The Roles And Molecular Mechanisms Of Autophagy And EMT In Malignant Transformation Of Human Hepatocytes Induced By Arsenite

Arsenic is an established human carcinogen that widely found in the environment.There are two main forms of arsenic,arsenate and arsenite,in which sodium arsenite（NaAsO₂）is the most important one.It has been reported that long-term exposure to arsenic can cause a variety of diseases.In arsenic pollution areas,the incidence of liver cancer was significantly higher than other places,which threaten the lives and health of local residents.Many mechanisms have been proposed for arsenic carcinogenesis.Recently,many studies have shown that autophagy and EMT are closely related to the development and progression of human hepatocellular carcinoma induced by NaAsO₂.However,the specific process and molecular mechanism need to be further explored.Autophagy is an important metabolic mechanism of eukaryotic cells to maintain cell homeostasis.Autophagic dysfunction is an important factor in the development of tumors.The epithelial-to-mesenchymal transition（EMT）is involved in a variety of pathophysiological processes and plays an important role in the development of tumors.Long-term exposure to arsenic can lead to cell autophagic disorder,which can not be normal decomposition of metabolic harmful components,leading to accumulation of intracellular EMT transcription factors,promoting the occurrence of EMT,and then causing malignant transformation of cells.Studies have been reported that miRNAs are involved in NaAsO₂-induced autophagy by regulating downstream signaling pathways.miR-21 is one of the earliest discovered carcinogenic miRNAs.In the previous study,high expression of miR-21 were obsevered in various cells treated with NaAsO₂,including human normal hepatocytes（L-02）.It hasn’t been reported that the molecular mechanism of miR-21 involved in NaAsO₂-induced autophagy and the regulation of autophagy and EMT in malignant transformation of hepatocytes.ObjectivesThe effects of NaAsO₂ on miR-21 were investigated by variety of molecular biology methods,as well as the role of miR-21 in the regulation of autophagy and further study the role of autophagy and EMT in NaAsO₂-induced malignant transformation of hepatocytes and their molecular mechanisms.These results provide a scientific basis for further study on the molecular mechanism of arsenic carcinogenesis and offer molecular markers of arsenic-induced hepatocellular carcinoma.Methods1.Effects of acute treatment of arsenite on autophagy in L-02 cellsL-02 cells were exposed to 0.0,1.0,2.0,4.0 or 8.0μM NaAsO₂ for 24 h,or treated with 0.0 or 2.0μM NaAsO₂ for 0,3,6,12 or 24 h,or exposed to 2.0μM NaAsO₂ or 100 nM Rapamycin for 24 h respectively.Western blot was used to detect the protein levels of mTOR,beclin 1 and LC3 Ⅱ/Ⅰ.The expression level of LC3 was detected by immunofluorescence assay.The number of autophagy was detected by transmission electron microscopy.We were going to observe the effect of NaAsO₂ on autophagy of L-02 cells.2.The role of ERK signaling pathway in the autophagy activation induced by arsenite in L-02 cellsL-02 cells were exposed to 0.0,1.0,2.0,4.0 or 8.0μM NaAsO₂ for 24 h,or treated with 0.0 or 2.0μM NaAsO₂ for 0,3,6,12 or 24 h,or exposed to 10μM MEK/ERK inhibitor U0126 for 3 h,then were exposed to 0.0 or 2.0μM NaAsO₂ for 24 h.Western blot was used to detect the expression levels of p-ERK,ERK,PTEN,mTOR,Beclin 1 and LC3Ⅱ/Ⅰ.To observe the role of ERK signaling pathway in the activation of autophagy induced by NaAsO₂ in L-02 cells.3.Effects of arsenite on miR-21 level and its target proteins in L-02 cellsL-02 cells were treated with 0.0,1.0,2.0,4.0 or 8,0μM NaAsO₂ for 24 h,or treated with 0.0 or 2.0 μM NaAsO₂ for 0,3,6,12 or 24 h.The levels of miR-21 target proteins PTEN,PDCD4 and Spry 1 were detected by Western blot.We were going to observe the effect of NaAsO₂ on the level of miR-21 and expression of its target proteins in L-02 cells.4.The role of miR-21 in autophagy induced by arsenite in L-02 cellsL-02 cells were transfected with 100 nM anti-miR-21 and anti-miR-nc respectively,then were exposed to 0.0 or 2.0μM NaAsO₂ for 24 h.The level of miR-21 was detected by qRT-PCR.The levels of PTEN,ERK,beclin 1,LC3 II/I and mTOR were detected by Western blot.These were used to observe the effect of miR-21 on PTEN,ERK and autophagy-related protein levels.5.Effects of PTEN on ERK and autophagy-related proteins in L-02 cells treated with arseniteL-02 cells were transfected with pCDNA-PTEN and pCDNA for 24 h,then treated with 2.0 μM NaAsO₂ for 24 h,while the other two groups of L-02 cells were exposed to 0.0 or 2.0μM NaAsO₂ for 24 h respectively.Western blot detects levels of PTEN,p-ERK,mTOR,beclin 1 and LC3 II/I.To observe the effect of PTEN on ERK and autophagy-related proteins in L-02 cells treated with sodium arsenite.6.miR-21 through PTEN involved in arsenite-induced L-02 cells ERK activation and autophagy activationL-02 cells were co-transfected with 100 nM anti-miR-21 or anti-miR-nc and 25 nM PTEN siRNA or Con siRNA for 24 h,then treated with 2.0μM NaAsO₂ for 24 h.Western blot was used to detect the levels of PTEN,P-ERK,ERK,mTOR,beclin 1 and LC3.The number of autophagy in L-02 cells was detected by transmission electron microscopy.To observe the effect of miR-21 on the ERK activation and autophagy activation of L-02 cells induced by sodium arsenite by PTEN.7.Effects of chronic treatment of arsenite on autophagy of L-02 cellsL-02 cells were chronic treated with 0.0 or 2.0μM NaAsO₂ 30 passages（about 15 weeks）for transformation,getting control L-02 cells（L-02）and transformed L-02 cells（T-L-02）.The levels of autophagy-related proteins p62,beclin 1,LC3 II/I and ATG5 of L-02 cells and T-L-02 cells were detected by Western blot.The autophagic flux level was detected by mRFP-GFP-LC3 double adenovirus.L-02 cells were treated with 100 nM of Rapamycin for 24 h as positive control,then treated with L-02 cells and T-L-02 cells were observed by transmission electron microscopy.To observe the effect of chronic treatment on autophagy of L-02 cells.8.Effects of chronic treatment with arsenite on EMT in L-02 cellsL-02 cells were treated with 0.0 or 2.0μNaAsO₂,and L-02 cells were treated with L-02 cells（about 15 weeks）to obtain 0,10,20 and 30 control L-02 cells and arsenic transformed T-L-02 cells.The morphology of the cells was observed under microscope.The expressions of EMT proteins were detected by Western blot and immunofluorescence assay.To observe the effect of NaAso2 on EMT in L-02 cells.9.To investigate the effect of autophagic flux on EMT in L-02 cells treated with arseniteT-L-02 cells were treated with 2.0μM NaAsO₂ with 25 nM ATG5 siRNA or Con siRNA for 24 h,or treated with 100 nM Rapamycin for 24 h,then were treated with 0.0 or 2.0μM NaAsO₂ for 24 h.Autophagy-related proteins and EMT levels were measured by Western blot.To observe the effect of autophagy on the treatment of EMT in L-O₂ cells treated with NaAsO₂.10.p62 through Snail involved in arsenite induced EMT and malignant transformation in L-02 cellsT-L-02 cells were treated with 2.0μNaAsO₂ with 25 nM ATG5 siRNA or Con siRNA for 24 h.The expression of p62,Snail,E-cadherin and vimentin were detected by Western blot and immunofluorescence assay.The expression of p62,Snail,E-cadherin and vimentin were detected by immunohistochemistry.Soft agar colony experiments were used to detect the maligant degrees of T-L-02 cells.Transwell assay was used to detect invasion and metastasis properties of T-L-02 cells.L-02 cells were treated with 0.0 or 2.0μM NaAsO₂ for 24 h,and the combination of p62 and Snail in L-02 cells treated with NaAsO₂ was measured by Co-IP assay.The binding ability of p62 protein to Snail protein was detected by Co-IP assay in L-02 cells and T-L-02 cells.T-L-02 cells were observed by immunofluorescence co-localization to detect the expression of Snail and p62.These were to observe p62 control Snail in NaAsO₂-induced L-02 cells EMT and malignant transformation of the role.Results1.Effects of acute treatment of arsenite on autophagy level in L-02 cellsThe results showed that the levels of mTOR protein were significantly decreased in a concentration and time dependent manner in L-02 cells treated with NaAsO₂.The autophagy-related proteins beclin 1 and LC3 Ⅱ/Ⅰ increased gradually with a dose-and time-dependent effect.2.0μM NaAsO₂ increased the expression of LC3 and the number of autophagic vacuoles in L-02.The results indicate that NaAsO₂ can activate autophagy in L-02 cells.2.The role of ERK signaling pathway in the autophagy activation of L-02 cells induced by arseniteThe results showed that the level of p-ERK protein increased in a concentration and time dependent manner in L-02 cells treated with NaAsO₂.The levels of p-ERK,beclin 1 and LC3 II/I in L-02 cells treated with 2.0 μM NaAsO₂ were significantly higher than those in control L-02 cells,while the levels of PTEN and mTOR decreased,which indicate U0126 could reverse the changes of these proteins after arsenite treatment in L-02 cells.The results show that U0126 could down-regulate the autophagy-related proteins of L-02 cells induced by NaAsO₂,suggesting that ERK signaling pathway was involved in NaAsO₂-induced autophagy.3.Effects of arsenite on miR-21 level and its target proteins in L-02 cellsThe levels of miR-21 were significantly increased with the increase of NaAsO₂ concentration and time,while the levels of target proteins of miR-21 PTEN,PDCD4 and Spry 1 were significantly decreased with the increasing of NaAsO₂ in a dose and time effect.The results indicate that NaAsO₂ can upregulate miR-21 levels and inhibit the expression of its target proteins.4.The role of miR-21 in autophagy activation of L-02 cells induced by arseniteResults showed that the up-regulation of miR-21 could inhibit arsenite induced the increase of miR-21 beclin 1,LC3 II/I and p-ERK and the decrease of PTEN and mTOR.The results suggest that downregulation of miR-21 by anti-miR-21 can block the activation of ERK pathway and the increase level of autophagy induced by NaAsO₂ in L-02 cells,suggesting that miR-21 plays an important role in NaAsO₂-induced autophagy.5.Effects of PTEN on ERK and autophagy-related proteins in L-02 cells treated with arseniteThe results showed that overexpression of PTEN could significantly reverse the decrease of PTEN and mTOR and the increase of beclin 1,LC3 II/I and p-ERK induced by 2.0μM NaAsO₂ in L-02 cells.The results show that high expression of PTEN could inhibit the activation of ERK induced by NaAsO₂ and activate mTOR pathway and inhibit the expression of autophagy-related proteins,suggesting that PTEN plays an important role in the regulation of autophagy caused by NaAsO₂.6.miR-21 through PTEN involved in arsenite-induced L-02 cells ERK activation and autophagy activationThe results showed that down-regulation of miR-21 could block the decrease of PTEN and mTOR and the increase of beclin 1 and LC3II/I of ERK and autophagy-related proteins caused by arsenite.The knockdown of PTEN could reverse this process.Anti-miR-21 can reduce the number of autophagosomes,and the combination of PTEN siRNA can reverse this process.Results indicating that downregulation of miR-21-induced decrease levels of autophagy can be blocked by the PTEN siRNA,suggesting that the high expression of miR-21 inhibits PTEN and activates ERK,plays an important role in NaAsO₂-treated L-02 cell autophagy.7.Effects of chronic treatment of arsenite on autophagy of L-02 cellsThe results showed that the expression level of beclin 1,LC3 II/I and ATG5 in T-L-02 cells was significantly higher than that in control group（P<0.05）,and the expression of autophagic substrate protein p62 in T-L-02 cells was significantly higher than that control T-L-02 cells.The fluorescence intensity in T-L-02 cells were significantly higher than those of L-02 cells,and the ratio of yellow fluorescent spots and red fluorescent spots in T-L-02 cells was significantly lower than that in control cells.The ratio of autophagosome was increased while the ratio of autolysosome decreased in T-L-02 cells.The results indicating that inhibition of lysosome formation and block of autophagic flux in NaAsO₂-treated T-L-02 cells.8.Effects of chronic treatment with arsenite on EMT in L-02 cellsThe results showed that 2.0μM NaAsO₂ treated with 30-generation malignant transformation of L-02 cells,with the treatment time（malignancy degree increased）,the cell morphology gradually changed from the circular epithelial cells to the long spindle-like stromal cell-like cells.The expression of E-cadherin decreased and the level of vimentin was significantly increased after treatment,and the expression of EMT transcription factor Snail was also significantly increased,which indicating that NaAsO₂ chronic treatment induced EMT in L-02 cell.9.To investigate the effect of autophagic flux on EMT in L-02 cells treated with arseniteThe results showed that ATG5 siRNA inhibited the expression of beclin 1 and LC3 II/I in T-L-02 cells,but increased the level of p62 and blocked autophagic flux,which induced the increase of Snail and vimentin in T-L-02 cells and the decrease of E-cadherin.The expression of mTOR protein in T-L-02 cells was decreased by Rapamycin treatment,while the levels of beclin 1 and LC3 II/I were increased.The expression of p62 was inhibited,which indicates the block of autophagic flux.And the expression of Snail and vimentin were thereby inhibited,raising the level of E-cadherin.These results indicate that autophagic flux plays a key role in the NaAsO₂-induced EMT in L-02 cells.10.p62 by Snail involved in arsenite induced L-02 cells EMT and malignant transformationThe results showed that the protein expression and fluorescence intensity of p62,Snail and vimentin were decreased after p62 siRNA treatment,while the level of E-cadherin was increased.The colony formation number and cell invasion and metastasis of T-L-02 cells in p62 siRNA were significantly lower than the control T-L-02 cells.The binding of p62 and Snail protein was observed in 2.0μM NaAsO₂ acute treated L-02 cells and chronic treated T-L-02 cells.Those results indicate that p62 through Snail involved in NaAsO₂-induced L-02 cells EMT and malignant transformation.Conclusions1.miR-21 via PTEN activating of ERK signaling is involved in arsenite-induced autophagy in human hepatic cells.2.Arsenite-induced the blockage of autophagic flux,which induces the increases of p62 level and the accumulation of Snail,plays an important role in EMT and the malignant transformation of L-02 cells induced by arsenite.