| Background and purposeHepatocellular carcinoma(HCC)is one of the malignant tumors that seriously threaten human health,its occurrence and development is related to multiple factors,stages,steps and abnormal changes of various genes,the specific molecular mechanism has not been elucidated so far.Cytochrome P450 oxidase(CYP)1A2 is an important liver drug metabolism enzyme.At present,the research of CYP1A2 mainly focuses on the metabolism of pre-carcinogens and gene polymorphism,there are few studies on its role in tumorigenesis and development.Our previous studies found that the expression of CYP1A2 in liver cancer tissues was significantly decreased,and its low expression was related to the malignant biological characteristics of liver cancer,but whether CYP1A2 is involved and through what mechanism in the progression of liver cancer is still unclear.Reactive oxygen species(ROS)is a regulatory molecule for cell signal transduction and gene expression.It has a regulatory effect on cell growth,proliferation,migration,differentiation and apoptosis.It has always been considered that ROS is an important factor in inducing tumors.Recent studies have found that ROS exerts anti-tumor effects by inducing apoptosis.Studies have shown that CYP is involved in oxidative stress and is one of the important sources of ROS,but there are few reports about the role of CYP1A2 and ROS in liver cancer.The subject mainly studied:(1)The expression and clinicopathological significance of CYP1A2 in human liver tissues;(2)The effect of CYP1A2 on the malignant biological behavior of liver cancer cells and its mechanism.Methods1 The expression and clinicopathological significance of CYP1A2 in human liver tissues1.1 RT-q PCR and Western Blotting were used to detect the expression levels of CYP1A2 m RNA and protein in paracarcinoma and tumor tissues.1.2 Mann-Whitney U test was used to analyze the relationship between the expression of CYP1A2 and the clinicopathological characteristics of liver cancer.2 The effect of CYP1A2 on the malignant biological behavior of liver cancer cells and its mechanism2.1 Construction of lentivirus-mediated CYP1A2 overexpression liver cancer cell lines.2.2 The effects of CYP1A2 on the proliferation,migration and invasion of liver cancer cells in vitro were detected by using CCK-8,clone formation,scratch and transwell assay.2.3 The effect of CYP1A2 on the expression level of Gsk3β-Axin-JNK signal axis proteins was detected by Western Blotting.2.4 The fluorescent probe DHE was uesd to detected the effect of CYP1A2 on the content of ROS in cells.2.5 CYP1A2 inhibitor fluvoxamine and antioxidant NAC were used to explore the mechanism of CYP1A2 mediating Gsk3β-Axin-JNK signal axis and EMT through ROS.Results1 The expression and clinicopathological significance of CYP1A2 in human liver tissues1.1 Expression of CYP1A2 m RNA and protein in human liver tissue samplesAmong the 88 paired samples,the expression level of CYP1A2 m RNA in 80 tumor tissues was lower than that in paracarcinoma tissues(P<0.0001),and the low expression rate was 90.9%.Among the 36 paired samples,the expression of CYP1A2 protein in 33 tumor tissues was lower than that in paracarcinoma tissues,and the low expression rate was 90.5%.1.2 The expression level of CYP1A2 m RNA in tissue samples from patients with different types of liver cancerAccording to clinical diagnosis,100 cases human liver tissue samples were divided into 5 categories,including 74 cases of HBV-related hepatocellular carcinoma(HBV-HCC),10 cases of HBV-related recurrent hepatocellular carcinoma(HBV-RHCC),4 cases of non-HBV-related primary hepatocellular carcinoma(HCC),7 cases of primary cholangiocarcinoma(Intrahepatic cholangiocarcinoma,ICC)and 5cases of metastatic liver cancer(MLC).In tumor tissues of HBV-HCC patients,the expression level of CYP1A2 m RNA was lower than that in paracarcinoma tissues(P=0.0142),64 cases of low expression in 71 paired samples,and the low expression rate was 90.1%.In tumor tissues of HBV-RHCC patients,the expression level of CYP1A2 m RNA was significantly lower than that in paracarcinoma tissues(P=0.002),7 paired samples were all low-expressed.All 4 samples of HCC patients were paired,the expression level of CYP1A2 m RNA in tumor tissues was significantly lower than that in paracarcinoma tissues(P=0.021).The expression level of CYP1A2 m RNA in ICC and MLC samples was lower than that in paracarcinoma tissues,but it was not statistically significant difference(P>0.05).1.3 The expression level of CYP1A2 m RNA in tissue samples from HBV-HCC patients with different pathological characteristicsAnalysis of the expression of CYP1A2 m RNA in tumor and paracarcinoma tissues of 74 HBV-HCC patients found,tumor number,tumor size,envelope integrity,metastasis,vascular invasion,embolus,cirrhosis grade,tumor differentiation,AFP,CEA and CA199 all affect the expression level of CYP1A2 m RNA,the expression level of CYP1A2 m RNA in tumor tissues was significantly lower than that in paracarcinoma tissues(P<0.01).2 The effect of CYP1A2 on the malignant biological behavior of liver cancer cells and its mechanism2.1 CYP1A2 overexpression inhibits the proliferation,migration and invasion of liver cancer cells in vitroThe CCK-8 assay was used to detect the proliferation ability of the cells,compared with the GFP group,the proliferation ability of CYP1A2 overexpression group was reduced.The clone formation assay was used to detect the clone formation ability of the cells,compared with the GFP group,the clone formation ability of CYP1A2 overexpression group was reduced.The scratch assay was used to detect the migration ability of the cells,compared with the GFP group,the migration ability of CYP1A2 overexpression group was slower.The transwell assay was used to detect the invasion ability of the cells,compared with the GFP group,the invasion ability of CYP1A2 overexpression group was weakened.2.2 Overexpression of CYP1A2 activates Gsk3β-Axin-JNK signal axisWestern Blotting was used to detect the expression level of cellular pathway proteins.The results showed that compared with GFP group,the expression of Gsk3β-Axin-JNK signal axis proteins p-Gsk3β,Axin,MEKK1,MKK7,p-JNK and p-c-Jun was increased in CYP1A2 overexpression group(P<0.05).After treatment with furafylline,compared with the CYP1A2 overexpression group,the ability of cells proliferation,migration and invasion was recovered,and the expression of signal axis proteins was reversed(P<0.05).2.3 CYP1A2 regulates Gsk3β-Axin-JNK signal axis through ROSFluorescence microscope and flow cytometry were used to detect the ROS content of the cells.The results showed that compared with the GFP group,the ROS content of CYP1A2 overexpression group increased significantly(P<0.05).After treatment with NAC,compared with the CYP1A2 overexpression group,the content of ROS decreased,the ability of cell proliferation,migration and invasion was recovered,and the expression of signal axis proteins was reversed(P<0.05).2.4 CYP1A2 regulates EMT of liver cancer cells through ROSCompared with the GFP group,the cell morphology changed from elongated to oval after CYP1A2 overexpression,the protein expression of epithelial marker E-cadherin was increased,the proteins expression of mesenchymal markers N-cadherin,Vimentin and the key transcription factors Twist,Slug and Snail was decreased(P<0.05).After treatment with furafylline and NAC,compared with the CYP1A2 overexpression group,the expression of EMT-related proteins was reversed(P<0.05).Conclusion1.The low expression of CYP1A2 in human liver cancer tissues is related to the clinicopathological characteristics such as tumor size,vascular invasion and differentiated degree.2.CYP1A2 has diagnostic value for liver cancer with normal level of AFP,CEA and CA199,and the combined application may have a better diagnostic complementarity.3.Overexpression of CYP1A2 inhibits the proliferation,clone formation,migration and invasion of liver cancer cells in vitro.4.Overexpression of CYP1A2 activates Gsk3β-Axin-JNK signal axis by increasing the level of ROS in liver cancer cells,while inhibiting EMT,thereby inhibiting the progression of liver cancer. |
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