| Part ?,Clinical significance of CYP1B1 in colorectal cancerObjective:To investigate clinical significance of CYP1B1 in colorectal cancerMethods:Choose the postoperative pathology of 231 patients to construct tissue microarrays,who were diagnosed colorectal cancer(CRC)at the Affiliated Hospital of Jiangnan University and underwent radical resection there.Immunohistochemistry staining was performed to examine the expression of CYP1B1 in tumor tissues and corresponding adjacent normal tissues and the most appropriate value for CYP1B1 score was obtained according to the Youden index so as to analyze the relationship between the expression of CYP1B1 and clinical and pathological factors.The Kaplan-Meier method was used to plot OS curve and the multivariate survival analysis was performed by Cox proportional hazards model to investigate the prognostic value of CYP1B1 in CRC.Results:The positive staining of CYP1B1 was predominantly localized to the cytoplasm of tumor cells according to immunohistochemistry results.Compared with corresponding adjacent normal tissues,the expression of CYP1B1 in CRC tissues was significantly increased,which shows statistical difference(P<0.01).The cutoff values defining the expression level of CYP1B1 were 4.75 by Youden index.We found that 92 cases had high expression of CYP1B1 and the expression of CYP1B1 was only related to gender,which shows statistical difference(P<0.05).According to the survival analysis,CYP1B1 is a poor prognostic factor for colorectal cancer.Patients with high expression of CYP1B1 had poor survival rate,which shows statistical difference(P<0.01).According to multivariate Cox regression analysis,the expression of CYP1B1(P<0.05)was one of the independent poor prognostic factors,which shows statistical difference.Conclusion:CYP1B1 is a poor prognostic factor for colorectal cancer.High expression of CYP1B1 is significantly associated with worse prognosis in CRC patients.The expression level of CYP1B1 is one of the independent poor prognostic factors.Part ?,To study the biological functions of CYP1B1 in colorectal cancerObjective:To investigate the effects of CYP1B1 on proliferation,migration and invasion,EMT and the correlation with the Wnt/p-catenin pathway in colorectal cancer cells.Methods:In different human colorectal cancer cell lines(SW480,SW620,HCT8,HCT116,CaCo-2,DLD-1,LOVO),Real-Time PCR and Western Blot were used to detect the mRNA and protein expression of CYP1B1.Two human CRC cell lines with relatively high expression of CYP1B1 would be screened as cell lines for subsequent experiments.In two human CRC cell lines with relatively high expression of CYP1B1,siRNA was used to down-regulate the expression of CYP1B1.1.Differences in OD at 450 nm were detected by CCK8 so as to detect the number of proliferation of CRC cells and differences in PCNA protein expression were tested by Western Blot;2.Scratch test and transwell assays were used to detect the migration and invasion ability of different cancer cells and Western Blot was used to detect the differences in the protein expression of MMP2 and MMP9;3.Western Blot was used to test the differences in the protein expression of E-cadherin,N-cadherin and Vimentin;4.Western Blot was used to test the differences in the protein expression of ?-catenin and CyclinDl in nuclear nucleus.XAV939 was used to inhibit the Wnt/?-catenin pathway in two CRC cell lines with relatively high expression of CYP1B1,Western Blot was used to test the differences in the protein expression of CYP1B1,E-cadherin,N-cadherin,Vimentin and ?-catenin,CyclinD1 in nuclear nucleus.Results:The results of Real-Time PCR and Western Blot showed that the mRNA and protein expression of CYP1B1 were relatively high in HCT8 and CaCo-2 cell lines.In human CRC HCT8 and CaCo-2 cell lines,1.CCK8 showed that through down-regulation of CYP1B1,the proliferation of colorectal cancer cells could be reduced,which shows statistical difference(P<0.05).Western Blot showed that the down-regulation of CYP1B1 could reduce the protein expression level of PCNA,which shows statistical difference(P<0.01).2.The scratch test and Transwell assays showed that down-regulation of CYP1B1 could weaken the migration and invasion ability of CRC cells,which shows statistical difference(P<0.05),and Western Blot showed that the protein expression level of MMP2 and MMP9 could be reduced after the down-regulation of CYP1B1,which shows statistical difference(P<0.01).3.Western Blot showed that down-regulation of CYP1B1 promoted the expression of E-cadherin,which shows statistical difference(P<0.01)and inhibited the expression of N-cadherin and Vimentin,which shows statistical difference(P<0.01).while the expression of Vimentin decreased with the down-regulation of CYP1B1,which shows statistical difference(P<0.01).4.Western Blot showed the protein expression level of ?-catenin and Cyclin D1 in the nucleus could be reduced after the down-regulation of CYP1B1,which shows statistical difference(P<0.01).Western Blot showed the protein expression levels of ?-catenin and Cyclin D1 in the nucleus decreased,which shows statistical difference(P<0.01),the expression of E-cadherin increased,which shows statistical difference(P<0.01),and the expression levels of N-cadherin and Vimentin decreased,which shows statistical difference(P<0.01)while the protein expression level of CYP1B1 did not change significantly after inhibiting the Wnt/?-catenin pathway in human CRC HCT8 and CaCo-2 cells,which shows no statistical difference(P>0.05).Conclusion:Down-regulation of CYP1B1 expression can inhibit the proliferation of colorectal cancer cells,weaken their migration and invasion ability and inhibit EMT in CRC cells.Down-regulation of CYP1B1 expression may affect EMT by inhibiting the Wnt/?-catenin signaling pathway in biological behavior.Part ?,To investigate the regulation mechanism of CYP1B1 in colorectal cancerObjective:To investigate the regulation mechanism of CYP1B1 in colorectal cancerMethods:Choose the postoperative pathology of 231 patients to continue to be further tested using tissue microarray technology who were selected in the first part of the study.Immunohistochemistry staining was performed to examine the expression of B7-H3 in tumor tissues and corresponding adjacent normal tissues and the most appropriate value for B7-H3 score was obtained according to the Youden index.We analyzed the relationship between the expression of CYP1B1 and B7-H3 in the pathological tissue samples of the same patient.The Kaplan-Meier method was used to plot OS curve and the multivariate survival analysis was performed by Cox proportional hazards model to investigate the prognostic value of combined expression of B7-H3 and CYP1B1 in CRC.Real-time and Western Blot were used to detected the difference in mRNA and protein expression of B7-H3 and CYP1B1 in B7-H3 high and low expression stably transformed human CRC cells(SW480-B7-H3/SW480-NC,CaCo-2-shB7-H3/CaCo-2-NC).After the expression of CYP1B1 was down-regulated by using RNAi in SW480-B7-H3-and CaCo-2-NC cells,Real-Time PCR and Western Blot were used to detect the differences in mRNA and protein expression of CYP1B1 and B7-H3.In SW480-B7-H3/SW480-NC,CaCo-2-shB7-H3/CaCo-2-NC cells,Western Blot was used to detect the differences in protein expression of B7-H3 and p-Akt.After using P13K-Akt inhibitor LY294002 to restrain PI3K-Akt signaling pathway and PI3K-Akt agonist IGF to activate PI3K-Akt signaling pathway in the human CRC SW480 cell lines,the differences in protein expression of CYP1B1,p-Akt,Akt and B7-H3 were tested by Western Blot.Results:Positive expression of B7-H3 was primarily localized to the cytoplasm and membrane of tumor cells.Compared with corresponding adjacent normal tissues,the expression of B7-H3 in CRC tissues was significantly increased,which shows statistical difference(P<0.01).The cutoff values defining the expression level of B7-H3 were 2.75 by Youden index.The expression of CYP1B1 was positively correlated with the expression of B7-H3 according to immunohistochemistry results.According to the survival analysis,Patients with high expression of both CYP1B1 and B7-H3 had the poorest OS compared to patients with low expression of both CYP1B1 and B7-H3 and other patients,including patients that were either CYP1B1-high and B7-H3-low or CYP1B1-low and B7-H3-high,which shows statistical difference(P<0.01).In SW480-B7-H3/SW480-NC and CaCo-2-shB7-H3/CaCo-2-NC cells,the results of Real-Time PCR and Western Blot showed that the mRNA and protein expression of CYP1B1 was positively correlated with B7-H3 expression.The down-regulation of CYP1B1 led to the decrease of its mRNA and the protein expression,which shows statistical difference(P<0.01),while the mRNA and protein expression of B7-H3 did not change significantly in SW480-B7-H3 and CaCo-2-NC cells,which shows no statistical difference(P>0.05).Western Blot analysis showed that the expression of p-Akt was positively correlated with the expression of B7-H3 in SW480-B7-H3/SW480-NC and CaCo-2-shB7-H3/CaCo-2-NC cells.In human CRC SW480 cell lines,Western Blot showed that B7-H3 could increase the protein expression of CYP1B1 and p-Akt,which shows statistical difference(P<0.01).PI3K-Akt pathway inhibitor LY294002 could reduce the expression of CYP1B1 and p-Akt,which shows statistical difference(P<0.01),while the expression level of B7-H3 and Akt had no significant changes,which shows no statistical difference(P>0.05).Western Blot showed PI3K-Akt pathway agonist IGF-1 could increase the expression of CYP1B1 and p-Akt,which shows statistical difference(P<0.01),while the expression level of B7-H3 and Akt had no significant changes,which shows no statistical difference(P>0.05).Conclusion:There is a positive correlation between the expression of CYP1B1 and B7-H3 both in vivo and in vitro in CRC.B7-H3 may regulate the expression of CYP1B1 via the PI3K-Akt signaling pathway in CRC.Full text conclusion:1.CYP1B1 is a poor prognostic factor for colorectal cancer.High expression of CYP1B1 is significantly associated with worse prognosis in CRC patients.The expression level of CYP1B1 is one of the independent poor prognostic factors.2.Down-regulation of CYP1B1 expression can inhibit the proliferation of colorectal cancer cells,weaken their migration and invasion ability and inhibit EMT in CRC cells.Down-regulation of CYP1B1 expression may affect EMT by inhibiting the Wnt/?-catenin signaling pathway in biological behavior.3.There is a positive correlation between the expression of CYP1B1 and B7-H3 both in vivo and in vitro in CRC.B7-H3 may regulate the expression of CYP1B1 via the PI3K-Akt signaling pathway in CRC. |
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