The Roles And Molecular Mechanisms Of Exosomal CircRNA₁00284 In Malignant Transformation Of Human Hepatocytes Induced By Arsenite

Arsenic is a naturally existing,toxic metalloid that is often found as a contaminant in drinking water,and it is believed that there are harmful effects from arsenic even when levels are below the drinking water standard.Long-term exposure to arsenic is associated with lung,bladder,and skin cancer,and with noncancerous disorders such as cardiovascular disease,diabetes,and skin lesions.Carcinogenesis induced by arsenic is related to multiple mechanisms.Researchs have recently found that circular RNAs?circRNAs?and exosomes play important roles in the manlignant transformation and hepatocellular carcinomas induced by sodium arsenite?NaAsO₂?of hepatocytes,but the specific molecular mechanism remains undefined.Circular RNAs?circRNAs?are a novel kind of non-coding RNAs?ncRNAs?,and unlike linear RNA generate a closed annular structure.Several circRNAs serve as biomarkers in the development of hepatocellular carcinomas and regulate gene expression by acting as“miRNA sponges”.Exosomes are cell-derived vesicles that are present in many eukaryotic fluids,including blood,and urine,and in the culture medium of cells.Exosomes carry messenger RNAs?mRNAs?,miRNAs,and double-stranded DNA.Exosomes are involved in cell-to-cell signaling and may influence processes in normal cells because they can merge with and release their contents into cells that are distant from their cell of origin and play an important role in the tumor microenvironment.In exosomes,circRNAs have been found enriched and stable recently and participate in the malignant transformation induced by arsenite.However,the functions of exosomal circRNAs in hepatocellular carcinomas induced by arsenite remain undefined.Therefore,based on the malignant transformation model of human L-02 cells induced by chronic exposure to NaAsO₂ which had been pre-conducted,investigated various molecular biology methods was used to investigate the roles and molecular mechanisms of exosomal circRNA₁00284 derived from arsenite-transformed cells on the cell cycle and cell proliferation together with the malignant transformation of normal liver cells.It provides a scientific basis for elucidating the carcinogenic mechanism of NaAsO₂ and establishing a rapid,accurate and sensitive screening system of liver cancer induced by arsenite.ObjectiveTo investigate the role of exosomal circRNA₁00284 in the accelerated cell cycle and promoted proliferation together with the malignant transformation of normal liver cells induced by chronic exposure to arsenite.MethodsL-02 cells were exposed to 0.0 or 2.0?M NaAsO₂ for 0,3,6,12 or 24 h,or for 0,10,20 or 30 passages.Normal L-02 cells or L-02 cells which had not been transformed were exposed to medium?T-CM?from arsenite-transfomed L-02 cells?T-L-02?or exosomes isolated from T-CM?T-CM-exo?.CircRNA₁00284 siRNA was used to down-regulate the levels of circRNA₁00284 siRNA in T-L-02 cells and exosomes derived from T-L-02 cells.PLCDH-circRNA₁00284 was used to up-regulate the levels of circRNA₁00284.MiRNA-217 mimic was used to up-regulate the levels of miR-217 in L-02 cells or miR-217 inhibitor was used to down-regulate the levels of miR-217 in L-02 cells.The levels of circRNA₁00284 were detected by qRT-PCR.The expressions of EZH2 and cyclin D1 were measured by western blot.Flow cytometry was used to evaluate the situation of cell cycle.CCK8 was used to detect the cell proliferation.Colony formation assay and subcutaneous tumorigenicity test in nude mice were used to detect the degree of malignancy;Transwell assay was used to detect migration and invasion of cells.bioinformatics tools was used to predict the miRNA binding to circRNA₁00284.Transient transfection technique to investigate the role of exosomal circRNA₁00284 in promoting normal L-02 cell cycle and abnormal proliferation induced by T-L-02 cells via miR-217.Serum exosomes was isolated from population exposed to arsenite or normal population,qRT-PCR was used to detect the levels of circRNA₁00284.Results1.The effects of circRNA₁00284 on the neoplastic capacity of arsenite-transformed L-02 cells?1?The effects of chronic exposure to arsenite on the malignant transformation of L-02 cells and the levels of circRNA₁00284The results showed that L-02 cells formed more colonies in agar after exposure to 2.0?M arsenite for 30 passages compared to the passage-control cells?C-L-02?;the tumor volumes for the group implanted with arsenite-transformed cells were greater than those in control group.after exposure to 2.0?M NaAsO₂ for 6 h or after10 passages of chronic exposure to 2.0?M NaAsO₂,with longer times of exposure to arsenite,the levels of circRNA₁00284 showed a rising trend and levels of circRNA₁00284 were increased in a time-dependent manner compared cells before exposure or passage-control cells.The circRNA₁00284 was resistant to RNase R in contrast to the linear isoform mRNA GCLM in T-L-02 cells and was enriched in the cytoplasm.These results indicate that exposure to arsenite induces the malignant transformation of normal liver cells as well as up-regulation in L-02 cells.?2?The effects of circRNA₁00284 on the neoplastic capacity of arsenite-transformed L-02 cellsThe results showed that the level of circRNA₁00284 decreased after transfecting cells with siRNA compared with that in arsenite-transformed L-02 cells;arsenite-transformed L-02 cells transfected with circRNA₁00284 siRNA formed fewer colonies in agar than those transfected with control siRNA;silencing of circRNA₁00284 in arsenite-transformed cells inhibited cell invasion and migration.The expression of circRNA₁00284 were upregulated after transfection with pLCDH-circRNA₁00284 in non-transformed cells;non-transformed cells transfected with pLCDH-circRNA₁00284 formed more colonies in agar than those transfected with pLCDH control together with enhanced invasion and migration.Thus,circRNA₁00284 is involved in the neoplastic and metastatic capacity of arsenite-transformed L-02 cells.2.The effects of media from T-L-02 cells on the levels of circRNA₁00284,cell cycle,and proliferation of normal L-02 cells.The results showed that the levels of circRNA₁00284 in normal L-02 cells were elevated when they were exposed to media from arsenite-transformed L-02?T-CM?cells relative to the media from passage-control L-02 cells?CM?.Normal L-02 cells treated with medium from arsenite-transformed L-02 cells showed elevated levels of EZH2 and cyclin D1 as well as accelerated cell cycle and promoted cell proliferation.These results indicate that,for normal liver cells,media from arsenite-transformed L-02 cells induced the increases of circRNA₁00284 levels,an accelerated cell cycle,and abnormal cell proliferation.3.The effects of blocking the formation of exosomes on the elevated levels of circRNA₁00284 as well as cell cycle and proliferation in L-02 cells induced by T-CMThe results showed that blocking of exosomes derived from arsenite-transformed L-02 cells by GW4869 inhibited the expression of circRNA₁00284,the expressions of EZH2 and cyclin D1 and the accelerated cell cycle compare to cells treated with T-CM.These results indicate that T-L-02 cells may influent the expressions of circRNA₁00284 and cell cycle of L-02 cells through exosomes.4.The effects of exosomes derived from T-CM on the levels of circRNA₁00284 in L-02 cellsThe results showed that exosomes isolated from C-L-02 cells?CM-exo?or T-L-02 cells?T-CM-exo?revealed typically rounded shapes sizing around 100 nm.Western blot analysis confirmed the presence of exosome marker proteins CD81 and CD9 in both types of exosomes.The levels of circRNA₁00284 were higher in exosomes from arsenite-transformed cells and could be uptaken by L-02 cells which induced the increases of circRNA₁00284 in normal L-02 cells.these data show the transfer of circRNA₁00284 derived from arsenite-transformed L-02 cells into normal liver cells via exosomes.5.The effects of exosomes derived from T-CM on the cell cycle and cell proliferation of normal L-02 cellsThe results showed that the T-CM-exo accelerated the cell cycle and promoted the proliferation of normal L-02 cells.Exposure of L-02 cells to 0,10,20,50,or 100?g/mL of T-CM-exo for 24 h,but not CM-exo,induced increases of EZH2 and cyclin D1.These results indicate that exosomal circRNA₁00284 derived from arsenite-transformed L-02 cells have an influence on the cell cycle and proliferation of normal L-02 cells.6.The effects of exosomal circRNA₁00284 derived from T-CM on the cell cycle,cell proliferation and malignant transformation of L-02cellThe results showed that knockdown of circRNA₁00284 blocked the increases of protein levels of EZH2 and cyclin D1 induced by T-CM-exo as well as the accelerated G1/S transition.These results confirm that exosomal circRNA₁00284derived from arsenite-transformed L-02 cells is involved in the abnormal cell cycle of normal L-02 cells.7.The effects of exosomal circRNA₁00284 derived from T-CM on the cell cycle and cell proliferation of normal L-02 cells via regulating miR-217The results showed that there were binding sites between circRNA₁00284 and miR-217.Overexpression of miR-217 attenuated the up-regulation of EZH2 and cyclin D1 induced by T-CM-exo.miR-217 inhibitor reversed the effects of exosomes from arsenite-transformed L-02 cells transfected with circRNA₁00284 siRNA on the levels of EZH2 and cyclin D1 in normal L-02 cells.The inhibited G1/S transition induced in normal L-02 cells by exosomes from arsenite-transformed L-02 cells transfected with circRNA₁00284 siRNA was also restored by an miR-217 inhibitor.Moreover,exosomes derived from cells transfected with circRNA₁00284 siRNA inhibited proliferation of normal L-02 cells,but this effect was reversed by an miR-217 inhibitor.These results indicate that exosomal cicrRNA₁00284 accelerates the cell cycle and promotes cell proliferation via miR-217 in normal liver cells.8.The effects of exosomal circRNA₁00284 derived from T-CM on the cell cycle,cell proliferation and malignant transformation of L-02cellThe results showed that inhibition of exosomal circRNA₁00284 impeded the enhanced abilities of colony formation,tumorigenic in nude mice,invasion and migration in non-transformed L-02 cells induced by T-CM-exo.These results confirm that exosomal circRNA₁00284 derived from arsenite-transformed L-02 cells is involved in the malignant transformation of non-transformed L-02 cells.9.Changes in the levels of circulating exosomal circRNA₁00284 in people exposed to arsenite.The results showed that there were higher expressions of exosomal circRNA₁00284 in the sera of people exposed to arsenite compared with those in sera of paired healthy volunteers.These results indicate that exosomal circRNA₁00284 can be used as a potential biomarker of arsenite exposure.Conclusions1.CircRNA₁00284 is involved in the malignant transformation of liver cells induced by arsenite and can be transferred into normal liver cells.2.Exosomal circRNA₁00284 derived from arsenite-transformed L-02 cells induces the acceleration of the cell cycle and promoted proliferation via regulating miRNA-217 and plays an important role in the malignant transformation of liver cells induced by arsenite.3.Exosomal circRNA₁00284 was up-regulated in the sera of people exposed to arsenite.