| Purpose and SignificanceColorectal cancer is one of the most common malignant tumors of digestive tract in our country,the incidence and mortality of colorectal cancer were 23.03/10million and 11.11/10 million,in recent years,with the accelerated pace of life,diet and environment changes,there is an rising trend in the incidence of colorectal cancer in China.The formation of colorectal cancer is a complex process of long term,multiple attacks and multiple gene mutations.The complexity of the mechanism of the occurrence and development of colorectal cancer determines the individualized treatment demand and prognosis evaluation.Restricted by social economic development and diagnosing methods,many patients did not get individualized and accurate treatment,benefit less and missed the best time for treatment,which increased the burden on the family and wasted of social costs.Therefore,it is of great clinical significance and social benefits to find the key genes in the development of colorectal cancer,and to study its functional mechanism,to establish a simple and convenient individualized treatment and prognosis evaluation model.SETDB1 gene located on human chromosome 1q21.3,a length of about 38.6Kb,encoding SETDB1 protein,mainly involved in the process of trimethylated histone H3K9,usually caused gene silencing or transcription inhibition.SETDB1 protein was found early to treat Huntington disease.In recent years,it has been found that the abnormal expression of SETDB1 protein is closely related to the occurrence and development of many tumors.In neuroblastoma,The SETDB1 protein effect the proliferation and differentiation of neuroblastoma cells by maintaining the "STEM" of tumor cells,in prostate cancer,the expression of SETDB1 protein has obvious differences in hormone dependent,and can affect prostate cancer cell proliferation,differentiation and apoptosis.At present At present,there is no study on the expression of SETDB1 protein in colorectal cancer,our previous results found that SETDB1 protein had high expression in colorectal cancer,suggesting that the expression of SETDB1 is associated with the occurrence and development of colorectal cancer,but its molecular mechanism is still not clear.NOTCH1 gene mapped to human chromosome 9q34.3,a length of about 51.4Kb,encoding NOTCH1 protein,was one of the receptor in NOTCH signaling pathway.Activated NOTCH1 protein was involved in many types of cells differentiation,proliferation and apoptosis.Abnormal activation of NOTCH pathway was also involved in the occurrence and development of a variety of tumors.The earliest evidence for abnormal activation caused by mutations in the NOTCH1 gene was acute lymphocytic leukemia T cells,and subsequent studies had found that abnormal activation of NOTCH was an important factor in the occurrence and development of breast cancer,colorectal cancer,pancreatic cancer and other solid tumors.NOTCH gene could be used as target for tumor therapy.The activation of NOTCH pathway cuold also inhibit the process of tumor formation.Results of further study suggested that this inhibitory effect in colorectal cancer may be associated with histone methylation inhibition and interaction between NOTCH/WNT pathway and other pathways,but the molecular mechanism is not clear.Therefore,this study will further investigate the expression of SETDB1 and NOTCH1 protein in colorectal cancer tissues and explore the molecular mechanism of its function to provide theoretical basis for individualized treatment of colorectal cancer.The objectives of the study are as follows:1.Detection of SETDB1 expression in colorectal cancer tissues and cells,and analyze its relationship with clinical prognosis,2.To investigate the expression of SETDB1 in colorectal cancer and to explore the molecular mechanism of SETDB1 in the carcinogenesis and progression of colorectal cancer,3.The co-expression of NOTCH1 and SETDB1 was detected and the relationship between the expression and clinical prognosis was analyzed,4.To investigate the different expression state of NOTCH1 and SETDB1 in colorectal cancer and to explore its molecular mechanism in the development and progression of colorectal cancer.Materials and Methods1.The expression of SETDB1 protein in colorectal cancer cell lines,fresh tissues and paraffin sections was detected by Western blot,real-time fluorescence quantitative PCR and immunohistochemistry,and the relationship between SETDB1 expression and prognosis was analyzed;2.The colorectal cancer cell lines with SETDB1 protein stable overexpression and interference were established.Using CCK8,colony formation assay and Xenograft model in nude mice detected the proliferation of established cell lines in vivo and in vitro,Cell scratch assay and transwell assay was performed to detect changes of migration and invasion in established cell lines in vitro,the changes ofapoptosis and cycle in established cell lines were detected by flow cytometry;3.The colorectal cancer cell lines were induced apoptosis by 5-fluorouracil(5-Fu),and using flow cytometry assay detected changes of anti-apoptotic ability in SETDB1 interference and overexpression cell lines,the changes of apoptosis related genes(TP53?BCL2?BAX)were detected by western blot,the promoter SETDB1 binding sites of TP53 gene were detected by CHIP-PCR;4.The expression of SETDB1 protein and cNOTCH1 protein in colorectal cancer cell lines and paraffin sections was detected by Western blot and immunohistochemistry,and the relationship between SETDB1 and cNOTCHl co-expression and prognosis was analyzed;5.The cell lines with different SETDB1 protein expression were treated by NOTCH1 activator(doxycycline)or inhibitor(gamma secretase inhibitor,DAPT),and the changes of proliferation in vitro were measured by CCK8 and clone formation assay,cell scratch assay and transwell assay was performed to detect changes of migration and invasion ability in treated cell lines;6.Gene chip technique was used to investigate the changes of genes in NOTCH activated colorectal cancer cell lines after SETDB1 interference,Western blot technology was used to verify the expression of STAT1.Result1.The expression of SETDB1 protein in colorectal cancer1.1 Western blot,real-time fluorescence quantitative PCR results showed that:compared with the normal colonic epithelial cell line(NCM460 cell line),the expression of SETDB1 in colon cancer cell lines were higher,the expression level of SETDB1 protein in 8 cases of colorectal cancer tissues was higher than that of matched adjacent tissues;1.2 The results of immunohistochemistry showed that SETDB1 positive expression was mainly localized in the nucleus,showed different degrees of brown.In 46.7%(14/30)colorectal cancer tissues showed strong positive expression,among them,the strong positive ratio of high differentiation group reached 55%,and the positive proportion of low differentiation group was 30%;1.3 The expression of SETDB1 protein was detected in 102 cases of colorectal cancer,and the survival analysis was measured by Kaplan-meier method.The results showed that the overall survival rate of SETDB1 expression in colorectal cancer tissues was significantly higher than that in the high expression group,and the difference was statistically significant(p<0.05).2.The functional role of SETDB1 in colorectal cancer in vitro and in vivo2.1 The SETDB1 over-expression and interference lentiviral vectors were successfully constructed.Useing these lentiviral vectors,SETDB1 overexpression and interference stable colorectal cancer cell lines were established,and the over-expression and interference efficiency were detected by Western blot,fluorescence real-time quantitative PCR;2.2 Overexpression of SETDB1 significantly increased the proliferation and invasion ability of colorectal cancer cells.CCK8 assay showed that the cell growth speed in SETDB1 over-expression group was significantly faster than the control group(P<0.05),colony formation assay showed that the cell clone formation rate of SETDB1 overexpression group was higher than that of control group(P<0.05),the results of subcutaneous tumor formation experiment showed that,compared with the control group,the xenografts of SETDB1 over-expression group grew faster,larger(P<0.05),Transwell assay results showed that the number of pass-through cells in SETDB1 over-expression group was higher than that of control group(P<0.05),cell scratch test showed that the migration ability of cells in SETDB1 over-expression group was stronger than the control group(P<0.05);2.3 The proliferation and invasion ability of SETDB1 knockdown colorectal cancer cell decreased significantly.CCK8 assay showed that the cell growth speed in SETDB1 interference group was significantly slower than the control group(P<0.05),colony formation assay showed that the cell clone formation rate of SETDB1 interference group was lower than that of control group(P<0.05),the results of subcutaneous tumor formation experiment showed that,compared with the control group,the xenografts of SETDB1 interference group grew slower,smaller(P<0.05),Transwell assay results showed that the number of pass-through cells in SETDB1 interference group was lower than that of control group(P<0.05),cell scratch test showed that the migration ability of cells in SETDB1 interference group was weaker than the control group(P<0.05).3.Effect of SETDB1 expression on apoptosis of colorectal cancer cells3.1 Compared with the control group,the apoptosis rate of SETDB1 overexpression and interference group were not significantly changed(P>0.05).The cells in the SETDB1 interference group had significant G2 phase arrest,compared with the control group,and the cell cycle of the SETDB1 over-expression group did not change significantly;3.2 After 5-Fu induced apoptosis,the apoptosis rate of SETDB1 over-expression group was significantly lower than that of control group(P<0.05),accordingly,the expression of TP53 protein was less than that of the control group,while the interference group was significantly increased(P<0.05),and the expression of TP53 increased significantly.BAX and BCL-2 did not change significantly;3.3 CHIP-QPCR results showed that there were SETDB1 binding sites in TP53 promoter region.4.Proliferation and invasion ability of SW480 cells significantly increased after SETDB1 interference.CCK8 assay showed that the cell growth speed in SETDB1 interference group was significantly faster than the control group(P<0.05),colony formation assay showed that the cell clone formation rate of SETDB1 interference group was higher than that of control group(P<0.05).5.The co-expression of SETDB1 and cNOTCH1 in colorectal cancer5.1 The expression of cNOTCHl was detected in 102 cases paraffin sections with the same sample of SETDB1 expression.The results showed that the number of positive cases of SETDB1 was 79 cases,and the number of positive cases of cNOTCH1 was 47 cases,accounting for 46.08%in all cases,and there was a negative correlation between cNOTCHl expression and prognosis;5.2 According to the expression of two proteins in colorectal cancer cases,we divided all cases into 4 group,SETDB1(+)cNOTCH1(+),SETDB1(+)cNOTCH1(-),SETDB1(-)cNOTCH1(+),SETDB1(-)cNOTCHl(-),and then observe the relationship between expression and with clinical prognosis.The results showed that SETDB1(+)cNOTCHl(-)and SETDB1(-)cNOTCH1(-)were negatively correlated with prognosis;5.3 DLD1 cell line was expressed as SETDB1(-)cNOTCHl(-),and SW480 cell line was expressed as SETDB1(+)cNOTCH1(+).6.Changes of cell proliferation and migration ability after NOTCH1 activator(doxycycline)and inhibitor(gamma secretase inhibitor,DAPT)treatment6.1 The application of NOTCH1 activator(doxycycline)increased expression of intracellular HES1 in treated cells.In DLD1 cells,CCK8 and clone formation assay showed that the growth rate and the number of colony formation in treated group higher than that in untreated group(P<0.05),while DLD1-SETDB1 cell line showed reverse results.In DLD1 cells,compared with the untreated group,transwell assay and cell scratch test showed that the migration ability of DLD1 cells was significantly increased,while DLD1-SETDB1 cells showed reverse results;6.2 The application of NOTCH1 inhibitor(gamma secretase inhibitor,DAPT)decreased the expression of HES1 in treated cells.In SW480 cells,CCK8 and clone formation assay showed that the growth rate and the number of colony formation in treated group higher than that in untreated group(P<0.05),while SW480 interference cells showed reverse results.In SW480 cells,compared with the untreated group,transwell assay and cell scratch test showed that the migration ability of SW480 cells was significantly increased,while SW480 interference cells showed reverse results.7.Gene chip detectionCompared with the control group,279 genes were up-regulated and the expression of the 204 genes was down regulated in the SW480 SETDB1 interference cell line.8.The expression of STAT1 in SW480 SETDB1 interference cellsThe results of Western blot showed that the expression of STAT1 were significantly down regulated after SETDB1 interference in SW480 cells.Conclusion1.SETDB1 was up-regulated in colorectal cancer tissues and cells,and its expression level was correlated with clinical prognosis;2.Overexpression of SETDB1 significantly increased the proliferation,invasion and migration of colorectal cancer cells.The proliferation and migration of colorectal cancer cells were significantly decreased after SETDB1 interference;3.The expression of SETDB1 decreased the expression of TP53 in colorectal cancer cells and promoted the development of colorectal cancer;4.The expression of SETDB1 is influenced by the activation of NOTCH1 protein,and the combination expression of the two proteins can influence the proliferation and invasion of colorectal cancer cells;5.Under the condition of NOTCH1 protein activation,SETDB1 inhibited the invasion of colorectal cancer cells by up-regulating the expression of STAT1. |
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