Promotion Of Liver Tumor Progression By Microcystin-LR In Early Developmental Stage Of Kras^V12 Transgenic Zebrafish

With the increasing frequency of cyanobacterial blooms,the health risk of humans exposed to Microcystin（MC）,a secondary metabolite of cyanobacterial,has gradually increased.Epidemiological investigations have shown that long term low-dose MC exposure increased higher risk of developing hepatocellular carcinoma.However,the direct evidence involved in the role of MC exposure in the development of hepatocellular carcinoma remains unclear.Liver tumor promotion of MC still needed to be verified in different life stages among more species.Kras^V12 zebrafish was an experimental model that could achieve high expression of oncogenic kras and green fluorescent gene EGFP in the liver under the induction of Doxycycline（Dox）.This study focused on the ability of liver tumor-promoting of MC.MC-LR,the most toxic isomer of MC was chosen in this study.Acute and sub-acute tumor-promoting effect of MC-LR on kras^V12 transgenic zebrafish in the early life stage was observed.The molecular mechanisms of liver tumor-promoting of MC were explored through several indexes such as accumulation of MC-LR in the liver,the value of m RNA and protein-related HCC,and the basic morphological indicators of zebrafish.Provide experimental bases and theoretical support for clarifying the relationship between the high incidence of hepatocellular carcinoma and MC exposure.1.Dox-induced kras^V12 transgenic zebrafish embryos were exposed to 0,0.1,1 mg/L MC-LR for 3 d.The results demonstrated that MC-LR could affect the development of kras^V12 transgenic zebrafish larvae and increase the liver fluorescence mean gray value（intensity/area）of kras^V12 transgenic zebrafish.In addition,histopathological sections showed that the zebrafish liver in the highest concentration group showed the characteristics of early hepatocellular adenoma.The Real-time PCR results showed that the m RNA expression of kras,mapk3,c-jun,prmt5,hdac3,and hspa9 was up-regulated.However,the value of m RNA expression of tp53 was significantly down-regulated in MC-LR（1μg/L）+Dox group.Furthermore,the expression level of protein PRMT5 was increased in the highest concentration group.2.PRMT5 inhibitor（CMP5）could effectively inhibit the increase in fluorescence mean gray value（intensity/area）of kras^V12 transgenic zebrafish liver induced by MC-LR.The histopathological showed that the degree of hepatocytes in the MC-LR+Dox+CMP5group was less atypia.Compared MC-LR+Dox group,the expression of the prmt5 and kras was up-regulated.3.The kras^V12 transgenic zebrafish juveniles were exposed to environmentally relevant concentrations（0,0.1μg/L MC-LR+20 mg/L Dox,1μg/L MC-LR+20 mg/L Dox and 1μg/L MC-LR）15 d.The results demonstrated that MC-LR exposure could promote the proliferation of hepatocytes,resulting in increased HSI and the accumulation of MC-LR in the liver.Compared to the Dox group,the HSI in the highest concentration group was increased by 1.6 folds.The histopathological results showed that the typical features of HCC in the highest concentration group（1μg/L MC-LR+20 mg/L Dox）.However,normal liver structure with a little number of inflammatory cells was observed in the single MC-LR treated group.The results of q RT-PCR showed that gene kras,mapk3,c-jun,prmt5,hdac3,hspa9,tp53 were up-regulated in the highest concentration group.These results indicated that MC-LR may have the ability to accelerate the malignant proliferation of hepatocytes but not a carcinogen.The molecular mechanism may be caused by the MAPK pathway activated by the high expression of the proto-oncogene kras and the activation of PRMT5.The progression of liver tumors could be inhibited by CMP5 through suppression of the m RNA expression of prmt5 and kras in kras^V12 transgenic zebrafish larvae.