The Role Of MiRNA-21/PTEN/mTOR Pathway In Gspe Inhibiting Arsenic-Induced Malignant Transformation Of L-02 Cells

Objective:Arsenic?Arsenic,As?can affect the proliferation of hepatocytes by regulating the expression of miRNA-21 gene and changing the activity of PTEN/mTOR pathway;grape seed proanthocyanidins?Grape seed proanthocyanidin extract,GSPE?can inhibit the oxidative stress toxicity caused by arsenic.However,it has not been reported whether proanthocyanidins can inhibit the malignant proliferation of hepatocytes induced by arsenic through miRNA-21/PTEN/mTOR pathway.Therefore,in this study,grape seed proanthocyanidins and arsenic were used to interfere with liver L-02 cells to verify the regulation of proanthocyanidins on miRNA-21/PTEN/mTOR pathway and the role of miRNA-21/PTEN/mTOR pathway in proanthocyanidins inhibiting arsenic-induced hepatocyte malignant proliferation.To explore whether proanthocyanidins antagonize the malignant proliferation of hepatocytes induced by arsenic through miRNA-21/PTEN/mTOR pathway,so as to provide theoretical basis for improving the mechanism of proanthocyanidins.Methods:In this experiment,L-02 cells were treated with As³⁺?2?mol/L?,GSPE?50mg/L?,miRNA-21 inhibitor,and Pten siRNA for 24 hours.The expressions of miRNA-21,mTOR,PTEN,PI3K,AKT,and PCNA,Ki-67 were measured.The CCK-8 analysis of cell viability,transwell analyses,and cell stripes was used to analyze the effects of arsenic on cell proliferation and apoptosis.QRT-PCR was used to detect the expression of miRNA-21 in cells by GSPE,miRNA-21,and PTEN siRNA inhibitors;Western Blot was used to detect the expression of mTOR,Pten,PI3K,AKT,PCNA,and Ki-67.Gap analysis was used to compare the differences between groups and the interaction between GSP and GSP.Several comparisons between groups were made using the Dunnett method.Results1.Effects of GSPE and As on cell morphology and activity.In the intervention experiment of different concentrations of As or GSPE,2?mol/L?146.866±2.957?and 6?mol/L?95.850±16.174?As could change the cell activity,increase the cell quantity and increase the cell activity.GSPE at 25 mg/L and 50 mg/L had no significant effect on cell morphology and activity,but cell activity was significantly reduced after treatment with 75mg/L?54,456±13.870?GSPE for 24 hours.2.Compared with the control group?46.333±7.234?,GSPE and As could significantly increase the invasive ability of hepatocytes treated with NaAsO₂?93.333±9.292?,but there was no significant difference between the GSPE treated group?57.333±2.309?and the control group?46.333±7.234?.Compared with the group treated with NaAsO2 alone?93.333±9.292?,the invasive ability of the cells treated with NaAsO₂ plus GSPE?59.333±7.094?was significantly decreased?P<0.05?.The results also showed that compared with the control group?63.333±6.658?,NaAsO₂ treatment alone?90.333±11.590?could significantly increase the migration ability of liver Lmur0₂ cells?P<0.05.The?;GSPE treatment group?51.667±6.028?had no significant effect on the migration ability of liver L-02 cells?P>0.05?.Compared with the group treated with NaAsO₂ alone?90.333±11.590?,the ability of cell migration decreased significantly in the group treated with NaAsO₂ plus GSPE?57.333±12.741?.3.The effect of GSPE and As on malignant proliferation of cells.The results showed that the scratch healing rate of NaAsO₂ group?18.249±5.050?was significantly higher than that of control group?4.638±2.85?after 12 hours treatment,but there was no significant change in GSPE group?11.277±6.024?.However,compared with the NaAsO₂ group?18.249±5.050?,the scratch healing rate of the NaAsO₂ plus GSPE group?7.248±4.829?was significantly lower than that of the control group?P<0.05?.After 24 hours of treatment,compared to the control group?26,431±4.517?,there were no significant changes in the healing rate of cells treated only with NaAsO₂?20.709±15.083?and GSPE?17.665±7.383?,whereas the single NaAsO₂ treatment group?20.709±15.083?,the NaAsO₂+GSPE combined treatment group?21.945±15.083?had no significant effect.After 48 hours of treatment,compared with the control group?30.997±2.421?,the scratch healing rate of the NaAsO₂ group?38.053±7.396?had no significant effect,but the results showed that the scratch healing rate of the GSPE group?25.809±8.469?had no significant effect on the scratch healing rate of the cells,while the scratch healing rate of the control group?38.053±7.396?had no significant effect on the scratch healing rate,while the scratch healing rate of the control group?25.809±8.469?had no significant effect on the scratch healing rate.Compared with the NaAsO₂ group?38.053±7.396?,the scratch healing rate of the NaAsO₂+GSPE group?24.909±3.741?was significantly lower than that of the control group?P<0.05?.4.The regulation of miRNA-21/mTOR/PTEN pathway by GSPE and As.Compared with the control group,As increased the expression of miRNA-21 mRNA,and GSPE decreased the expression of miRNA-21 mRNA.Compared with the As treatment group,the co-intervention group of As and GSPE decreased the expression of miRNA-21 mRNA?1.61±0.1??P<0.05?.Compared with the control group,miRNA-21 inhibitor decreased the expression of miRNA-21?0.3±0.05?,PTEN siRNA had no significant effect on the expression of miRNA-21).Compared with the control group,miRNA-21 inhibitor decreased the expression of miRNA-21,PTEN siRNA had no significant effect on the expression of miRNA-21),and the co-intervention group of As and PTEN siRNA had no significant effect on the expression of miRNA-21 compared with the group treated with As alone.Compared with the co-intervention group of As and GSPE,the co-intervention group of As,GSPE and miRNA-21 inhibitor significantly decreased the expression of miRNA-21?0.2±0.01??P<0.05,As?.The co-intervention group of GSPE and PTEN siRNA had no significant effect on the expression of miRNA-21?P>0.05?.Compared with the co-intervention group of As,GSPE and miRNA-21 inhibitor,the co-intervention group of As,GSPE,miRNA-21 inhibitor and PTEN siRNA had no significant effect on the expression of miRNA-21.Compared with the control group,As increased the expression of mTOR,PTEN and p-AKT,but had no significant change in the expression of PI3K and AKT.Compared with the simple As intervention group,the co-intervention group of As and GSPE inhibited the activities of mTOR,PTEN and p-AKT,but had no significant effect on the expression of PI3K and AKT.Compared with the co-intervention group of As and GSPE,the co-intervention group of As,GSPE and miRNA-21 inhibitor inhibited the expression of mTOR,PTEN,PI3K and p-AKT?P<0.05?.The co-intervention group of GSPE and PTEN siRNA inhibited the expression of mTOR,PTEN,PI3K and p-AKT?P<0.05?.5.Effects of GSPE and As on PCNA and Ki-67 in cells.Compared with the normal control group,the expression of PCNA and Ki-67 in As treated group increased?P<0 05?.Compared with the group treated with As alone,the co-intervention group of As and GSPE decreased the expression of PCNA and Ki-67?P<0.05?.Compared with the co-intervention group of As and GSPE,the co-intervention group of As,GSPE and miRNA-21 inhibitor decreased the expression of PCNA?P<0.05?,but did not change the level of Ki-67?P>0.05?.The co-intervention group of);As,GSPE and PTEN siRNA decreased the expression of PCNA?P<0.05?.ConclusionArsenic can up-regulate the activity of miRNA-21/PTEN/mTOR pathway,increase the expression of PCNA and Ki-67,thus induce malignant proliferation of hepatocytes,while proanthocyanidins can inhibit the expression of miRNA-21.When the expression of miRNA-21 is down-regulated,it further reduces the activity of PTEN/mTOR pathway,and finally achieves the purpose of antagonizing arsenic-induced malignant proliferation of hepatocytes.These results suggest that proanthocyanidins can antagonize the malignant proliferation of hepatocytes induced by arsenic through miRNA-21/PTEN/mTOR pathway.