bowel disease,and Sdcl may play a maintaining role through its HS.Due to the similar characteristics of the heparin binding sites,so it can be combined with IFN?and TNF? and inhibit its activity,and even heparin can replace a variety of biological functions of HSPG.Research on the relationship between Sdcl and EMT is relatively small.Expression of Sdcl in the cell membrane to maintain the normal intestinal mucosa of intestinal gland epithelial cell morphology,Sdcl in exfoliated cells of normal the epithelial morphology and function change,and in the process of EMT,polarized epithelial cells lose normal shape,connections between cells reduced,then transform into mesenchymal like cells morphology,which occurred in epithelial cell phenotype transition to mesenchymal cell phenotype.Studies have shown that the shedding of Sdcl can induce the EMT occurrence of mammary epithelial cells,and SDC1 has a positive correlation with the expression level of E-cadherin while decreased E-cadherin expression is the most important sign of change to the EMT occurrence.Fibroblasts are also involved in the expression of Syndecan-1 and influence the EMT occurrence.Therefore,Sdc1 and EMT are both involved in changing epithelial cells and the intercellular adhesion and connection,the loss of Sdcl may lead to EMT,but no research has been conducted in colorectal cancer occurrence mechanism.Materials and Methods:1.Materials:Rat crypt epithelial cells rats IEC-6(ATCC,Manassas,VA),rat monoclonal anti-E Cadherin antibody(Abcam,USA),rabbit monoclonal anti-Vimentin antibody(Abcam,USA),rabbit monoclonal anti-Sdcl antibody(Abcam,USA),rabbit anti-smad2antibody(CST,USA),transwell chambers and cell culture plate(Coming,USA),CCK-8 kit(Dojindo,Japan),ethidium bromide(PI)(Sigma-Aldrich,USA),male Balb/c mouse(Animal Experiment Center of Southern Medical University,Guangzhou,China),Lenti-sdc 1-shRNA(Genechem,Shanghai,China),azoxymethane(Sigma-Aldrich,USA),Dextran sodium sulfate(MP biomedicals,USA),Clexane(enoxaparin sodium)(SANOFI WINTHROP INDUSTRIE,France).2.Methods2.1 Morphological change of IEC-6 under the stimulation of TGF-? in different concentrations and EMT phenomenon.IEC-6 cells are cultured in 6 well plates prefilled with medium containing 1%serum.Apply TGF-? to every well at the concentration gradient of 0,10,20,30,40 and 50 ng/ml.48-hours reactions.The experiments are repeated 3 times.Observe the morphological changes of cells under microscope.The expression of E-cadherin,vimentin and syndecan-1 are tested by Western blot,immunofluorescence methods.2.2 Transfection of two different sequence Lenti sdcl-shRNA and blank lentivirus in IEC-6 cells,observation of morphological change and EMT phenomenon.The IEC-6 cells were grown in 96 well plates at the density of approximately 1000cells/well.Each well are given 0.5ul,1u1,5ul,10ul lentivirus diluent.Transfected IEC-6 with two lentivirus with different knockout sequence sdc1-shRNA1 and sdc1-shRNA2 according to cell ratio.Then extracting cell proteins.Observe the morphology of cells under microscope,the expression level of syndecan-1 of IEC-6 respectively?Expression of sdc1,E-cadherin and vimentin are tested in IEC-6 Transfected by sdcl-shRNAland sdcl-shRNA2 was investigated by westernblot,fluorescence quantitative PCR.2.3 Detection of invasive ability of IEC-6 transfected by lenti-sdcl-shRNA by transwell assayDetect the invasive ability of IEC-6 transfected by lenti-sdcl-shRNA by transwell chambers prefloored with matrigel.1×104cells were put in each upper chamber,and the reaction was terminated at 12h,24h,36h 0.1%crystal violet was used to stain cells,and then calculate the total number of cells in lower chambers by counting crystal violet staining under microscope.2.4 Detection of cell-cycle of Lenti-sdcl-shRNA transfected IEC-6 by PI testIEC-6 cells were respectively transfected by Lenti-sdcl-shRNA and control virus and were put at the cell culture plates the day before the experiment.Cells were fixed overnight at 4? and analyze the next day.2.5 Detection of proliferation of Lenti-sdcl-shRNA transfected IEC-6 using CCK-8 kitThe proliferation of IEC-6 cells transfected by Lenti-sdcl-shRNA and control lentivirus respectively at 12h,24h,36h,48h,was tested using CCK-8.2.6 Wound healing test for migration ability of cells infected with lenti-sdc1-shRNA and control lentivirusIEC-6 transfected by lenti-sdcl-shRNA and control lentivirus were culture until cell confluence reached 100%,and the percentage of wound width in each group at different time were compared2.7 Effect of inhibitation of sdcl downregulation by LMWH in EMT processWhen cell confluence is 60%in 6 well plates,add 0 ng/ml,10ng/ml TGF?,20ng/ml TGF(3,20ng/ml TGF?+20ul LMWH,20ng/ml TGF?+20ulLMWH and 20ng/ml TGF(3+20ulLMWH into the wells respectively.After 48h,collect protein to test the expression level of syndecn-1,Ecadherin,Vimentin,p-smad2,smad2 by western blot.2.8 Detection of invasive ability of IEC-6 stimulated by TGF ? and LMWH by transwell assayThe IEC-6 were plated in six-well plates,and were divided into the following three groups of 20ng/ml TGF?,20ng/ml TGF?+30ul LMWH and the control group.In 48 hours,cells were respectively plated in the transwell chambers(10,000 cells per well).The cell invasion ability was determined in 30 hours by counting the stained cells by he crystal violet.2.9 Detection of migration ability of IEC-6 stimulated by TGF ? and LMWH by wound healing testThe IEC-6 were plated in six-well plates,and were divided into the following three groups of 20ng/ml TGF?,20ng/ml TGF?+30ul LMWH and the control group.Whe the cell fusion rate reached 100%,the width of wounds in different groups were compared.2.10 Effect of inhibitation of sdcl detachment by LMWH in EMT progress in IBD related colorectal cancer mouse models57 male Balb/c mice of 6?8w are randomly divided into blank control group(12 mice),cancer control group(21 mice),the early heparin treatment group(12 mice)and the late heparin treatment group(12 mice).Except for mice in the balnk contol group,which were injected with the same amount of saline,all the other groups are intraperitoneal injected with AOM(12.5 mg/kg).After the administration of AOM,mice were feed with clean water for 1 week,and 3%DSS for 1 week,DDS was replaced every 3 days,and change to drink water again for 2 weeks.Repeat for 3 cycles according to the above protocol.After the administration of DSS,3-6 mice were randomly sacrificed at day 0 and week 3,week 6,week 12,week 16 in cancer control group,2-4 were sacrificed at day 0 and week 3,week 6,week 12,week 16 in other groups.Disease activity indexs are scored weekly.Intestinal tissues were collected,and the number of tumors,the length and the weight of intestine were recorded.The intestinal tissues were obtained for western blot,HE staining and immunohistochemical staining.2.11 Statistical software SPSS13.0 was used for statistical analysisCell proliferation was studied via factorial analysis,invasion and metastasis by using independent t test,DAI comparison by using repeated measures univariate analysis,and multiple independent samples were compared through employing univariate analysis of variance(one way ANOVA)for statistical processing,taking P<0.05 as significant difference.3.Results:3.1 In this experiment,we stimulated the IEC-6 cells with TGF-?concentration gradient of 0 ng/ml,10 ng/ml,20 ng/ml,30 ng/ml,40 ng/ml and 50 ng/ml,then observed under the microscope after 48 hours,it is found that as the concentration increased,the morphology of cells change from normal circular,spherical to the spindle,and cell morphology varies,some cells extend out of the reach,estimate the percentage rate of deformation of cells under the microscope are 0%,20%,70%,70%,70%,80%respectively when the concentration was up to 20 ng/ml,the proportion of deformation cells did not become significantly higher.3.2 Using Western blot to detect the protein expression levels of syndecan-1,E-cadherin and Vimentin.It is found that as the TGF-? concentration increased,the expression of E-cadherin syndecan-1 as the TGF-? stimulation concentration gradient increased,the expression levels of sdcl and epithelial cell surface marker E-cadher decreased(F=259.199,P<0.001;F=109.846;P<0.001);E-cadherin and syndecan-1 in the TGF-? concentrations of 20 ng/ml and untreated cells decreased significantly(P<0,001).3.3 Testing cells stimulated with 20 ng/ml TGF-? by immunofluorescence staining.When the exposure time was consistent,it can be noticed that the fluorescence intensity of E-cadherin and syndecan-1 in IEC-6 cells was weak,after TGF-? stimulation,fluorescence intensity decreased.Fluorescence intensity of vimentin in the cytoplasm of IEC-6 was strong,the intensity increased after the TGF-? stimulation.3.4 Two groups of IEC-6 were transfected with sdcl shRNAl and sdc1-shRNA2 plasmid packaged lentivirus,observed cell morphology under the microscope,cell transfected with Lenti sdc1-shRNA1 and Lenti sdc1-shRNA2 cell morphological changes,themorphology of cells transferred with the latter lentivirus changed significantly.3.5 After the IEC-6 transfected Lenti-sdcl-shRNA2,the sdcl expression was significantly reduced(F=597.410,P<0.001),while no significant down-regulation of the expression of sdc1 who transfected Lenti-sdc1-shRNA1,was found.3.6 Fluorescence quantitative PCR(Quantitative Real-time PCR)verification transfected Lenti-sdc1-shRNA2 of IEC-6 cells sdc1,E-cadherin and Vimentin mRNA levels,it is found that IEC-6 cells,Lenti-sdcl-shRNA2 of sdcl of mRNA levels were significantly lower than the control group(t=7.263,P<0.005vs.control),and E-cadherin mRNA levels were significantly lower than the control group(t=3.631,P<0.05vs control),Vimentin mRNA level was significantly higher than that of in the blank control group(t=9.900,P<0.001 vs control).3.7 Number of lower cell Lenti-sdcl-shRNA-transfected after IEC-6 cells increased compared with the control group,the invasion capacity strengthened.Significant differences exist between the two groups(t=13.229,P<0.001),Lenti-sdcl-shRNA-transfected IEC 6 cell invasion capacity become stronger,about 1.5 times that of the control group.3.8 CCK-8 tested the cell proliferation capacity of the IEC-6 transfected with Lenti-sdcl-shRNA and blank lentivirus,it is found that the proliferation ability differs significantly(p<0.001)between the IEC-6 cells transfected with Lenti-sdc1 and the blank lentivirus control group.The IEC-6 cell proliferation capacity of Lenti-sdc1-shRNA and blank lentivirus presented no obvious differences in 12 hours and 24 hours(12 hours:F=0.576,p=0.465;24 hours:F=1.219,p=0.295);after 24 hours,the proliferation of sdcl knockdown group increased significantly(36 hours:F=109.557 p<0.001;48 hours:F=6.235,p=0.032).3.9 Wound healing test examined the cell migration capacity of IEC-6 transfected with Lenti-sdcl-shRNA and blank lentivirus.When the test started(0 hour),the width was similar between the cells in the control group and cells transfected with Lenti-sdcl-shRNA;36 hours later,the scratch width of cells transfected with Lenti-sdcl-shRNA lentivirus was narrower than that of in the control group.Lenti-sdcl-shRNA cells compared with cells in control group,the scratch width ratio(scratch after 36h and 0h)became significantly lower(t=8.436,P=0.001).3.10 Under the background of 20ng/ml TGF ? stimulation,10ul,20ul,30ul and 40ul low molecular weight heparin(LMWH)were added.TGF ? at the concentration of 20ng/ml can successfully induce the occurrence of EMT,E-cadherin downregulation(F=272.730,P<0.001)and increased expression of Vimentin(F=33.855,P<0.001),and with the increasing concentration of heparin liter high protein,sdcl has tended to increase;meanwhile,in the hole in which 20ug/ml TGF ? and 40ul LMWH were added,the protein expression of sdcl was significantly higher than that of merely 20ng/ml TGF ? stimulation group(F=635.490,P<0.001).E-cadherin and Vimentin increased and decreased respectively in the hole in which 20ug/ml TGF ? and 40ul LMWH were added at the same time(F=272.730,P<0.001;F=33.855,P<0.001).3.11 Smad2 phosphorylation was the most significant when 20ng/ml of TGF ?cells were purely added.Smad2 phosphorylation gradually strengthens as TGF ?concentration(1 Ong/ml,20ng/ml)increases;while with the increase of the concentration of low molecular weight heparin intervention,Smad2 phosphorylation gradually reduces(F=249.713,P<0.001).3.12 DAI score comparison of AOM/DSS-induced colitis-associated colorectal cancer in a mouse model,and repeated measurements showed:from 1st week to 12th week,there are significant DAI differences at different time points in each group(F=10.240,P<0.001);there were significant differences(F=94.358,P<0.001)among the same group at different time points,there are also differences(F=48.055,P<0.001)at the same time point with different groups.During 13-16 weeks,significant statistical differences existed at the same time point among different groups(P<0.05);pairwise comparison,there was a significant difference between the control group and the treatment of advanced colorectal cancer group DAI score with the control group(P<0.05),while there was no significant difference between the early treatment group and the control group DAI score.The cancerization rate was 100%at 12th week and 16th week and 68.6%is located in distal colon,25.7%in the middle of the colon.3.13.After the mice with colon tissue HE staining paraffin sections under the microscope after HE staining of paraffin sections of colon tissue under the microscope mouse model,compared with the model group tissue cancer in different time periods and found that the longer modeling time,intestinal performance 3 to 12 weeks(Figure 3-3)consists of mild dysplasia-moderate dysplasia-severe dysplasia-adenocarcinoma change.HE intestinal epithelial cells seen in 6-8 weeks mainly in highly atypical hyperplasia,10 weeks after cell atypia,disorganized,compact form multi-layered,enlarged nuclei were pleomorphic,occupy most of the cell,cancer nests distributed,no obvious glandular formation,splitting the nucleus phase increases,the formation of adenoid irregularities.Mice swollen colon pathology.Categories with well-differentiated adenocarcinoma.3.14.For each group of mice were detected by immunohistochemistry sdc-1,vimentin and expression of E-cadherin,we found that the control group and the early treatment group 1 syndecan-and Ecadherin expression of mouse intestinal epithelial cells are cancerous than the control group high(syndecan-1:F=26.684,P<0.001;Ecadherin:F=29.320,P<0.001),and expressed mainly in epithelial membrane position expressing cells and a few cell cytoplasm;at the same time,the control group of mice and early treatment group Vimentin expression of intestinal epithelial cells are compared with the control group of cancer(F=38.933,P<0.001),vimentin expression in the lower Vimentin the control and treatment groups,mainly located in the cytoplasm of epithelial cells,and in cancer control group,we can see Vimentin expressed mainly in cancer cells,and the expression of high strength.3.15.Sdcl Western blot detection of early detection and treatment of cancer in the control group of mice,vimentin,E-cadherin and p-smad2 expression levels.Figure 3-5 and Table 3-6,6 weeks ago,E-cadherin expression level of intestinal cancer in the control group of mice were unchanged and higher than six weeks after the decreased expression of E-cadherin,to 12-16 weeks The expression level of the lowest,and 16 weeks in the early treatment group,the expression of E-ecaderin gradually increased(F=43.010,P<0.001).Six weeks ago,intestinal cancer Vimentin protein levels in the control group of mice were weak,6-9 weeks after the vimentin is gradually increased,and 16 weeks in the early treatment group vimentin expression is decreased(F=11.099,P<0.001).In colorectal cancer in the control group,the expression of mouse intestinal Sdcl loss and decline in the first three weeks,the expression level rises early treatment group 16 weeks(F=29.158,P<0.001).Control mice intestinal p-smad2 weak expression in cancer control group with modeling prolonged expression of p-smad2 gradually increased to 9-16 weeks increased expression of the most obvious,were significantly different from the control group(P<0.001),in the early treatment group 16 weeks of mouse intestinal p-smad2 expression of colorectal cancer compared with the control group of mice modeling weak 9-16 weeks(F=156.968,P<0.001).Conclusions4.1.Syndecan-1 expression knockdown can induce normal intestinal epithelial cells IEC-6 appear EMT phenomenon;E-cadherin down,Vimentin expression regulation of cell morphology,knockdown Syndecan-1 expression changes;accompanied by cell invasion,migration and proliferation force enhance.4.2.Low molecular weight heparin can inhibit syndecan-1 expression,thereby inhibiting the occurrence of EMT,this process may have indirect or direct relation with LMWH blocking smad2 pathway.4.3.Low molecular weight heparin can inhibit the invasion and migration abilities of IEC-6 cells after the occurrence of EMT.4.4.In AOM+DSS animal model experiments of colitis-related cancer,DAI and pathological scores in early heparin treatment group are better than those of control group and late heparin treatment group;early use of low molecular weight heparin can reduce the number of tumors,lower levels of intestinal inflammation,inhibit EMT occurrence and the activation of smad2 pathway. |
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