| Neuroblastoma is a malignant common in young children 15 years old people in solid tumors.According to statistics,because the tumor is prone to metastasis,the tumor resection rate of domestic children is less than 50%,although the new chemotherapy regimen makes the survival rate of children slightly improved,but the disease-free survival rate is still less than 30%.Finding a new approach to anti neuroblastoma or targeting drugs with low side effects has become a key problem to be solved.Glutathione peroxidase is a series of peroxidase activity in widespread,it can be iros too much（ROS）reduced to non-toxic compounds catalyzed by H2O2,and decomposed into water,thereby reducing oxidative damage to cell peroxidase.GPX4 is the most important subunit of this enzyme system.It distributes various membrane structures in the cell and eliminates various lipid peroxides.Recent studies have shown that down regulation of GPX4expression can induce tumor cell growth by increasing the sensitivity of tumor cells to chemotherapeutic drugs by inducing ferroptosis to occur.Ferroptosis is a newly found iron dependent programmed cell death pattern,which is significantly different from apoptosis.Erastin ferroptosis is a small molecule activator,via downregulation of GPX4 resulted in intracellular accumulation of lipid peroxide induced cell death,and this process by ferroptosis inhibitor,but does not affect the apoptosis of tumor cells induced by chemotherapeutic drugs.At present,most chemotherapy drugs still play an anti-tumor role by inducing tumor cell apoptosis,but the tumor cells will become resistant to the increase of drug use time.The discovery of Ferroptosis will provide a new direction for further study of antitumor pathway.Cisplatin is a non periodic wide anticancer spectrum and strong effect of antitumor drugs,combined with a variety of chemotherapy drugs had no cross resistance,has been used as first-line chemotherapy in neuroblastoma in combination chemotherapy.The study found that cisplatin could induce the formation of ferroptosis in human colon cancer cells and non-small cell lung cancer cells.Ferroportin（Fpn）is the only iron export protein in cells.It plays an indispensable role in iron transmembrane output and regulates iron homeostasis.Alterations in Fpn expression may result in iron deficiency or iron overload in[4-6].The accumulation of intracellular iron is associated with the deficiency of Fpn and promotes the occurrence of ferroptosis by increasing the lipid ROS produced by Feton reaction.Fpn,like GPX4,is also an independent risk factor for cancer and is associated with reduced survival in patients with distant metastases.Previous studies have shown that Fpn expression is reduced in cancer cells.However,the potential role of Fpn in ferroptosis induced by erastin in neuroblastoma cells remains to be explored.It is not clear whether Fpn can play a synergistic anti-tumor role with GPX4.Whether the expression of GPX4 exists in neuroblastoma tissues of children,whether the down-regulation of GPX4 causes the death of human neuroblastoma cell lines,and how it plays a role in the way of death,and how it affects the sensitivity of tumor cells to cisplatin have not been discussed.Therefore,we intend to investigate the expression of GPX4 in human neuroblastoma tissues and cell lines in vitro,to study the effect of silencing GPX4 gene by RNA interference on the proliferation of human neuroblastoma cells and its mechanism,and to study whether downregulation of GPX4affects the sensitivity of cells to cisplatin.To investigate whether the combination of ferroptosis inducer and cisplatin can enhance the antitumor activity by down-regulating GPX4,and to further explore the role of Fpn in regulating GPX4 inhibitor erastin-induced neuroblastoma cell death,in order to provide a new target for the treatment of neuroblastoma in children.Part one Expression of GPX4 in human neuroblastoma tissues and cellsObjective:To observe the expression of GPX4 gene and protein in children neuroblastoma tissue and human neuroblastoma SK-N-SH cell line,and to explore whether neuroblastoma cells can be used as targets to induce the occurrence of ferroptosis.Methods:We collected 42 clinical specimens from children with neuroblastoma,used for paraffin section,and routinely cultured human neuroblastoma cell line SK-N-SH.The expression of GPX4 gene in tissues and cells was detected by RT-qPCR,and the expression of GPX4 protein in tissues and cells was detected by immunohistochemistry,immunocytoch-emistry and Western blot.Results:1.The results of immunohistochemistry showed that in the 42 specimens of the 42 cases of neuroblastoma,the cytoplasm of the tumor cells in 39specimens was filled with brown yellow granules,that was,the positive expression was GPX4 positive,the positive expression rate was 92.86%.The positive expression of GPX4 in the para cancerous tissues of the 25 specimens showed that the positive expression rate was 59.52%.2.Immunocytochemistry results showed that GPX4 was mainly expressed in cytoplasm of SK-N-SH cells.3.The results of Western Blot showed that the expression of GPX4protein was detected in 42 cases of neuroblastoma,while 19 cases in the paracancerous tissue did not detect the expression of GPX4 protein,and 23cases of para cancer tissues were low expression,obviously lower than the relative expression of tumor tissue,and the difference was statistically significant（P<0.01）.The expression of GPX4 target band was also detected in SK-N-SH cells.4.The results of RT-qPCR showed that the expression of GPX4 mRNA was detected in 42 cases of neuroblastoma,while 13 in the paracancerous tissue did not detect the expression of GPX4 mRNA,and the GPX4 mRNA in the adjacent tissues was low,and the relative expression was significantly lower than the relative expression of the tumor tissue,and the difference was statistically significant（P<0.01）.The expression of GPX4 mRNA was also detected in SK-N-SH cells.Summary:1.GPX4 mRNA and protein were expressed in human neuroblastoma SK-N-SH cell line and pediatric neuroblastoma tissue.2.The expressions of GPX4 mRNA and protein in pediatric neuroblastoma tissues were significantly higher than that in normal tissues.Part two Effect and mechanism of down-regulation of GPX4 onprolifera-tion of human neuroblastoma cellsObjective:To investigate the effect of down-regulation of GPX4 gene on the proliferation of neuroblastoma cell lines and its possible mechanism from the morphological and molecular biological levels of cell proliferation and apoptosis,intracellular lipid peroxidation and intracellular iron content.Methods:SK-N-SH cells were randomly divided into 7 groups,（1）blank control group:only adding transfection reagent;（2）negative control group:transfection reagent and control siRNA-A;（3）unrelated control group:no siRNA and transfection reagent;（4）positive control group:transfection reagent and beta-actin siRNA;experimental group:6.25 nM,12.5 nM and 25nM GPX4 siRNA transfection group.After collecting interference 24,48 and72h,the cells were collected,and RNA and protein were extracted.The expression of GPX4 mRNA in all cells after interference was detected by RT-qPCR;Western blot was used to detect the expression of GPX4 protein in each group of cells before and after transfection;the effective transfection concentration of GPX4 siRNA in subsequent experiment was determined by inverted fluorescence microscope,and the proliferation activity of each cell was detected by MTT method;flow cytometry was used to detect cell apoptosis;The cell apoptosis in each group was detected by TUNEL.Flow cytometry was used to detect the level of ROS in each group,and the activity of MDA,glutathione,iron ion and GPXs were detected by biochemical method,and the changes of mitochondria structure in the cells were observed by transmission electron microscope.Results:1.To evaluate the efficiency of RNA interference.⑴The results of real-time quantitative PCR detection showed that the expression of GPX4mRNA in the cells of the unrelated control group,the blank control group,the positive control group and the negative control group did not change obviously,and the expression of GPX4mRNA in the experimental A group cells with 6.25 nM GPX4 siRNA interference after 48 and 72 h was lower than that in the unrelated control group,the blank control group,the positive control group and the negative control group.In the control group,there was no significant difference（P>0.05）,and the expression of GPX4mRNA in the experimental group B with 12.5 nM GPX4 siRNA interference 24,48 and 72h was significantly lower than that in the unrelated control group,the blank control group,the positive control group and the negative control group.The expression of 25 nM GPX4 siRNA interference in the experimental C group was significantly lower than that of the unrelated control group,24,48 and72h.In the blank control group,the positive control group and the negative control group,the difference was statistically significant（P<0.05）,and the expression of GPX4mRNA was significantly lower than that of interference72 h after interference of 48 h in the experimental group（P<0.05）,and the expression of GPX4mRNA after interference of 48 h in the experimental C group was significantly lower than that of 72 h（72 h）,and the difference was statistically significant.（P<0.05）;the expression of GPX4mRNA in experimental B group after 48 h interference was higher than that in the experimental C group after 48 h interference,the difference was not statistically significant（P>0.05）.⑵The results of Western blot detection showed that there was no significant change in the expression of GPX4protein in the cells of the control group,the blank control group,the positive control group and the negative control group after interference 24,48 and 72 h（P>0.05）.The expression of GPX4 protein in the experimental A,B,C group cells after interference 48 and 72 h were lower than those in the unrelated control group and the blank control group.In the positive control group and the negative control group,the expression of GPX4 protein was significantly lower than that of interference 48 h after 72 h interference（P<0.05）,and the expression of GPX4 protein in the B group was significantly lower than that of the experimental A group after interference of 48 and 72 h（P<0.05）in the B group,and the protein expression in the experimental B group was lower than the interference 48 after 72 h.But the difference was not statistically significant（P>0.05）,and the expression of GPX4 protein in the experimental group C was significantly lower than that of the experimental B group after interference of 48 h and 72 h（P<0.05）.The expression of GPX4 protein in the experimental C group after 72 h was significantly lower than that of the interference 48 h.The difference was statistically significant（P<0.05）.Morphological observation showed that 12.5 nM GPX4 siRNA group and 25nM GPX4 siRNA group interfered for 72 h and the number of suspended cells increased.2.The results of MTT test showed that the transfection reagent had no obvious effect on the survival rate of the cells,and the cell survival rate decreased significantly after the transfection of GPX4 siRNA,and the survival rate of the cells in the experimental group was significantly lower than that in the unrelated and negative control groups（P<0.01）.3.The results of flow cytometry showed that the transfection reagent had no obvious effect on cell apoptosis,and the apoptosis rate of GPX4 siRNA was significantly increased after transfection of GPX4,and the apoptosis rate of the experimental group was significantly higher than that of the unrelated control group and the negative control group（P<0.05）.4.The results of TUNEL detection showed that the number of apoptotic cells increased significantly after transfecting GPX4 siRNA with GPX4,and the number of apoptotic cells in the experimental group was significantly higher than that in the unrelated control group and the negative control group（P<0.05）.5.The results of flow cytometry showed that the transfection reagent had no obvious effect on the intracellular ROS level,and the level of ROS in the cells increased obviously after the transfection of GPX4 siRNA with GPX4,and the level of intracellular ROS in the experimental group was significantly higher than that in the unrelated control group and the negative control group（P<0.05）.6.The transfection reagent had no obvious effect on the intracellular GSH content and GPXs activity.After transfection of GPX4 siRNA,the content of GSH in the cells decreased obviously.The content of GSH in the cells in the experimental group was significantly lower than that in the unrelated control group and the negative control group（P<0.05）.,and the GPXs activity in the cells decreased significantly.The activity of GPXs in the cells of the test group was significantly lower than that in the experimental group.Control group and negative control group（P<0.05）.7.The transfection reagent had no obvious effect on the intracellular MDA content,and the content of MDA in the cells increased significantly after transfecting GPX4 siRNA with GPX4,and the content of MDA in the experimental group was significantly higher than that of the unrelated control group and the negative control group（P<0.05）.8.The transfection reagent had no obvious effect on the iron content in the cells,and there was no obvious change in the iron content in the cells after transfection of GPX4 siRNA,and there was no significant difference in the content of iron in the experimental group from that of the unrelated control group and the negative control group（P>0.05）.9.The transfection reagent had no obvious effect on the morphology of mitochondria in the cells.After transfecting GPX4 siRNA,the mitochondria were obviously smaller,the mitochondrial membrane was thickened and the electron density increased.Summary:1.Down-regulation of GPX4 reduced the survival rate of SK-N-SH cells and increased the apoptotic rate.2.Down-regulation of GPX4 reduced GSH content in SK-N-SH cells,increased intracellular ROS and lipid peroxidation levels,decreased GPXs activity,and altered intracellular mitochondrial morphology.Part three Down regulation of GPX4 can enhance the sensitivity ofhuman neuroblastoma to cisplatinObjective:Firstly,the possible way of down-regulation of GPX4inducing SK-N-SH cell death was observed.Then,the sensitivity of SK-N-SH cells to cisplatin was observed by the intervention of cisplatin.Finally,GPX4inhibitor was combined with cisplatin to observe the synergistic anti-tumor effect and to explore whether down-regulation of GPX4 could improve the sensitivity of SK-N-SH cells to cisplatin.Methods:Ferroptosis inhibitor,inhibitor of apoptosis and inhibitor of autophagy were used to treat SK-N-SH cells.After 48 h,cells were collected for further experiments.The cells were divided into control group,vehicle group,z-VAD-fmk group,Fer-1 group,Necrostatin-1 group,3-MA group,group of control,vehicle,cisplatin and cisplatin+siRNA group,（3）control group,vehicle group,cisplatin group,Erastin group and cisplatin group.The cell proliferation activity was detected by MTT method,the cell death rate was detected by the kit method,and the lipid peroxidation level,glutathione level,iron ion content and GPXs activity in the cells were detected by biochemical method.Results:1.The death mode of SK-N-SH cells induced by down-regulation of GPX4:⑴The results of MTT assay showed that compared with group control,the cell proliferation rate of vehicle,z-VAD-fmk,Fer-1,Necrostatin-1 and3-MA groups decreased significantly（P<0.05）.Compared with the vehicle group,the cell proliferation rate of Fer-1 groups increased significantly（P<0.05）.Compared with z-VAD-fmk group,the cell proliferation rate of Fer-1 group increased significantly（P<0.05）.⑵Compared with the control group,the activity of Caspase-3 in vehicle,z-VAD-fmk,Fer-1,Necrostatin-1and 3-MA groups increased significantly（P<0.05）.Compared with the vehicle group,the cell Caspase-3 activity of z-VAD-fmk group was significantly lower than that in the vehicle group（P<0.05）.There was no significant difference in cell Caspase-3 activity（P>0.05）.⑶Compared with the control group,the lipid peroxidation level of vehicle,z-VAD-fmk and Fer-1 groups increased significantly（P<0.05）.Compared with the vehicle group,there was no significant difference in lipid peroxidation in the z-VAD-fmk group（P>0.05）,and the level of lipid peroxidation in the Fer-1 group decreased significantly.Low（P<0.05）;compared with z-VAD-fmk group,the level of lipid peroxidation in Fer-1 group was significantly lower（P<0.05）.⑷Compared with the control group,the iron content of vehicle,z-VAD-fmk and Fer-1 groups was not significantly different（P>0.05）.Compared with the vehicle group,there was no significant difference in the iron content in the z-VAD-fmk group（P>0.05）,and the iron content in the Fer-1 group decreased significantly（P<0.05）,compared with the z-VAD-fmk group.The iron content in group Er-1 was significantly decreased（P<0.05）.2.Effect of down-regulation of GPX4 on the sensitivity of cisplatin in SK-N-SH cells:⑴Compared with the control group,the cell mortality of cisplatin and cisplatin+siRNA group was significantly higher than that in the control group（P<0.05）.Compared with the vehicle group,the cell mortality of cisplatin group and cisplatin+siRNA group increased significantly（P<0.05）.Compared with cisplatin group,the cell mortality of cisplatin+siRNA group increased significantly（P<0.05）.⑵The lipid peroxidation level of cell lipid peroxidation showed that the lipid peroxidation level of cisplatin group and cisplatin+siRNA group increased significantly compared with control group（P<0.05）.Compared with group vehicle,the lipid peroxidation level of cisplatin group and cisplatin+siRNA group increased significantly（P<0.05）.Compared with cisplatin group,cisplatin+siRNA group was fine.The level of lipid peroxidation increased significantly（P<0.05）.⑶Compared with group control,the cell GSH content of cisplatin group and cisplatin+siRNA group decreased significantly（P<0.05）.Compared with group vehicle,the content of GSH in cisplatin group and cisplatin+siRNA group decreased significantly（P<0.05）Compared with cisplatin group,the GSH content of cisplatin+siRNA group was significantly lower than that of cisplatin group（P<0.05）.⑷Compared with the control group,the cell GPXs activity of cisplatin group and cisplatin+siRNA group decreased significantly（P<0.05）,compared with the group vehicle,the cell GPXs activity of cisplatin group and cisplatin+siRNA group decreased significantly（P<0.05）,and the cell GPXs activity of cisplatin+siRNA group was lower than that of cisplatin group compared with that of group vehicle,and the cell GPXs activity of cisplatin+siRNA group was significantly lower than that of the group of cisplatin and cisplatin（P<0.05）.3.Effect of erastin on the sensitivity of cisplatin in SK-N-SH cells:⑴Compared with the control group,the cell mortality of cisplatin group,Erastin group and cisplatin+Erastin group increased significantly（P<0.05）.Compared with the vehicle group,the cell mortality of cisplatin group,Erastin group and cisplatin+Erastin group increased significantly（P<0.05）.Compared with cisplatin group,the cell death of cisplatin+Erastin group was more than that of cisplatin group.The mortality rate increased significantly（P<0.05）.Compared with Erastin group,the cell death rate of cisplatin+Erastin group increased significantly（P<0.05）.⑵Compared with the control group,the lipid peroxidation level of cisplatin group,Erastin group and cisplatin+Erastin group increased significantly（P<0.05）.Compared with the vehicle group,the lipid peroxidation level of cisplatin group,Erastin group and cisplatin+Erastin group increased significantly（P<0.05）,and cisplatin group and cisplatin+Erastin group（P<0.05）.The level of lipid peroxidation in cisplatin+Erastin group increased significantly（P<0.05）,and the level of lipid peroxidation in cisplatin+Erastin group was significantly higher than that in group Erastin（P<0.05）.⑶Compared with the control group,the GSH content of cisplatin group,Erastin group and cisplatin+Erastin group decreased significantly（P<0.05）.Compared with the vehicle group,the GSH content of cisplatin group,Erastin group and cisplatin+Erastin group decreased significantly（P<0.05）.Compared with the cisplatin group,cisplatin+Erastin group was compared with that of cisplatin group.Content decreased significantly（P<0.05）.Compared with Erastin group,GSH content in cisplatin+Erastin group decreased significantly（P<0.05）.⑷The cell GPXs activity assay showed that the GPXs activity of cisplatin group,Erastin group and cisplatin+Erastin group decreased significantly compared with control group（P<0.05）.Compared with the vehicle group,the activity of GPXs activity of cisplatin group,Erastin group and cisplatin+Erastin group decreased significantly（P<0.05）.Compared with cisplatin group,cisplatin+Erastin group cells were compared with that of cisplatin group.The activity of GPXs was significantly decreased（P<0.05）,and the activity of GPXs in cisplatin+Erastin group was significantly lower than that in Erastin group（P<0.05）.Summary:1.Down-regulation of GPX4 can induce ferroptosis in SK-N-SH cells.2.Down-regulation of GPX4 can increase the sensitivity of SK-N-SH cells to cisplatin.3.The combination of GPX4 inhibitor and cisplatin can increase the sensitivity of SK-N-SH cells to cisplatin.Part four Downregulation of ferroportin can enhance the sensitivity ofhuman neuroblastoma to erastinObjective:To observe the role of Fpn in regulating the death of neuroblastoma cells induced by GPX4 inhibitor erastin,and to explore whether down-regulation of Fpn and down-regulation of GPX4 can play a synergistic anti-tumor role.Methods:1.After SK-N-SH cells were treated with 20 mu erastin for 6,12 and 24hours,the expression of Fpn mRNA was detected by real-time quantitative PCR,the expression of Fpn protein was detected by Western blot,and SK-N was treated with 20 mu erastin,ferrostatin-1,DFO,NAC,Z-VAD-FMK,Necrostatin-1 and 3-methyladenine respectively.After 12 h of-SH cells,the expression of Fpn mRNA in the cells of each group was detected by real-time fluorescence quantitative PCR.2.According to different intervention methods,the cells were divided into three groups:control group,erastin group,control siRNA group,ferroportin 1 siRNA group.After 24 hours of culture,Fpn mRNA expression was detected by real-time fluorescence quantitative PCR,cell death rate,iron content and lipid peroxidation were detected by kit,and ferroportin 1 siRNA+eras were used to detect the level of lipid peroxidation.SK-N-SH cells were treated with ferrostatin-1,DFO,NAC,Z-VAD-FMK,Necrostatin-1 and3-methyladenine for 24 hours respectively,and then the cell death rate was detected.3.The cells were divided into three groups according to the intervention method:control group,erastin group,erastin+ponasterone group.The expression of Fpn protein was detected by Western blot after 24 hours of culture.Cells were divided into four groups according to the intervention method:erastin group,erastin+ponasterone group,erastin+ponasterone+ferroportin 1 siRNA group,Fpn cDNA group.Cell death rate and iron content were detected after 24 hours of culture.Cells were divided into three groups according to the intervention method:control group,erastin group,erastin+Hepcidin group,and detected after 24 hours of culture.Cell death rate.Results:1.Effect of Erastin on the expression of Fpn in SK-N-SH cells After treated with erastin（20μM）,the Fpn mRNA began to decrease at 6 h,decreased significantly at 12 h（P<0.05）,and decreased significantly at 12 h and 24 h（P<0.05）.In addition,erasin decreased the mRNA and protein levels of Fpn in a time-dependent manner（P<0.05）.Ferrostatin-1,DFO and NAC increased the expression of Fpn（P<0.05）.However,the level of Fpn was not regulated by Z-VAD-FMK,Necrostatin-1 and 3-Methyladenine（P>0.05）.2.The effect of down regulation of Fpn on erastin sensitivity of SK-N-SH cells.SH-SY5Y cells were transfected with ferroportin-1 siRNA or control siRNA and co-incubated with erastin for 12 hours.The level of Fpn mRNA was effectively reduced by transfecting ferroportin-1 siRNA（P<0.05）.The downregulation of Fpn enhanced the growth inhibition induced by erastin in SH-SY5Y cells（P<0.05）.Down regulation of Fpn also increased the intracellular iron content and lipid peroxidation induced by erasin（P<0.05）.In addition,compared with the cells treated with erastin alone,the viability of the cells treated with ferrostatin-1,DFO and NAC was significantly increased（P<0.05）,while the cells treated with Z-VAD-FMK,Necrostatin-1 and 3-methyladenine did not affect the viability of the cells（P>0.05）.3.Effect of upregulation of Fpn on erastin sensitivity of SK-N-SH cells After incubated with ponasterone（10 M）for 24 h,the protein level of Fpn increased significantly（P<0.05）.Fpn inducer ponasterone decreased the growth inhibition and intracellular iron content induced by erastin（P<0.05）.On the contrary,FPN siRNA reversed Fpn and reversed the effect of ponasterone（P<0.05）.At the same time,up-regulation of Fpn by transfecting Fpn cDNA also significantly decreased the growth inhibition and iron content induced by erastin（P<0.05）.Hepcidin promoted erastin induced ferroptosis（P<0.05）.Summary:1.Down-regulation of Fpn can induce ferroptosis in SK-N-SH cells.2.Down-regulation of Fpn can increase the sensitivity of SK-N-SH cells to erastin.Conclusions:1.GPX4 mRNA and protein were expressed in human neuroblastoma SK-N-SH cell lines,neuroblastoma tissues and primary cultured neurobl-astoma cells.2.Down regulation of GPX4 can induce apoptosis and ferroptosis in SK-N-SH cells.3.Down regulation of GPX4 induces ferroptosis production by increasing lipid peroxidation level and decreasing GSH content in SK-N-SH cells.4.Down regulation of GPX4 can enhance the sensitivity of SK-N-SH cells to cisplatin.5.The combination of GPX4 inhibitor and cisplatin can improve the sensitivity of SK-N-SH cells to cisplatin.6.Downregulation of Fpn can induce ferroptosis in SK-N-SH cells.7.Downregulation of Fpn can enhance the sensitivity of SK-N-SH cells to erastin. |
PDF Full Text Request