Promotion Effect Of Liver Tumor At Environmentally Relevant Levels In Kras^V12 Transgenic Zebrafish

Cyanobacterial blooms and their harmful metabolites can cause serious harm to humans and the ecological environment,and have received extensive attention in recent years.Microcystin-LR（MC-LR）is the most toxic isomer of microcystin that is widely present in nature and has been reported to be hepatotoxic.Epidemiological surveys and laboratory studies have shown that long-term exposure to microcystins has a higher probability of developing liver cancer,MC-LR has tumorigenic effects in vivo and in vitro,but the mechanism of MC-LR promoting hepatocellular carcinoma（HCC）is still unclear..In this experiment,the inducible liver tumor transgenic zebrafish Kras ^V12was used as a model to explore the promoting effect of environmental doses of MC-LR on liver tumor progression.Through subacute exposure,the mechanism of MC-LR on the promotion of transgenic zebrafish Kras ^V12hepatocarcinogenesis was explored through several indexes such as MC-LR content,zebrafish liver pathological sections,PP2A activity,phosphorylated proteomics,gene and protein expression.The main results are as follows:1.Environmentally relevant levels of MC-LR（3μg/L）aggravated the development of liver tumors in female transgenic zebrafish Kras ^V12,but did not have the same effect on male transgenic zebrafish Kras ^V12.The 3μg/L MC-LR exposure group of female transgenic zebrafish Kras ^V12induced by the inducer Dox increased the HSI and the mean fluorescence intensity by 1.4 folds and 1.2 folds,respectively,compared with the Dox alone group.Histopathological sections showed obvious characteristics of hepatocellular carcinoma in female transgenic zebrafish Kras ^V12.The HSI and mean fluorescence intensity in male transgenic zebrafish Kras ^V12exposed to Dox+3μg/L MC-LR were increased by 1.3 and 1.2 folds,respectively,compared with the Dox alone group.There were no significant differences in pathological sections in male transgenic zebrafish Kras ^V12.2.Environmentally relevant concentrations（3μg/L）of MC-LR can reduce the activity of protein phosphatase 2A（PP2A）and down-regulate the transcription level of PP2A subnit gene in female transgenic zebrafish Kras ^V12,but 1μg/L MC-LR did not cause the same influence.The PP2A activity of the 1 and 3μg/L MC-LR exposure groups of female transgenic zebrafish Kras ^V12induced by the inducer Dox decreased to 92.3±0.6%and 91.0±1.1%of the blank control group.Statistical analysis results showed a significant difference between the blank control group and the Dox+3μg/L MC-LR exposure group.Compared with the Dox alone group,the transcript levels of PP2A subunit related genes ppp2caa,ppp2r2ba and ppp2r2bb in20 mg/L Dox+3μg/L MC-LR group were down-regulated by 1.8-,1.9-and 2.6-folds,respectively.3.Using TMT labeling quantitative phosphoproteomics in Dox alone group and the binary mixture of Dox and MC-LR（3μg/L）group,we found that the expressions of 1049 phosphopeptides were notably altered（fold change>1.2 or<0.83）with 508up-regulated and 541 down-regulated after MC-LR exposure.Subcellular localization analysis showed that most of these differentially phosphorylated peptides were localized in the nucleus（60.4%）.Domain analysis showed that Armadillo-type fold was dominant.Enriched pathways by KEGG analysis suggested that differentially phosphorylated proteins were mainly related to tight junction and Wnt signaling pathway.4.The m RNA and protein expression ofβ-Catenin,a key regulator of the Wnt signaling pathway,was up-regulated following exposure to Dox+3μg/L MC-LR.Compared with the Dox alone group,the transcription level and protein expression ofβ-Catenin in the Dox+3μg/L MC-LR exposure group were increased by 1.3 folds and3.0 folds,respectively.In conclusion,these results suggested that MC-LR inhibited the activity of PP2A,which in turn activated Wnt signaling,resulting in progression of liver tumor in female transgenic zebrafish Kras ^V12.