| Objective Inorganic arsenic compounds are identified human carcinogens,but due to animal carcinogenic models have not been successfully established,leading to the long-term unrevealed of its exact molecular cellular mechanism involved in arsenic toxicity.The in vitro cell model of malignant transformation of cells has been widely used for the oncogenic action and mechanism of inorganic arsenic compounds.In the hypothesis of many arsenic carcinogenic mechanisms proposed in recent years,the oxidative stress theory has received extensive attention.The previous study of our lab found that oxidation-anti-oxidation imbalance during the malignant transformation of cells,continuous expression of nuclear transcription factor Nrf2 in the anti-oxidation signal pathway Keap1-Nrf2/ARE,accelerating cell proliferation and resisting cell death during arsenic-induced malignant transformation Plays a very important role,but the specific molecular mechanism is not yet clear.In this study,a model of malignant transformation of immortalized human keratinocyte(Ha Ca T)cells was induced by the long-term exposure of 1.0 μmol/L sodium arsenite(Na As O2)to study the dynamic changes of autophagy during arsenic-induced malignant transformation of cells.And further explore the role of nuclear transcription factor Nrf2 on autophagy and its regulation mechanism,providing a new scientific basis for the molecular mechanism of arsenic carcinogenesis.Methods 1.Dynamic changes of autophagy during different passages of malignant transformation Ha Ca T cells induced by Na As O2:(1)Western blot was used to detect 0、1、7、14、21、28,and 35 generations of Ha Ca T cells exposure to Na As O2 at a concentration of 0.0 and 1.0 μM.The expression levels of the autophagy-related proteins Beclin1、LC3II/LC3 I and p62 were detected;(2)The RFP-LC3 was transfected with the lentiviral virus,and the dynamic changes of autophagic expression at 1、4 and 35 generations after exposure to 0.0 and 1.0 μM Na As O2 were observed by fluorescence microscope.(3) Western blot was used to detect T-Ha Ca T which transformed from normal Ha Ca T after treatment with 1.0μM Na As O2 for 35 generations.Interference with Bafilomycin A1(Baf A1),Rapamycin and Wortmannin were used to identified changes of autophagic flux.2.The may function of Nrf2 on Autophagy during Ha Ca T cells transformation:(1)Oxidative damage during the malignant transformation of Ha Ca T cells and related indicators of Keap1-Nrf2/ARE signal pathway detection: a 、 Detection of hydrogen peroxide,superoxide,4-HNE,MDA,and SOD by microplate reader Changes in 0、1、7、14、21、 28 and 35 passages of Ha Ca T cells treated with 0.0 μmol/L and 1.0 μmol/L Na As O2;b、The expression of Nrf2 and its downstream antioxidant protein HO-1 was detected by Western blot.(2)The possible role of nuclear transcription factor Nrf2 in the regulation of autophagy in Ha Ca T cells: a、Transfection with Nrf2 si RNA into T-Ha Ca T cells to reduces the expression of Nrf2 in cells,and the corresponding normal passage control group,malignant transformation control group and Con si RNA group were set as negative control group,positive control group and blank control group.Western blot was used to detect the expression level of Nrf2 and autophagy-related proteins Beclin1、LC3II/LC3 I and p62;b、in T-Ha Ca T cells after the autophagy activator Rapamycin was added,the expression levels of autophagy-related proteins Beclin1、LC3II/LC3I、p62 and Nrf2 were detected by Western blot.3.The possible mechanism of regulation of autophagy by the transcription factor Nrf2 during malignant transformation of Ha Ca T cells:(1)Nrf2 transcription factor regulates the initiation of autophagy through the Nrf2-m TOR signaling pathway: a、Western blot detection of 0.0 and 1.0 μM Na As O2 infected Ha Ca T cells 0、1、7、14、21、28,and 35 passages the expression of PI3K/Akt/m TOR signaling pathways in cells;b、Transfection with Nrf2 si RNA in T-Ha Ca T cells to reduce the expression of Nrf2 in cells and the corresponding normal passage control group,malignant transformation control group and Con si RNA group were set as negative control group,positive control group and blank control group respectively.Western blot was used to detect the expression levels of p-Akt、p-m TOR and autophagy marker proteins Beclin1、LC3II/LC3I、p62.c、PI3K inhibitor LY294002 was added to T-Ha Ca T cells to inhibit the expression of PI3 K,then the expression of p-Akt、p-m TOR、Nrf2、Beclin1、LC3II/LC3I、p62 were detected by Western blot.d、After adding m TOR inhibitor Rapamycin to inhibit the expression of m TOR in T-Ha Ca T cells,the expression changes of Nrf2、Beclin1、LC3II/LC3 I and p62 in the cells were detected by Western blot.(2)The transcription factor Nrf2 regulates the expression of autophagy by Nrf2-Bcl2-Beclin1: a、Western blot was used to detect the expression of Bcl2 in Ha Ca T cells of 0、1、7、14、21、28 and 35 th generations with 0.0 μmol/L and 1.0 μmol/L Na As O2;b、Transfection of Nrf2 si RNA in T-Ha Ca T cells,which reduced the expression of Nrf2 in cells,the corresponding normal passage control group,malignant transformation control group and Con si RNA group were set as negative control group,positive control group and blank control group,and then the expression levels of Bcl2 and autophagy markers Beclin1、LC3II/LC3 I and p62 were detected by Western blot in negative control group,positive control group and blank control group respectively.c.Transfection of Bcl2 si RNA into T-Ha Ca T cells decreased the expression of Bcl2 in the cells.At the same time,the corresponding normal passage control group,malignant transformation control group and Con si RNA group were set as negative control group,positive control group and blank control group.And blank control group,the expression levels of Nrf2 and autophagy markers Beclin1、LC3II/LC3 I and p62 were detected by Western blot.Results 1.Changes of autophagy during malignant transformation of Ha Ca T cells:(1)Results showed that compared the control group,the amount of autophagy marker proteins Beclin1,LC3II/LC3 I expressed in Ha Ca T cells with 1.0 μM Na As O2 at passages 1 to 14 were higher than normal passages(p<0.05),after 14 passages,the expression levels of Beclin1 and LC3II/LC3 I began decrease,and at the 35 passage the level of Beclin1 and LC3II/LC3 I were significantly lower when compare to the passage 0 cells(p<0.05).At the same time,the autophagic degradation protein p62 showed a tendency of increasing along with the Na As O2 exposure,after 14 passages,the expression level of p62 remained higher than the normal control cells(p<0.05).(2)The fluorescence spots of RFP-LC3 lentivirus showed that the LC3 fluorescence spots in the cells were higher than those of the normal passages control cells(p<0.05)aslo higher than 1 generation when the Ha Ca T cells were exposed to 1.0μmol/L Na As O2 for 14 generations(p<0.05);when the Ha Ca T cells were exposed to 1.0μmol/L Na As O2 for 35 generations,the numbers of LC3 fluorescence spots in the cells were significantly lower than the normal passages control cells and the passage 1 cells(p<0.05).b: In T-Ha Ca T cells and normal passage control cells,8 μmol/L Na As O2 was added at the same time,it was found that after adding Wortmannin,the expression of LC3II/LC3 I in T-Ha Ca T and normal passage control cells was significantly decreased(p<0.05);after adding Baf A1,the expression level of LC3II/LC3 I was significantly increased in both cells(p<0.05),but still lower in T-Ha Ca T cells when compared with normal control group(p<0.05).After adding autophagy activator Rapamycin,it was found that LC3II/LC3 I was significantly increased in both T-Ha Ca T and passaged control cells(p<0.05),still the expression of LC3II/LC3 I in T-Ha Ca T cells was lower than the passage control group(p<0.05).2.The may function of transcription factor Nrf2 in malignant transformation of Ha Ca T cells:(1)From 1st to 14 th generation,the activity of hydrogen peroxide and superoxide in the cells of the Na As O2 exposed group was significantly higher than that in the normal control group(p<0.05);after 14 generations,the exposure group of the hydrogen peroxide and superoxide gradually were decreased,and there was no significant difference in activity at the 35 th generation when compared with the normal passage control group.The MDA content peaked at the first passage after 1.0 μM Na As O2 treatment,and then gradually decreased after 14 passages,there was no significant difference compared with the normal passage control group.In the first generation of exposure,the intracellular 4-HNE content increased rapidly,reached its peak,and then decreased gradually,compared with the normal control group,the difference was not statistically significant after generation of 7.(2)a、The expression of Nrf2 and p62 in T-Ha Ca T cells transfected with T-Ha Ca T cells was significantly lower than that before transfection(p<0.05),and autophagy-related proteins Beclin1 and LC3 II /LC3 I expression increased(p<0.05);b、Intracellular autophagy-related proteins Beclin1 and LC3II/LC3 I were detected in T-Ha Ca T cells after treatment with autophagy activator Rapamycin,the level of Beclin1 and LC3II/LC3 I were significantly increased compared with untreatment T-Ha Ca T cells,while the expression of p62 and Nrf2 were not significant change.3.The possible mechanism of regulation of autophagy by the transcription factor Nrf2 during malignant transformation of Ha Ca T cells:(1)Along with the arsrnite exposurement,the p-PI3K/PI3K、p-Akt/Akt、and p-m TOR/m TOR protein were increased,after 14 generations they remained stable at higher levels and were all higher than normal control cells(p<0.05).After transfected with Nrf2 si RNA in T-Ha Ca T cells,the expression of p-PI3 K and p-Akt had no significant change compared with untransfection group,the protein level of p-m TOR and p62 expression were decreased(p<0.05),while the protein expression of Beclin1 and LC3II/LC3 I was significantly increased(p<0.05).After addition of PI3 K inhibitor LY294002 to T-Ha Ca T cells,compared with the untreated Ha Ca T group,the expression levels of p-Aktp-m TOR,and Nrf2 in the treated group were decreased(p<0.05),while the expression of Beclin1 and LC3II/LC3 I protein was increased(p<0.05)there was no significant difference in the expression of p62.After adding the m TOR inhibitor Rapamycin into T-Ha Ca T cells,the protein expression of Nrf2 and p62 in the treated group was not significantly changed compared with the untreated T-Ha Ca T cells,while the protein expression levels of Beclin1、LC3II/LC3 I were significantly increased.High(p<0.05).(2)After long-term exposure to 1.0 μM Na As O2,Bcl2 in cells in the 1.0 μmol/L Na As O2 exposure group continued to increase,and the expression level was significantly higher than that in the normal control group(p<0.05).After Nrf2 si RNA was transfected into T-Ha Ca T cells,the expression of Bcl2 and p62 in T-Ha Ca T cells was significantly decreased compared with that before transfection(p<0.05).The protein expression level of Beclin1 and LC3 II /LC3 I was significantly increased(p<0.05);after Bcl2 si RNA was transfected into T-Ha Ca T cells and Bcl2 was significant decreased compared with that un-transfected group,there was no significant change of the levels of Nrf2 and p62 protein in T-Ha Ca T cells of transfected group,but the protein expression of Beclin1 and LC3II/LC3 I was significantly increased after transfection(p<0.05).Conclusions 1.The autophagic defect during arsenite-induced Ha Ca T cells malignant transformation as characterized by a decrease in the activation ability of the autophagy initiation phase and a suppression of the autophagic flux degradation phase.2.The high expression of nuclear transcription factor Nrf2 during malignant transformation of arsenic-induced Ha Ca T cells is responsible for the abnormality of intracellular autophagy.3,Arsenite exposure activates PI3K/Akt signaling pathway,which leads to the continuous high expression of nuclear transcription factor Nrf2,then regulates cell autophagy through Nrf2-m TOR and Nrf2-Bcl2-Beclin1 signaling pathway. |
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