A Study On The Correlation Between CBX4 And The Prognosis Of AFB₁-related Hepatocellular Carcinoma

Objective:Aflatoxin B₁（AFB₁）is the most common carcinogen from aflatoxins,which mainly induces human hepatocellular carcinoma（HCC）.Epidemiological evidence has shown that AFB₁displays its carcinogenic roles by contaminating agricultural products and next entering human bodies.Mechanically,AFB₁is firstly transferred into exo-AFB₁（AFBO）by I-type detoxification enzyme cytochrome P450 in liver;whereas AFBO can bind to DNA and results in the formation of AFB₁-DNA adducts.Although the formation of AFB₁-DNA adducts is an important step of AFB₁-induced hepatocarcinogenesis,detailed mechanisms have been unclear.Recently,studies have shown that Chromo box 4（CBX4）,an important member of poly-comb repressive complex1（PRC1）,displays a vital role of the occurrence and development of HCC by regulating transcription and post-translational modifications.Our studies found that CBX4 has a role in regulating angiogenesis of HCC,but it has not been clear whether CBX4 is related to the prognosis of HCC associated with AFB₁.Therefore,this degree project aimed to explore whether CBX4 affects the prognosis of AFB₁-related HCC.Methods:（1）This project is a retrospective investigative study conducted in the high exposure areas of AFB₁.A total of 428 patients with HCC from the high exposure areas of AFB₁were recruited and the corresponding surgical resection specimens with tumors were collected.Additionally,basic clinical data consisting of gender,age,ethnicity,hepatitis B virus（HBV）and hepatitis C virus（HCV）infection,clinicopathological data（including cirrhosis,tumor size,tumor differentiation,and tumor stage）,and survival followed-up data were also collected at the same time.（2）Immunohistochemical staining of CD31 was used to evaluate microvessel density（MVD）in HCC tumor tissues.（3）AFB₁exposure level was elucidated using the amount of AFB₁-DNA adducts in cancer tissues and AFB₁-DNA adducts were detected by competitive enzyme-linked immunosorbent assay.（4）The expression level of CBX4 protein in cancer tissue samples was detected by immunohistochemical method.（5）The multivariate logistic regression model was used to analyze the relationship between CBX4 and the clinicopathological characteristics of HCC.The odds ratio（OR）and the corresponding 95%confidence interval（CI）was used to evaluate the specific effects of CBX4on the clinicopathological characteristics of HCC.Kaplan-Meier survival method was used to analyze the effects of different expression levels of CBX4 on the survival of HCC;whereas a multivariate Cox regression model was used to calculate the hazard ratio（HR）and evaluate the specific values of CBX4 on the prognosis of HCC.Results:（1）The results from the ELISA analyses showed that the average value of AFB₁-DNA adducts in HCC patients is 2.82±1.60μmol/mol DNA.Logistic regression model analyses show that compared to patients with low AFB₁exposure,these with the high AFB₁exposure have a larger tumor size and a higher MVD.The corresponding risk values are69.6（33.62～141.86）and 2.56（1.36～4.81）,respectively.（2）The results from Kaplan–Meier survival model show that the median survival time（MST）and median tumor reoccurrence-free survival time（MRT）of patients with low AFB₁exposure were respectively69.00（55.41–82.59）months and 70.00（44.93–95.07）months,respectively;whereas the MST and MRT for these high-exposure individuals are 20.00（13.04–26.96）months and 13.00（9.54-16.46）months,respectively.（3）Results from Kaplan–Meier survival analyses showed that the MST and MRT of patients with low CBX4 expression in their tumor tissues were69.00（52.75～85.25）months and 48.00（23.69～72.31）months,respectively;while MST and MRT for these patients with high CBX4 expression are 22.00（18.00～26.00）months and16.00（10.88～21.12）months,respectively.（4）Results from the combined analyses of AFB₁exposure and CBX4 expression exhibited that compared with HCC patients with both low AFB₁exposure and low CBX4 expression,these with both high AFB₁exposure and high CBX4 expression had shorter MST and MRT.Corresponding P values are 5.43?10^-15and3.51?10^-17.（5）Results from multivariate Cox regression survival model analyses further confirmed that both AFB₁and CBX4 are independent variables affecting the prognosis of HCC,and their corresponding death risks are 2.09（1.64～2.65）and 1.76（1.38～2.24）,respectively;the corresponding risk of tumor recurrence are 2.29（1.79～2.93）and 1.80（1.41～2.30）,respectively.Moreover,the effects of AFB₁and CBX4 on the prognosis of HCC has a multiplicatively interactive feature for HCC prognosis,with an interactive value of 1.98（1.61?2.59）for overall survival and 1.94（1.58?2.54）for tumor reoccurrence-free survival,respectively.Conclusion:Our findings show that both high AFB₁exposure and high CBX4expression in tumor tissues affects overall survival and tumor recurrence-free survival of HCC patients,which are independent variables leading to poor prognosis of this tumor.It is worth noting that the effects of AFB₁and CBX4 on the prognosis of HCC are multiplicatively interactive,and the predictive value of high CBX4 expression on the poor prognosis of HCC is more obvious under the conditions of high exposure of AFB₁.These results suggest that CBX4 is a poor prognostic biomarker for AFB₁-associated HCC.