Mechanistic Characterization Of TLR9/NF-?B Regulating The Occurrence And Development Of Colitis And Colitis-associated Colorectal Cancer

Background and objective:Colorectal cancer(CRC,also called large bowel cancer)mainly includes colon cancer and rectal cancer,is one of the most common cancers of the digestive tract in the world.The latest epidemiological data indicated that CRC was ranked the fourth and second in global cancer morbidity and mortality,and the prevalence of this cancer is increasing year by year,with 1.1041 million new cases worldwide in 2018.Inflammatory bowel disease(IBD)is a chronic,high recurrence rate digestive tract autoimmune disease of unknown etiology,including ulcerative colitis(UC)and Crohn's disease(CD),is regarded as an important precursor to CRC.The longer the disease duration,the higher the risk of canceration.The incidence of IBD-associated CRC in patients with UC was reported as a cumulative risk rate of 2% at 10 years,8% at 20 years,and 18% at 30 years of disease duration.Chronic inflammation is the leading cause of immune cell infiltration and proliferation and is believed to be a high-risk factor related to colitis-associated colorectal cancer(CAC).Studies have confirmed that the sequence of “inflammation-dysplasia-carcinoma” evolves in IBD,but the mechanism of IBD's carcinogenesis is unclear and there is still no defined clinical monitoring protocol and prevention methods.Therefore,it is important to elucidate the pathogenesis of CAC and develop effective CRC prevention and treatment strategies.Toll-like receptor 9(TLR9),a member of the Toll-like receptor(TLR)family,is located in the cytoplasm and intracellular endosomes and can be activated by unmethylated bacterial Cp G DNA.Activation of the TLR9 signalling pathway induces a Type 1 T helper(Th1)immune response and stimulates the proliferation of B lymphocytes,thus protecting the host from external microbial invasion.Various studies have revealed that abnormal TLR9 expression might be involved in the pathogenesis and progression of UC.In addition,abnormal expression of TLR9 also occurs during tumorigenesis and the development of colorectal cancer.The mechanism of TLR9's involvement in IBD and CRC and whether TLR9 is involved in IBD's “inflammation-dysplasia-carcinoma” process are not currently clear.Nuclear factor kappa light chain enhancer of activated B cells(NF-?B)is a transcription factor involved in various biological processes,such as inflammatory reactions,immune responses,apoptosis and proliferation.It is regarded as a molecular hub linking inflammation and cancer.NF-?B may play an important role in colorectal carcinogenesis by regulating matrix metalloproteinase-9(MMP-9).A variety of protein kinases or receptors are involved in the regulation of NF-?B,such as AMP-activated protein kinase(AMPK),Toll-like receptor,TNF receptor,etc.TLR9 might regulate the expression of interleukin(IL)-6,depending on My D88/NF-?B signalling.However,whether TLR9 regulates NF-?B in CRC and its related mechanisms have not been elucidated.Therefore,this project intends to explore the role of TLR9 in the development of colitis-colorectal cancer and its relationship with NF-?B via in vivo and in vitro studies,which may provide a new theoretical basis for the pathogenesis of colorectal cancer,especially colitis-associated colorectal cancer,offer early detection protocols or novel therapeutic molecular targets.Part I:TLR9 expression in IBD and human coloreatal cancer Objective : To detect the expression of TLR9 in IBD and human colorectal cancer,and to evaluate its correlation with clinical pathological features.Methods : 1.The colorectal lesion tissue and paralesional tissue from IBD patients(25 cases)were collected.The expression of TLR9 in IBD lesion tissue and paralesional tissue was detected by immunohistochemistry.2.The colorectal cancer and adjacent tissue samples(49 pairs)of different pathological stages were collected.The expression of TLR9 in colorectal cancer and adjacent tissue was detected by immunohistochemistry.Clinicopathological association of TLR9 expression in CRC patients was analyzed.Results:1.Immunohistochemical data showed that there was no significant statistical difference in the expression of TLR9 in IBD lesion tissue and paralesional tissue(P >0.05).While,the expression of TLR9 in 71.4%(35/49)of colorectal cancer tissue was significantly higher than that of adjacent normal tissue(P < 0.05).2.The expression of TLR9 protein with patients age,sex ratio,tumor site and degree of tumor differentiation were not significantly related(P > 0.05).3.TLR9 protein expression was related with tumor lymph node metastasis.TLR9 expression in colorectal cancer with lymph node metastasis was significantly higher than that in no lymph node metastasis group(P < 0.05).4.TLR9 protein expression was related with tumor TNM stage.TLR9 protein expression rate in patients with advanced(stage III-IV)colorectal cancer was significantly higher than that in patients with early(stage I-II)colorectal cancer(P < 0.05).Conclusions : 1.TLR9 was up-regulated in colorectal cancer tissues,which was significantly different from that in adjacent tissues(P < 0.05).2.The TLR9 expression was related to lymph node metastasis and TNM staging in colorectal cancer patients,but no related to age,gender,site of onset,and tumor differentiation degree.Part II:Construction of colitis-related carcinogenesis animal model and the expression and correlation of TLR9 and NF-?B in colitis-carcinogenesisObjective : To construct a colitis-related carcinogenesis animal model and to investigate whether TLR9 was involved in the occurrence of colitis-related carcinogenesis,the expression of NF-?B and the correlation.Methods : 1.BALB/c mouse colitis-related carcinogenesis animal model was induced by AOM/DSS.The body weight,disease activity index,large intestine length,inflammation index and the number of tumors were recorded.2.The colorectal mucosal lesions in mice was detected by H&E method.The expression of TLR9,NF-?B and Ki67 in colorectal mucosal tissues were detected by immunohistochemistry,and the correlation was analyzed.Results: 1.We constructed a BALB/c mouse colitis-related carcinogenesis animal model induced by AOM/DSS.The modeling time was 23 weeks.The average weight of mice in the model group(AOM + DSS)and DSS group decreased with time.The average weight of the mice in the model group decreased from 21.6 ± 1.23 g at the beginning of the experiment to 19.5 ± 0.7 g at week 23(P < 0.05),the average weight of DSS mice decreased from 21.6 ± 1.3 g at the beginning of the experiment to 20.8 ±0.8 g at week 23(P < 0.05),and the DAI index,which reflects the severity of colitis,increased significantly.2.The lengths of the large bowels in model group and C were shorter than those in the other groups,which could be clearly seen starting in the 3rd week after treatment(P < 0.05).However,the severity and extent of inflammation were increased in model group compared with those of DSS group in the later stages.3.After the 12 th week,colorectal tumours were observed only in the mice of model group.Pathological results further revealed that the mice in model group presented with acute inflammation(AIF),chronic inflammation(CIF),adenoma and adenocarcinoma of the colorectum at weeks 3,6,12 and 18,respectively.4.TLR9 was significantly upregulated in AIF,CIF,adenoma and adenocarcinoma tissues compared with that of corresponding tissues from normal control mice(P < 0.05).Furthermore,the protein expression of TLR9 was significantly upregulated in adenocarcinoma compared with that in acute inflammation,chronic inflammation and adenoma(P < 0.05).5.The IHC results showed that the expression of NF-?B was apparently higher in the AIF,CIF,adenoma and adenocarcinoma tissues than in the corresponding tissues from normal control mice(P < 0.05).Moreover,NF-?B was significantly elevated in adenocarcinoma compared with that in AIF and adenoma tissues(P < 0.05).6.The IHC results showed that Ki67 expression gradually increased in the intestinal lesions,including in AIF,CIF,adenoma,and adenocarcinoma tissues(P < 0.05),compared with the expression in corresponding tissues from normal control mice.7.The expression levels of TLR9 and NF-?B were significantly positively correlated(rho = 0.8236,P < 0.001).In addition,there was also a significant positive correlation between TLR9 and Ki67 expression(rho =0.5515,P < 0.0001)as well as between NF-?B and Ki67 expression(rho = 0.5103,P< 0.01).Conclusions : 1.We have successfully established a colitis-related carcinogenesis animal model.2.TLR9 and NF-?B were involved in the development of colitiscolitis-related carcinogenesis.3.There was a correlation between TLR9 and NF-?B in the process of colitis-colitis-associated colorectal cancer.Part III:The role of TLR9 in colorectal cancer in vitroObjective : To investigate the expression of TLR9 in human colorectal cancer cell lines and to explore the effect of TLR9 on the biological behavior of human colorectal cancer cells and the related mechanism.Methods : 1.The expression of normal intestinal mucosa epithelial cells FHC and colorectal cancer cell lines SW620,Caco2,HCT116,HT29 and Lovo was detected by Western blot.2.The effects of chloroquine,an inhibitor of TLR9 and transfection of TLR9 interfering plasmid si RNA-TLR9 on proliferation,migration ability and clone formation ability of colorectal cancer cell lines SW620,HT29 were detected by MTT method,cell scratch test and clone formation experiment.3.The expression of TLR9,MMP9,PCNA and Bcl-xl protein in colorectal cancer SW620 or HT29 cells treated with chloroquine and si RNA-TLR9 was detected by Western blot.Results:1.Western blot data indicated that TLR9 protein expression was present in normal intestinal mucosal epithelial cells FHC and colorectal cancer cell lines SW620,Caco2,HCT116,HT29,and Lovo,and the expression of TLR9 in each colorectal cancer cell line was significantly higher than that of FHC(P < 0.05).In colorectal cancer cell lines,HT29 and SW620 had higher TLR9 expression levels.2.MTT assay,cell scratch test and clone formation experiment indicated that chloroquine,an inhibitor of TLR9,could inhibit the proliferation,migration and colony forming ability of HT29 and SW620 cells in a dose and time dependent manner.3.MTT assay,cell scratch test and clone formation experiment indicated that transfection of TLR9-interfering plasmid si RNA-TLR9 can inhibit the proliferation,migration and clone formation ability of SW620 cells.4.Western blot results showed that the expression of TLR9,MMP9,PCNA and Bcl-xl protein decreased in SW620 and HT29 cells treated with chloroquine.5.Western blot data showed that transfection of SW620 cells with si RNA-TLR9 reduced the expression of TLR9,MMP9,PCNA,and Bcl-xl proteins in a dose-dependent manner.Conclusions : 1.TLR9 may promote the proliferation,migration and colony formation of colorectal cancer.2.TLR9 is a potential target for colorectal cancer treatment.Changing TLR9 expression can change the downstream target genes MMP-9,PCNA and Bcl-xl.Part IV:The mechanism of TLR9 affecting biological behavior of colorectal cancer cells by regulating NF-?BObjective: To investigate the mechanism of TLR9 regulating NF-?B in colorectal cancer and to screen TLR9 related genes and tumor pathways in colorectal cancer by transcriptome sequencing.Methods : 1.The expression of NF-?B,My D88,p-Akt,and p-ERK1/2 in colorectal cancer cells SW620 or HT29 treated with chloroquine and si RNA-TLR9 were detected by Western blot.2.The interaction of TLR9 and NF-?B was detected by co-immunoprecipitation method.3.The localization of TLR9 and NF-?B in colon cancer cells and the expression of NF-?B after intervention with TLR9 were detected by immunocytochemistry.4.Total RNA of SW620 cells transfected with si RNA-TLR9 and si RNA negative control was extracted,and Illumina(Hi Seq 2500/4000)sequencing was performed to detect differentially expressed genes and GO and KEGG functions and enrichment analysis were performed on the differentially expressed genes.Results : 1.Western blot results indicated that the expression of NF-?B,My D88,p-Akt,and p-ERK1/2 decreased after treatment of SW620 or HT29 cells with chloroquine in a dose-and time-dependent manner.2.Western blot results showed that after transfection of si RNA-TLR9,the expression of NF-?B,My D88,p-Akt,and p-ERK1/2 in SW620 cells decreased in a dose-dependent manner.3.The results of co-immunoprecipitation showed that TLR9,NF-?B and My D88 existed as protein complexes in SW620 cells.4.Immunocytochemical experiments showed that TLR9 and NF-?B co-localized in SW620 cells.The expression of NF-?B in cytoplasm decreased after transfection of si RNA-TLR9.5.85 differentially expressed genes were screened by transcriptome sequencing,including 47 down-regulated genes and38 up-regulated genes;6.GO function analysis: differentially expressed genes involved in biological process mainly participate in cellular process,biological regulation and metabolism;differentially expressed genes involved in cellular component mainly participate in cell,cell parts and organelle;differentially expressed genes involved in molecular function mainly participate in binding,catalytic activity and transcription regulator activity.7.KEGG analysis: KEGG pathway classification and enrichment analysis showed that these differentially expressed genes participate in signal transduction pathway including MAPK signaling pathway and estrogen signaling pathway,longevity regulatory pathway.The main cellular processes involved by differentially expressed genes are cell transport and catabolism,cell growth and death.Conclusions:TLR9 interacts with NF-?B,and TLR9 may acts directly on NF-?B to activate downstream signals,thus affect the development of colorectal cancer.The GO and KEGG function and enrichment analysis of differential genes showed that these genes can participate in specific functions and signal transduction pathways,suggesting that TLR9 has a unique pathogenic role in the development of colorectal cancer.Transcriptome high-throughput sequencing provides an experimental basis and theoretical basis for further research on the mechanism of TLR9 in colorectal cancer.