| Inflammatory bowel disease(IBD)is a chronic and recurrent intestinal inflammation without clear cause,with the possibility of lifelong recurrence.IBD can be divided into two major disorders: ulcerative colitis(UC)and Crohn’s disease(CD).Many studies have shown that IBD is one of the high-risk conditions for colorectal cancer,with the canceration risk of 1.2%,3.6% and 14.4% for patients who have suffered from UC for 10 years,20 years,and 30 years respectively.Patients with IBD are twice to four times more likely to develop colorectal cancer than the general population and colorectal cancer is one of the most serious complications of IBD.Inflammatory colorectal cancer differs from sporadic colorectum in pathogenesis,clinical features and pathology,so colorectal cancer caused by colitis is also called colitis-associated colorectal cancer(CAC).CAC has the molecular pathobiology of progression from inflammation,dysplasia to cancer.Tumor necrosis factor(TNF),a main inflammatory mediator,will also cause malignant tumor besides inflammatory diseases and immunological diseases.Studies have proved that TNF has dual characters in tumorigenesis.In the early stage of inflammation,it can promote cell apoptosis,prevent tumor angiogenesis and inhibit tumor.If the inflammation is prolonged,TNF can lead to genetic mutations,inhibit apoptosis and stimulate angiogenesis,and TNF and other inflammatory factor constitute the inflammatory microenvironment,which releases a large number of reactive oxygen,nitric oxide and other active substances,further damaging DNA and promoting tumor angiogenesis.TL1A,a member of tumor necrosis factor superfamily,is a susceptibility gene factor IBD.TL1 A can be combined with DR3 to regulate the adaptive immunity of Th1,Th2,Th17 and other effector cells,to promote T cell proliferation and differentiation,and to gather inflammatory cells to the lesion site to increase the development of inflammation.To stop TL1 A combining with DR3 can obviously reduce the inflammatory response and reduce or control the occurrence and development of IBD.However,there is few studies about the role of TL1 A in colorectal cancer.our team confirm that,the expression of TL1 A,DR3,DcR3 has been significantly elevated in sporadic colorectal cancer,and the excessive expression of DcR3 in tumor tissues will hamper the combination of TL1 A with DR3,inhibit the body’s normal immune surveillance,inhibit the apoptosis of cancerous cells,and promote the occurrence and development of colorectal cancer.But the role of TL1 A in CAC has not yet been studied.The experiment uses lymphocytes over-expressed TL1 A transgenic mice and wild typy mice to establish a model of CAC,observe whether TL1 A is related to CAC.It also uses lentivirus technology to knockdown or increase the expression of TL1 A to observe the impact of TL1 A on the colorectal cancer cell line biological behavior and discuss the mechanism of the occurrence development of TL1A-associated CAC.Part I The role of TL1 A in CAC model in miceObjective:To investigate the effects of TL1 A in the studying whether TL1 A is involved in the occurrence and development of CACMethod:1.Using real-time Q-PCR method to extend TL1 A DNA fragment and agarose gel electrophoresis to identify CK-CD2-TL1A-GFP-transgenic mice.2.Dividing lymphocytes over-expressed TL1 A transgenic mice(Tg)and C57BL/6 wild type mice(WT)into the control group and AOM + DSS group by injecting AOM into the enterocoelia the mice and making them drink 2% DSS,with four groups in total and 10 for each group.Mice in the control group have normal diet,while the AOM+DSS group drink 2% DSS water for 7 days following intraperitoneal injection of AOM(12.5mg/Kg),and then drink distilled water for 14 days.This is a circle,and all of the mice will be killed in the 84 th day.3.Evaluation of colon inflammation in mice: including weight,DAI,colon length,changes of morphological colon and pathological grade.4.Evaluation of tumor formation rate in mice,including the number,diameter,and dysplasia.Results:1.By amplifying PCR in mice,the gel imaging shows a strong fluorescence signal in thevicinity of 192 bp,so they are transgenic mice.2.Compared with the mice in control group,the body weight of the Tg mice and WT mice from the AOM+DSS group reduced but DAI grade significantly increased.The AOM+DSS/Tg group has more serious inflammation than the AOM+DSS/WT group,with lighter weigh.The DAI score is increased(2.3±0.76 vs 1.28±0.47,P<0.05),and the length of the colon is shortened(7.00cm±0.97vs5.85±0.78 cm P<0.05).3.Compared with the mice in control group,in AOM+DSS group the size of cells is different,the level of the cells is disordered,the polarity disappeared,the goblet cells decreased,the nuclei increased,and the nuclear fission increased.The normal structure disappears and the abnormal hyperplasia is obvious.The AOM+DSS/Tg had a higher score of dysplasia than AOM+DSS/WT(2.75±0.5 vs 1.70±0.8,P<0.05).4.The AOM+DSS/Tg group compare with AOM+DSS/WT group,it has more colon tumors(3.50±1.19 vs 5.62±1.30,P<0.05)and has higher tumor formation rate(88.9% vs 66.7%).Part II The effect of TL1 A on the biological behavior of colorectal cancer cellsObjective:To observe the impact of TL1 A on colorectal cancer cell lines biological behavior by using lentivirus technology to knockdown or increase the expression of TL1 A and to observe the effect of TL1 A on colorectal cancer cell lines subcutaneous transplantation tumor by the nude mice subcutaneously tumor experimentMethod:1.The expression of TL1 A in colorectal cancer cell line detected by real-time PCR and select appropriate cell lines for cell functional experiment.2.Using lentivirus technology to transfect colorectal cancer cell lines and screening the efficiency of transfection by using real-time PCR.3.To verify the effect of TL1 A on the proliferation of colon cancer cell lines by CCK-8 and cell clone experiment.4.To observe the effect of TL1 A on the invasion and migration of colorectal cancer cell lines by transwell migration,invasion experiment and scratch experiment.5.To observe the volume of the transplanted tumor and evaluate the proliferation capacity of tumor tissues.by nude mouse subcutaneous planting tumor model.Results:1.Using Real-time PCR to test the expression of TL1 A in HCT15,SW480,DLD-1,LOVO,HT29 and HCT116.It shows that the expression of TL1 A in HCT116 and HT29 is relatively high,while lower in SW480.Therefore,the expression of TL1 A in HCT116 and HT29 knockdown and that in SW480 increased.2.TL1 A promotes the proliferation of colorectal cancer cellsThe CCK-8 confirms that while knockdown the expression of TL1 A,the proliferation ability of HCT116 and HT29 decreases(P<0.05),and that of SW480 increases after increasing the expression of TL1A(P<0.05).Cell cloning experiment also confirms that while knockdown TL1 A gene,the proliferation ability of HCT116 and HT29 decreases(P<0.05),and that of SW480 increases after increasing the expression of TL1A(P<0.05).3.TL1 A promotes the invasion and migration of colorectal cancer cellsThe transwell experiment show that the invasion and migration ability of HCT116 and HT29 decreases after knockdown the expression of TL1A(P<0.05),the invasion and migration ability of SW480 increases after increasing the expression of TL1A(P<0.05).The results of scratch test showed that the wound healing rate decreased significantly after knocking down the TL1 A gene(P<0.05),and the wound healing rate was significantly accelerated after SW480 overexpression of TL1 A gene(P<0.05).4.TL1 A promotes the growth of HCT116 cells transplanted subcutaneously in nude miceIn the experiment of subcutaneous tumor in nude mice,the average volume of subcutaneously transplanted tumor in TL1 A knockdown group is smaller than that in the control group(P<0.05).The average tumor volume of the group with knockdown TL1 A gene is smaller than that in the control group(P<0.05),and the Ki-67 test shows that the positive rate of Ki-67 in the group with LV-si-TNFSF15 is higher than the control group(P<0.05).Part III TL1 A promotes inflammation associated colorectal cancer through Wnt/β-catenin pathwayObjective: Discussing the possible molecular mechanism of TL1 A in promoting colorectal proliferation.Method:1.Using the intestinal structure of mice to detect immunohistochemistry and immunofluorescence to compare β-catenin and expression of the group with higher TL1 A and analyze the changes of the downstream target genes.2.To knockdown or highlight the expression of TL1 A in colorectal cancer cell lines by using lentivirus technology and using western blot method to find the changes of β-catenin and downstream target genes.3.Inhibiting the express of β-catenin to observe the effect of TL1 A on β-catenin and downstream target genes.Results:1.It shows that β-catenin in the AOM + DSS/Tg group is higher than that in the AOM + DSS/WT group(P < 0.05),and the expression of its downstre-am target genes c-myc,cyclin D1 also increases.2.After knockdown the expression of TL1 A in HCT116 and HT29,the expression of β-catenin,c-myc,cyclin D1 decreases(P < 0.05),but increases after overexpressing of TL1 A in SW480(P < 0.05).3.After knockdown the expression of TL1 A in HCT116,HT29 and using WIKI4,the expression of downstream target gene c-myc and cyclin D1 decreases.However,after highlighting the expression of TL1 A in SW480,the expression of c-myc,cyclin D1 rises and then decreases.(P < 0.05).The effect of TL1 A increasing c-myc,cyclin D1 can be offset by WIKI4.Conclusions:1.In the AOM+DSS model,the tumor rate of lymphocytes over-expressed TL1 A transgenic mice has more serious inflammation,higher score of dysplasia and more colon tumors and higher tumor formation rate,suggesting that TL1 A is involved in the occurrence and development of colorectal cancer.2 TL1 A can promote the proliferation,invasion and migration of colorectal cancer cell lines.3 TL1 A affect the occurrence and development of CAC through the Wnt/β-catenin pathway. |
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