The Role And Mechanism Of ALX4 During The Process Of Microcystin Induced Hepatocellular Carcinoma

BACKGROUND:The eutrophication of surface water and algae pollution in China has been very serious.Microcystins(MCs)are the most common algae toxins after eutrophication of freshwater bodies.The contamination of MCs with different degrees has been detected in the water bodies of the Yellow River,the Yangtze River,Dianchi Lake,Chaohu Lake,and Taihu Lake.The current drinking water safety problem has been a serious threat to human health,and contamination with MCs has been detected in drinking water and aquatic products.Since current tap water treatment processes and conventional food heating are not effective in removing MCs and their toxicity,MCs contamination has become an important threat to people's drinking water safety and food safety.MCs have selective toxicity to the liver.A large number of studies have shown that MCs is one of the main factors causing liver cancer.In China,primary hepatocellular carcinoma has been a serious threat to human life and health.The mortality and morbidity of liver cancer has remained high and the damage is very serious.Therefore,it is very important to study the factors that cause liver cancer in the environment.A large number of studies have confirmed that epigenetics,including DNA methylation,histone modifications and non-coding RNAs,plays an important role in the development of tumors.The occurrence of CpG islands methylation of these tumor-associated genes makes the expression of related genes abnormal and closely related to the occurrence of tumors.Therefore,the search for changes in the methylation of certain related genes during the process of microcystin-induced hepatocarcinogenesis will provide new ideas for the subsequent study of microcystin-induced liver cancer.In previous studies,we established a model of hepatocytes cell malignant transformation of induced by MCs and screened them for methylation chips and expression microarrays.We found that ALX4 gene is a novel hypermethylation gene.However,there are few studies on the genesis of HCC induced by MCs.The specific molecular mechanism is still unclear.Therefore,this study intends to study the epigenetic changes and mechanisms of ALX4 gene during the development of HCC induced by MCs,and to explore the main role,function and molecular mechanism of ALX4 gene during the development of microcystin-induced HCC.It will provide valuable biological information for the in-depth study of the mechanism of ALX4 gene during the development of microcystin-induced liver cancer,and provide valuable information for the subsequent effective treatment of microcystin-induced liver cancer.OBJECTIVE:1.To investigate the epigenetic changes and mechanisms of ALX4 gene during the process of HCC induced by MCs.2.To explore the main function and molecular mechanism of ALX4 gene during the occurrence and development of HCC induced by MCs.METHOD:1.The expression of ALX4 gene in MCs-induced malignant transformation L02 cells was detected by Q-PCR and WB.Meanwhile,the expression of ALX4 gene in microcystin-infected animal models was detected.2.MSP and BSP were used to detect the occurrence of ALX4 gene methylation during MCs-induced L02 cells,and further verified by demethylation experiments.3.The cell model of high expression and low expression of ALX4 gene was established and used to analyze the function of ALX4 gene in MCs-induced liver cancer.The proliferation of cells was detected by CCK8.Colony formation ability of cells was detected by colony formation assay.Cell cycle and apoptosis were detected by flow cytometry.The migration and invasion of L02 cells induced by microcystin was analyzed by wound healing assay and Transwell assay.4.The effect of ALX4 gene on tumor growth in vivo was analyzed using a nude mouse tumorigenesis assay.Proliferation was detected by Ki67,and apoptosis was detected by TUNEL.5.The molecular mechanism of the role of ALX4 gene in MCs-induced hepatocellular carcinoma was examined by Q-PCR and WB.RESULT:1.The ALX4 gene was gradually down-regulated with the increase of L02 cell generation of MCs treatment.Compared with the control group,the expression of the poisoned group was significantly down-regulated in the animal model of microcystin toxin exposure,and the difference was statistically significant(P<0.01).2.ALX4 gene was hypermethylated in microcystin-induced L02 cells.It may be due to DNA methylation led to low expression of ALX4 gene in MC-induced liver cancer.3.ALX4 overexpression promoted cell apoptosis and induced G1/S phase arrest,and inhibited proliferation,colony formation,invasion and metastasis of MC-induced malignant transformation L02 cells.4.Knockdown the ALX4 expression in ALX4-expressed L02 cells promoted proliferation,colony formation,and invasion and metastasis of MC-induced malignant transformation L02 cells.5.ALX4 gene significantly inhibited the growth of xenograft tumor in nude mice.At the same time,it was also clearly demonstrated that ALX4 gene inhibited the proliferation of microcystin-infected cells and promoted the apoptosis of MC-induced malignant transformation L02 cells in nude mice.6.In the MC-induced liver cancer,the high expression of ALX4 gene could promote the expression of P53 gene and inhibit the expression of C-MYC and MMP9.We also found that ALX4 expression regulated P53 downstream target molecule Bax and Bcl-2.Whereas,knockdown the expression of ALX4 inhibited the expression of P53 gene and promote the expression of C-MYC and MMP9.CONCLUSION:The low expression of ALX4 gene in MCs-induced hepatocellular carcinoma may be due to its hypermethylation.ALX4 inhibits the proliferation,invasion,and metastasis of microcystin-induced hepatocellular carcinoma cells,and induces G1/S phase arrest.ALX4 gene plays a tumor suppressing role in microcystin-induced liver cancer related to the P53 pathway and C-MYC and MMP9.This study made us have a certain understanding of the mechanism of microcystin-induced liver cancer and the role of ALX4 in live cancer.