Expression And Function Of MicroRNAs In The Hepatotoxicity Of Microcystin-LR

Due to the eutrophication increasing and global climate change,healthy risk of human being and aquatic animals from the exposure to cyanotoxins has become an increasingly serious global problem.Among cyanotoxins,microcystins(MCs)are one of the most widely distributed cyclic heptapeptide toxins.They have hepatotoxicity and tumor-promoting activity on humans,and are among the most common and dangerous groups.These cyanotoxins(MC-LR as 2B carcinogen by IARC)are effective tumor promoters,and there are signs that they may also act as tumor initiators.Epidemiological studies in some areas of China have shown that MCs are one of the risk factors for the high incidence of primary liver cancer(PLC).Both acute and chronic exposure to low or high doses of MC-LR can activate the apoptotic pathway,and longterm exposure to low concentrations of MC-LR increases the risk of cancer.Existing research showed that micro RNAs(miRNAs)have received great attention in environmental toxicology.miRNAs may be dysregulated in expression in a variety of toxic-related diseases and they may be involved in many physiological processes,including apoptosis,immune protection,nervous system development,and cancer pathogenesis.In addition,many studies have suggested that miRNAs may play a key role in the cell's response to MC-LR exposure.However,little is known about the relationship between miRNAs and MC-LR hepatotoxicity.In this study,high-throughput sequencing was used to screen known miRNAs that are differentially expressed in MC-LR-induced hepatotoxicity,and through bioinformatics to analyze the biological processes and important signal pathways of MC-LR hepatotoxicity which may be involved in the differentially expressed known miRNAs,while the expression screening of miRNA targets in MC-LR toxic effects as well as possible molecular mechanisms involved in the study,to find new molecular marker of MC-LR hepatotoxicity of new drug targets and treatment of various diseases provides new theoretical support.The following experimental results were obtained:(1)The expressional profile of miRNAs in the liver of silver carp following MC-LR exposureIn order to screen for known miRNAs that are differentially expressed in MC-LR-induced hepatotoxicity,small RNA transcriptome sequencing analysis was used in this study.High-throughput sequencing results showed that the expressions of 53 miRNAs and 319 miRNAs in the livers of silver carp of the treatment group(50 ?g/kg and 200 ?g/kg MC-LR)changed significantly compared with the control.When compared X with the 50 ?g/kg treatment group,the expression of 203 miRNAs in the liver was significantly up-regulated,while the expression of 163 miRNAs was down-regulated in the 200 ?g/kg treatment group.In addition,this study found that MC-LR exposure promoted the expression levels of miR-2187-3p,miR-2779,miR-2478,miR-16,miR-144-5p,miR-181a-3p,miR-223,MiR-451 and miR-499,while the expression levels of miR-146,miR-92,miR-203 and miR-98 were suppressed.This result further confirmed the above result by q PCR determination,indicating that these miRNAs may be related to the hepatotoxicity of MC-LR.Target site identification was then performed on the differentially expressed known miRNAs through target information prediction software through bioinformatics.We annotated miRNAs and their target genes through GO analysis and KEGG pathway analysis.GO enrichment analysis showed that these target genes were related to biological processes such as metabolic processes,cellular processes and single biological processes.In addition,KEGG pathway analysis showed that the target genes of differentially expressed miRNAs in the liver were mainly involved in insulin signaling pathway,PPAR signaling pathway,m RNA monitoring pathway,Wnt signaling pathway,and transcriptional abnormalities.(2)Effect of MC-LR exposure on miRNAs expressions in different tissues of sliver carpBy analyzing the result of high-throughput sequencing,miR-16,miR-181a-3p,miR-223,and miR-451 were screened as the miRNAs for the further study.Therefore,we examined the expression levels of these four miRNAs in different tissues(liver,spleen,kidney,and intestine)of silver carp after MC-LR exposure.The results showed that MC-LR promoted the expression levels of miR-16,miR-181a-3p,miR-223,and miR-451 in the fish liver.However,the expression of these miRNAs in the spleen,kidneys,and intestines varied greatly.These results indicated that the expression levels of miRNAs were tissue-specific due to differences in the function of organs.By reviewing relevant literatures and combining the results of this study,we inferred that miR-16 and miR-181a-3p might synergistically participate in MC-LR-induced inflammation.Meanwhile,miR-223 and miR-451 may become the the potential biomarkers for liver cancer due to the fact that miR-223 and miR-451 were involved in the hepatotoxicity of MC-LR by regulating the cell cycle and a variety of signaling pathways.In summary,we speculated that these four miRNAs may be involved in the toxic effect of MC-LR on multiple organs.(3)Expression of miR-16 in the liver of zebrafish following MC-LR exposureThe result of bioinformatics analysis showed that the seed sequence of miR-16 was found to be basically the same by comparing the mature sequences of human and zebrafish miR-16 and their precursor sequences.In addition,the mature sequence of miR-16 in many species was compared,and phylogenetic tree was constructed to determine the conservation of miR-16.It was found that miR-16 was conserved among different species.The expression and molecular mechanism of miR-16 in the liver of zebrafish following MC-LR exposure was performed,the result indicated that miR-16 may be involved in the regulation of MCLR-induced cell cycle,apoptosis,and inflammatory response.(4)Micro RNA-16 participates in the cell cycle alteration of HepG2 cells induced by MC-LRThe mechanism of miR-16 on hepatotoxicity,apoptosis and cell cycle changes in HepG2 cells induced by MC-LR exposure was explored in vitro using human liver cancer cell HepG2 cells.The effects of MCLR exposure on HepG2 cell viability,cell cycle,apoptosis,and the expression of miR-16 in the cells were investigated.The results showed that low-dose MC-LR(10 ?M)could promote cell viability indicated by determination the G1/S cell cycle progression of HepG2 cells,but did not affect the apoptosis of HepG2 cells.The expression result of miR-16 showed that low concentration MC-LR exposure could inhibit miR-16 expression.Therefore,we speculated that miR-16 might be involved in MC-LR-induced cell cycle change.To confirm this conjecture,the effects of miR-16 overexpression and expression inhibition on MC-LR-treated HepG2 cell viability,cell cycle,apoptosis,and related genes and proteins were examined.The result of cell viability showed that miR-16 overexpression inhibited the viability of MC-LR-treated HepG2 cells,while miR-16 inhibition promoted the viability of MC-LR-treated HepG2 cells.The cell cycle results detected by flow cytometry showed that overexpression of miR-16 induced an increase in G0/G1 phase cells and a decrease in S phase cells.The result of apoptosis determination by flow cytometry showed that after MC-LR exposure,miR-16 overexpression or inhibition did not change the apoptosis of HepG2 cells.In addition,miR-16 expression inhibition down-regulated p53 expression in MC-LR-treated HepG2 cells,while overexpression of miR-16 up-regulated p53 expression.Moreover,the protein level of P53 was consistent with the m RNA expression level as described above,suggesting that miR-16 may participate in the cell cycle arrest by regulating p53 induced with MC-LR exposure.Studies also revealed that the miR-16 family triggered the accumulation of G0/G1 cells by simultaneously silencing multiple cell cycle genes rather than a single target gene.Therefore,combined with the result of this study,it is reasonable to believe that miR-16 may participate in changes in the cell cycle process by regulating multiple cell cycle genes(including CDK6).In this study,when miR-16 was overexpressed or inhibited,the expressions of PTEN and c-myc in MC-LRtreated HepG2 cells was also disturbed,but the relationship between miR-16 and c-myc and PTEN is still unclear and needs to be studied in future work.Based on the above results,we think that miR-16 may be involved in the proliferation and cell cycle regulation of MC-LR treated HepG2 cells by changing the expression of p53,CDK6,PTEN and c-myc.The main conclusions of this study:(1)miRNA may play an important negative regulatory role in the hepatotoxicity of MC-LR.We found four miRNAs(miR-16,miR-181a-3p,miR-223,and miR-451)that might also have important regulatory roles in MC-LR liver toxicity.(2)After MC-LR exposure,the transcription levels of 4 miRNAs(miR-16,miR-181a-3p,miR-223,and miR-451)in the liver,spleen,kidney and intestine of silver carp were changed,and the expression was tissue-specific.(3)The result of bioinformatics analysis showed that miR-16 was highly conserved among different species.We found that miR-16 might be involved in the cell cycle,apoptosis and inflammatory response in the liver of zebrafish following MC-LR-exposure.(4)Low-dose MC-LR exposure can promote the viability of HepG2 cells and inhibit the expression of apoptosis-related genes.In addition,MC-LR exposure can also induce down-regulation of miR-16 expression.Further studies showed that miR-16 overexpression can inhibit the MC-LR induced viability of HepG2 cells by changing the expression of p53,CDK6,PTEN and c-myc.These results suggest that miR-16 may have a regulatory effect on the MC-LR-induced cell cycle change.