| Microcystins(MCs)produced by some species of cyanobacteria are a family of cyclic heptapeptide hepatotoxins which can induce hepatitis and promote liver cancer and have been a great threat to human health.In the present study,we aimed to elucidate the toxiciological mechanism of MCs via determining the expressions of proto-oncogenes and anti-oncogene in MC-LR-treated HepG2 cells by using qPCR and western blot to provide a basic data for evaluating the MC-related human health risk including hepatitis and liver cancer.We first conducted a conventional cytotoxicity test to determine the cytotoxicity of MC-LR on HepG2 cells by using MTT method.Then the noncytotoxic concentrations of MC-LR,i.e.,10 nM,50 n M,0.5 ?M,10 ?M and 50 ?M were obtained for subsequent MC-LR-exposure in HepG2 cells according to the result of above test.Meanwhile,the exposure time intervals were designed to be 8,24,48,and 72 h,respectively.After MC-LR-exposure at various time intervals,HepG2 cells were collocted and total RNA was isolated for the subsequent qPCR to determine the expressions of proto-oncogenes such as c-Met,c-fos,c-jun,c-myc and N-ras and anti-oncogene PTEN in HepG2 cells.Meanwhile western blot was used to determine protein contents of these genes.The main results obtained in this study were as following description.1.The cytotoxicity of MC-LR on HepG2 cellsThe result of MTT determination showed that no difference in cellular viability between the treatment and control groups was found in HepG2 cells after 8-72 h of MC-LR-exposure.However,cellular viability in 100 ?M of MC-LR group was significantly lower than that of control group after 48 h of MC-LR-exposure.2.Effects of MC-LR-exposure on mRNA levels of c-Met,c-fos,c-jun,c-myc,N-ras and PTEN in HepG2 cellsThe result of qPCR showed that the mRNA levels of c-Met in all treatment groups were remarkably higher than that of control group throughout the period of exposure,indicating that MC-LR-exposure significantly promoted the transcription of c-Met.We also fund that transcription promotion of c-Met by MC-LR-exposure was in a concentration and exposure-time dependent pattern.Regarding the expressions of these immediate early genes c-fos,c-jun and c-myc in HepG2 cells,MC-LR-exposure also up-regulated their transcriptions in similar way to c-Met,suggesting that immediate early genes c-fos,c-jun and c-myc might be involved in the cytotoxicity of MC-LR.MC-LR-exposure promoted the expression of N-ras in HepG2 cells also in a concentration and exposure-time dependent pattern described as above.The results of qPCR also showed that the mRNA levels of PTEN in almost all treatment groups were significantly lower than that of control group throughout the period of exposure except for 10 nM group at 72 h,suggesting that MC-LR-exposure suppressed the transcription of PTEN.It is also notable that transcription suppression of PTEN by MC-LR-exposure was in a concentration-dependent pattern.3.Effects of MC-LR-exposure on protein levels of c-Met,c-Fos,c-Jun,c-Myc,N-Ras and PTEN in HepG2 cellsThe results of western blot showed that MC-LR-exposure significantly increased protein levels of c-Met,c-Fos,c-Jun,c-Myc,N-Ras while decreased PTEN protein level in HepG2 cells,which is consistent with the result of transcription.This result suggests that MC-LR-exposure can enhance the protein level of pro-oncogene but decrease that of anti-oncogene gene.In conclusion,our results reveal that MC-LR-exposure at non-cytotoxic concentration can promote the expressions of pro-oncogene while suppress anti-oncogene expression in HepG2 cells at transcription and protein levels,respectively.This result suggests that these pro-oncogenes and anti-oncogene may be involved not only in the cytotoxicity of MC-LR but also in human hepatitis and liver cancer induced by MC-LR.Our results might be useful to understand the molecular mechanism of human hepatitis and liver cancer caused by MC-LR and might provide a basic data to evaluate the risk assessment of MC-LR for human health. |
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