| BackgroundHepatocellular carcinoma(HCC)is the fourth malignant tumor in the world with a high mortality rate,but the molecular mechanism of the development of HCC has not been systematically elucidated so far.Cytochrome P450 oxidase(CYP)2E1 is an important liver drug metabolism enzyme,mainly involved in the metabolism of precarcinogens and environmental toxins.Previous studies on the role of CYP2E1 in liver cancer mainly focused on the activation of procarcinogens such as ethanol,nitroso amine by CYP2E1,leading to gene mutation and cancer.However,with the progression of liver cancer,the expression of CYP2E1 in the cancer tissues gradually decreases,and the low expression of CYP2E1 is related to the malignant biological characteristics of liver cancer and the short survival time of patients.It is not clear whether CYP2E1 plays a specific biological role in the malignant progression of liver cancer.The occurrence of liver cancer is closely related to the dysregulation of various signal transduction pathways.Wnt/?-catenin signal transduction pathway is the most common dysregulated signal pathway in liver cancer,and the abnormal activation of the Wnt signal is closely related to the initiation,development,invasion,metastasis,and recurrence of liver cancer.Few studies have been reported on the relationship between CYP2E1 and Wnt/?-catenin signaling pathways.Objective1.The expression of CYP2E1 in liver cancer tissues and its effect on the malignant biological behavior of liver cancer cells;2.The mechanism of CYP2E1 inhibiting cell proliferation and the invasion and metastasis of liver cancer.Methods1 The expression of CYP2E1 in liver cancer tissues and its effect on the malignant biological behavior of liver cancer cells1.1 The expression levels of CYP2E1 mRNA and protein in liver cancer and para-carcinoma tissues were detected by qRT-PCR and Western Blotting.1.2 Mann-Whitney U test was used to analyze the relationship between the expression of CYP2E1 and the clinicopathological features of liver cancer.1.3 Kaplan-Meier method was used to analyze the influence of CYP2E1 expression on the overall survival of liver cancer patients.1.4 The lentivirus-mediated overexpression of CYP2E1 hepatocellular carcinoma cell lines was constructed,and the effects of CYP2E1 on cell migration,proliferation and invasion were detected by using scratch repair,CCK-8,and Transwell assay.1.5 Subcutaneous tumor formation,lung metastasis,and orthotopic liver transplantation tumor models were constructed in nude mice to study the effect of CYP2E1 on the proliferation,invasion,and metastasis of liver cancer cells in vivo.2 Mechanism of CYP2E1 inhibiting cell proliferation and the invasion and metastasis of liver cancer2.1 The effect of over-expression of CYP2E1 on the Wnt/?-catenin signaling pathway was investigated by using the dual-luciferase reporter gene and Western Blotting technique.2.2 The effects of CYP2E1 on ubiquitination degradation of Dvl-2 mediated by E3 ubiquitin ligase KLHL12 were studied by qRT-PCR,proteasome inhibitors,Western Blotting,in vitro ubiquitination experiment and Co-Ip technology.2.3 Fluorescence probe ROS detection kit,Western Blotting,Co-Ip technology,and antioxidants were used to explore the mechanism of CYP2E1 inhibited Wnt/?-catenin pathway through ROS.Results1 The expression of CYP2E1 in liver cancer tissues and its effect on the malignant biological behavior of liver cancer cells.1.1 The expression level of CYP2E1 in liver cancer tissues was significantly lower than that in para-cancer tissuesAmong the 81 paired samples,the transcription level of CYP2E1 in 74 HCC tissues was significantly lower than that in para-carcinoma tissues(P<0.0001),with a low expression rate of 91.36%.In the 36 pairs of paired samples,the expression level of CYP2E1 protein in liver cancer tissues was significantly lower than that in its paired para-cancer tissues,with a low expression rate of 77.8%.1.2 Low expression of CYP2E1 in liver cancer tissues is related to smoking,alcohol consumption,tumor diameter,vascular invasion and degree of differentiationThe effects of demographic factors and different pathological features on the expression level of CYP2E1 mRNA in liver cancer tissues were analyzed.The results showed that the expression level of CYP2E1 mRNA in liver cancer patients with smoking was significantly lower than that of non-smoking patients(P=0.033).The mRNA expression level of CYP2E1 in liver cancer patients with alcohol consumption was lower than that of those without alcohol consumption(P=0.020).The expression of CYP2E1 mRNA was significantly decreased in group with large tumor size(>5cm)than that in group with small tumor size(?5 cm)(P=0.049).CYP2E1 mRNA in the vascular invasion group was significantly down-regulated compared with that in the non-vascular invasion group(P=0.046).The expression level of CYP2E1 mRNA in the liver cancer tissues of the poorly differentiated group was lower than that of the moderately differentiated group(P>0.05),and the expression level of CYP2E1 mRNA in the moderately differentiated group was significantly lower than that of the highly differentiated group(P=0.006).1.3 Low expression of CYP2E1 in liver cancer tissues is associated with poor prognosis of patients1.4 Overexpression of CYP2E1 inhibits the in vitro migration,proliferation,and invasion of liver cancer cellsCCK-8 results showed that compared with the control group,the proliferation rate of MHCC-97H and SMMC-7721 cells with over-expression of CYP2E1 was significantly reduced.Transwell and scratch repair experiments showed that the invasion and migration ability of liver cancer cell lines with over-expression of CYP2E1 was significantly weaker than that of control cells.1.5 Over-expression of CYP2E1 inhibits the proliferation and invasion and metastasis of liver cancer cells in vivoSubcutaneous tumorigenesis results of nude mice showed that nude mice in the control group had macroscopic tumors within 5 days,while the MHCC-97H-CYP2E1 group had macroscopic tumors on the 17th day,and the tumor volume was significantly smaller than that of the control group.The lung metastasis model of nude mice which was observed with live animal imager shows that compared with the control group,the fluorescence intensity of the lungs in MHCC-97H-CYP2E1 and SMMC-7721-CYP2E1 groups decreased significantly.H&E staining of lung tissue sections showed that there was obvious lung metastasis of liver cancer cells in the lung tissues of nude mice in the control cell group,while almost no lung metastasis was found in nude mice in the CYP2E1 overexpression group.The results of the tumor model of orthotopic liver transplantation in nude mice showed that the liver fluorescence intensity of nude mice in the MHCC-97H-CYP2E1 group was significantly weaker than that in the control group.After taking tumor tissues,it was found that 3 nude mice in the overexpressed CYP2E1 group develop tumors(5 in total),and all 5 mice in the control group developed tumors,and the volume of liver tumors in the overexpressed CYP2E1 group was significantly smaller than that in the control group.2 Mechanism of CYP2E1 inhibiting cell proliferation and the invasion and metastasis of liver cancer2.1 Over-expression of CYP2E1 inhibits Wnt/?-catenin signaling pathwayThe TOPFlash dual-luciferase reporting experiment showed that compared with the control group,the luciferase activity of SMMC-7721 and MHCC-97H cells with overexpression of CYP2E1 significantly decreased(P<0.01).Western Blotting results showed that overexpression of CYP2E1 significantly reduced the protein expression levels of Dvl-2,?-catenin,TCF4,LEF1,and c-Myc,which were important members of the Wnt/?-catenin pathway.After transfection with the Dvl-2 expression plasmid,the cell proliferation capacity and protein expression level of the over-expressed CYP2E1 cells was significantly restored.2.2 CYP2E1 promotes the ubiquitination and degradation of Dvl-2Overexpression of CYP2E1 decreased the expression level of Dvl-2 protein but had no effect on mRNA expression level.Besides,western Blotting showed that proteasome inhibitor MG132 could significantly up-regulate the expression of Dvl-2,so it was speculated that Dvl-2 might be degraded by the proteasome pathway.In vitro ubiquitination experiments showed that the ubiquitination level of Dvl-2 in the CYP2E1 group was significantly increased.2.3 CYP2E1 enhances the interaction between KLHL12 and Dvl-2 to promote the ubiquitination of Dvl-2KLHL12 is an E3 ubiquitin ligase that degrades Dvl-2.The Co-Ip results showed that there was an interaction between Dvl-2 and E3 ubiquitin ligase KLHL12,and the binding amount of KLHL12 and Dvl-2 in the CYP2E1 overexpression group was significantly increased compared with the control group,indicating that CYP2E1 promoted the degradation of Dvl-2 by enhancing the interaction between KLHL12 and Dvl-2.2.4 CYP2E1 inhibits Wnt/?-catenin signaling pathway by ROSFluorescence probe results showed that,compared with the control group,overexpression of CYP2E1 significantly increased intracellular ROS levels.After treatment with CYP2E1 inhibitor(CMZ)and antioxidant(NAC),ROS content and important molecular protein expression of Wnt/?-catenin signal were found to be reversed.2.5 Over-expression of CYP2E1 inhibits EMT in liver cancer cellsAfter over-expressing CYP2E1 in MHCC-97H and SMMC-7721 cells,it was observed that the cell morphology changed from elongated to goose oval.EMT marker proteins E-cadherin,N-cadherin and Vimentin were detected,and it was found that E-cadherin expression increased and expression of N-cadherin and Vimentin are decreased in CYP2E1-overexpressed HCC cells.At the same time,the transcription factors Twist,Snail,and Slug,,which regulate EMT were detected,and it was found that compared with the control group,the protein expressions of Twist,Snail and Slug in overexpressed CYP2E1 HCC cells were significantly reduced.Conclusion1.Low expression of CYP2E1 in liver cancer tissues is associated with tumor diameter,vascular invasion,tumor differentiation and poor prognosis of patients.2.Over-expression of CYP2E1 inhibits the proliferation,invasion,and metastasis of liver cancer cells in vitro and in vivo.3.CYP2E1 inhibits Wnt/?-catenin signaling pathway by promoting the ubiquitination and degradation of Dvl-2.4.CYP2E1 regulatess the Wnt/?-catenin signaling pathway through ROS,thereby inhibiting the progression of liver cancer.Innovative1.CYP2E1 inhibits invasion and metastasis of liver cancer.2.CYP2E1 inhibits invasion and migration of liver cancer cells by regulating the Wnt/?-catenin signaling pathway through ROS.3.CYP2E1 regulates the ubiquitination and degradation of Dvl-2. |
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