The Roles And Molecular Mechanisms Of MiR-155 And Exosome In Malignant Transformation Of Human Hepatocytes Induced By Arsenite

Arsenic is a commonly and widely distributed contaminant in the natural environment,that has been identified as a human carcinogen,affects the health of thousands of people worldwide.Environmental exposure and occupational exposure to arsenic compounds may cause the occurrence of a variety of tumors.The liver is the main target organ of arsenic,and the incidence of cirrhosis,ascites and hepatocellular carcinoma is significantly increased in arsenic-contaminated areas.At present,the specific role and molecular regulation mechanism of arsenide-induced malignant transformation of hepatocytes in the development and progression of hepatocellular carcinoma have not yet been fully elucidated.Cancer stem cells?CSCs?are subpopulation of tumor cells which have self-renewal and differentiation capacity to induce tumor,which play an important role in the occurrence,metastasis,recurrence and resistance of hepatocellular carcinoma.Exosomes are nanoscale membrane-structure vesicles secreted by cells into body fluids by cells.They play an important role in the development of liver cancer by transporting proteins,nucleic acids and lipids as well as other important molecules and participating in the signal transduction and substance exchange among cells.Various studies have shown that intracellular miRNAs and exosomal miRNAs play important roles in carcinogenesis induced by arsenic.mi R-155 is a miRNA that is abnormally expressed during the development of multiple tumors.However,the function of intracellular mi R-155 in the acquisition of CSCs-like phenotype induced by NaAsO2 as well as the role of exosomal miR-155 in the regulation of malignant transformation of liver cells have not been reported.Therefore,based on the malignant transformation model of human L-02 cells induced by chronic treatment of NaAsO2 which had been pre-conducted,investigated the effects of NaAsO2 on miR-155 were investigated by various molecular biology methods,as well as the role of miR-155 in the regulation in the acquisition of CSCs-like phenotype and further study the role of exosomal miR-155 derived from malignant transformed L-02 cells induced by NaAsO2 in the inflammation of liver cells and their molecular mechanisms.These results provide a scientific basis for futher study on the molecular mechanism of damage in hepatocytes induced by arsenic and offered diagnostic markers of arsenic exposure.Part ?.The roles of miR-155 via regulating QKI in arseinte-induced the acquisition of CSCs-like properties and malignant transformation of hepatic cellsObjectiveTo investigate the effect of arsenite on miR-155,and to explore the role of miR-155 in arsenite-induced the acquisition of CSCs-like properties and malignant transformation in L-02 cells.MethodsL-02 cells were chronic treated with 0.0 or 2.0?M NaAsO₂ for 0,10,20 or 30passages.The proportion of SP cells was measured by flow cytometry.Spheroid formation was detected by spheroid formation assay.The levels of CD90,EpCAM and miR-155 were detected by qRT-PCR.Western blot was used to detect the protein levels of SOX2,Bmi1 and QKI in the cells.Soft agar colony formation and nude mice tumorigenesis test are used to detect cell malignancy.Dual luciferase reporter assay was used to detect the effect of miR-155 on the expression of QKI.The characteristics of miR-155 and QKI in the acquisition of CSCs-like properties of L-02cells induced by arsenite were analyzed by transient transfection technique.Results1.Effects of chronic arsenite exposure on the acquisition of CSCs-like properties in L-02 cellsThe results showed that the proportion of SP cells and the number of spheroids in T-L-02 cells was higher than the control group.The levels of SOX2,Bmi1,CD90and EpCAM in spheroids were increased.Chronic exposure to NaAsO2 upregulated the protein levels of SOX2 and Bmi1,the mRNA levels of CD90 and EpCAM in L-02 cells with a time dependent manner.The results indicate that chronic exposure to NaAsO2 induce L-02 cells to acquire CSCs-like properties.2.The effects of arsenite exposure on the level of miR-155 and QKI in L-02 cellsThe results showed that the levels of miR-155 increased in a time dependent manner in L-02 cells acutely or chronically exposed to 2.0?M NaAsO₂.The levels of QKI in L-02 chronically treated with NaAsO2 decreased in a time dependent manner.The levels of mi R-155 in T-L-02-spheroid was higher than that in T-L-02 cells,while the levels of QKI was significantly decreased.The results indicate that NaAsO2treatment leads to the increase in the levels of miR-155 in L-02 cells as well as decrease in the levels of QKI in a time-dependent effect.3.The role of miR-155 on the expression of QKI in L-02 cells treated with arseniteThe results showed that the levels of miR-155 was significantly reduced while the levels of QKI was significantly increased in T-L-02 cells transfected with anti-miR-155.NaAsO2 significantly reduced the luciferase activity of QKI wild-type,which was significantly picked up after application of anti-miR-155,while the luciferase activity of cells transfected with mutant reporter plasmid showed no significant change.The results indicate that miR-155 regulates the expression of QKI in cells exposed to NaAsO2 via binding to the 3'-UTR of QKI.4.Effects of miR-155 on the acquisition of CSCs-like properties in L-02 cells induced by arseniteThe results showed that anti-miR-155 treatment significantly reduced the protein levels of SOX2 and Bmi1,the mRNA levels of CD90 and EpCAM in T-L-02 cells as well as the decreased colony number of suspension cells compared to the T-L-02 cells.The results indicate that miR-155 plays an important role in the acquisition of CSCs-like phenotype in L-02 cells induced by NaAsO2.5.Effects of QKI on the acquisition of CSCs-like properties in L-02 cells induced by arseniteThe results showed that pcD-QKI treatment significantly increased the expression of QKI and reduced protein levels of SOX2 and Bmi1,the mRNA levels of CD90 and EpCAM in T-L-02 cells as well as the decreased colony number of suspension cells.The results indicate that QKI plays an important role in the acquisition of CSCs-like phenotype in L-02 cells induced by NaAsO2.6.Effects of miR-155-regulated QKI on the acquisition of CSCs-like properties in L-02 cells induced by arseniteThe results showed that downregulating the expression of QKI after knockout of miR-155 blocked the inhibition of the acquisition of CSCs-like phenotype in T-L-02cells caused by anti-miR-155.The results indicated that miR-155 plays an important role in the acquisition of CSCs-like phenotype in L-02 cells induced by NaAsO2 via regulating the expression of QKI.7.Effects of miR-155 on the malignant transformation in liver cells induced by arseniteThe results showed that anti-miR-155 treatment significantly reduced the soft agar colony formation and nude mice tumorigenesis of T-L-02 cells compared to the control T-L-02 cells.These results show that miR-155 plays an important role in the malignant transformation of L-02 cells induced by NaAsO2.ConclusionNaAsO₂ induces the increased level of miR-155 in L-02 cells.miR-155 regulates the expression of QKI in L-02 cells.miR-155 plays an important role in the acquisition of CSCs-like phenotype and the malignant transformation in liver cells induced by NaAsO2.Part?.The roles of exosomal miR-155 in the inflammation and malignant transformation of hepatic cells induced by arseniteObjectiveTo investigate the role of exosomal mi R-155 in the inflammation and malignant transformation of normal L-02 cells induced by arsenite-transformed L-02 cells.MethodsL-02 cells were co-cultured with arsenite-transformed L-02 cells?T-L-02?or treated with T-L-02 cell cultures?T-CM?or treated with exosomes extracted from T-CM.qRT-PCR was used to detect the levels of miR-155,IL-6,and IL-8.Western blot was used to detect the level of p-STAT3.Soft agar colony formation and nude mice tumorigenesis test were used to detect cell malignancy.Transient transfection technique was used to investigate the role of exosomal miR-155 in the inflammation of normal L-02 cells induced by arsenite-transformed L-02 cells.Results1.Effects of T-L-02 cells on the levels of miR-155 and inflammatory factors in normal L-02 cellsThe results showed that the levels of miR-155,IL-6,IL-8 and p-STAT3 in the L-02 cells co-cultured with T-L-02 cells were significantly higher than those in L-02cells co-cultured with C-L-02.The inhibition of endocytosis in L-02 cells by CytoD blocked the elevated levels of miR-155 and inflammatory factors in L-02 cells caused by T-L-02 cells.These results indicate that the co-culture with T-L-02 cells induced by NaAsO2 upregulates the levels of miR-155 and inflammatory factors in normal L-02 cells.2.Effects of medium from T-L-02 cells on the levels of miR-155 and inflammatory factors in normal L-02 cellsThe results showed that the level of miR-155,IL-6,IL-8 and p-STAT3 in L-02cells treated with T-CM was significantly higher than those treated with the CM.The results demonstrate that the conditioned medium from malignant transformed L-02cell induced by sodium arsenite can increase the levels of miR-155 and inflammatory factors in normal L-02 cells.3.Inhibition of T-CM exosomes synthesis on the levels of miR-155 and inflammatory factors in normal L-02 cellsThe results showed that T-CM of which the synthesis of exosomes had been inhibited by GW4869 significantly reduced the levels of miR-155,IL-6,IL-8 and p-STAT3 in normal L-02 cells compared to those treated with T-CM.The results suggest that exosomes play an important role in the increased of miR-155 and inflammatory factors in normal L-02 cells.4.Effects of T-CM exosomes on the levels of miR-155 in normal L-02 cellsThe results showed that the exosomes isolated from CM and T-CM showed the uniform disk shapes under the transmission electron microscopy and expressed the exosome protein markers.The levels of miR-155 was significantly increased in the exosomes derived from T-CM.Exosomes were internalized by normal L-02 cells with a dose-dependent manner,while CytoD reduced the exosomes uptake by suppressing the endocytosis of the exosomes.A large amount of miR-155 can be transported into the normal L-02 cells via exosomes.The results suggest that T-L-02cells transporte miR-155 to the normal L-02 cells via exosomes.5.Effects of T-CM exosomes on the levels of miR-155 and inflammatory factors in normal L-02 cellsThe results showed that the levels of miR-155,IL-6,IL-8 and p-STAT3 in the L-02 cells treated with T-CM exosomes were higher than those treated with CM exosomes in a dose-dependent manner.The results suggest that T-CM exosomes elevate the levels of miR-155 and inflammatory factors in normal L-02 cells.6.Effects of miR-155 on the elevated levels of inflammatory factors in normal L-02 cells treated with T-CM exosomesThe results showed that the decreased level of mi R-155 in exosomes from T-CM by anti-miR-155 blocked the elevated levels of miR-155,IL-6,IL-8 and p-STAT3 in normal L-02 cells induced by exosomes from T-CM.These results indicate that miR-155 play an important role in the elevated levels of inflammatory cytokines in normal L-02 cells caused by exosomes derived from T-L-02 cells.7.Effects of exosomal miR-155 on malignant transformation of L-02 cellsThe results showed that when L-02 cells were treated with 2.0?M NaAsO₂ for20 passages?As-L-02-p20?,cells could not form colonies on soft agar and subcutaneous tumorigenicity in nude mice.T-CM exosomes treatment significantly increased the soft agar colony formation and nude mice tumorigenesis in As-L-02-p20 cells.The inhibition of miR-155 expression in T-CM exosomes by anti-miR-155 inhibits T-CM exosome-induced soft agar colony formation and subcutaneous tumor formation in nude mice.These results show that miR-155exosome plays an important role in the malignant transformation of L-02 cells induced by NaAsO2.ConclusionsThe level of miR-155 in the exosome derived from arsenite-transformed L-02cells is higher than those from unteansformed L-02 cells.Exosomes derived from malignant transformed L-02 cells induced by NaAsO2 transfer miR-155 to surrounding cells,which induces pro-inflammatory activity and malignant transformation of liver cells.In summary,the novel findings of this study are as follows:1.miR-155 via regulating QKI plays an important role in the acquisition of CSCs-like phenotype and malignant transformation in liver cells induced by arsenite2.Exosomal miR-155 is involved in arsenite-induced inflammation and malignant transformation of hepatic cells.