| Objective: Lung cancer is the leading cause of cancer death worldwide.Non-small cell lung cancer(NSCLC)is the main pathological type of lung cancer.NSCLC is a serious threat to human health because of its rapid progress and easy metastasis.Previous studies have found that estrogen promotes the progression,invasion and metastasis of NSCLC by activating estrogen receptors(ERs).However,GPER1,as a newly discovered ERs in recent years,its impact on NSCLC and its mechanism are still unclear.This study aims to determine whether GPER1 could promote the progress of NSCLC,explore whether GPER1 could promote the progress of NSCLC by inhibiting the ferroptosis of NSCLC and its specific mechanism,and whether inhibition of GPER1 could enhance the sensitivity of NSCLC to cisplatin,and provide new targets and new ideas for the treatment of NSCLC.Methods:The methods of Chapter 1 mainly include: the protein level of GPER1 in NSCLC tissue samples was investigated by western blotting(WB)and immunohistochemical staining(IHC).The correlation between the expression of GPER1 and the prognosis of NSCLC patients was analyzed using the public database.The methods of Chapter 2 mainly include: NSCLC cell line overexpressing and knocking down GPER1 was constructed by chronic disease transfection,and was verified by fluorescence quantitative PCR and WB.CCK8 experiment and clone formation experiment were used to detect the effect of expression and regulation of GPER1 and G1 on cell death induced by hydrogen peroxide(H2O2).The effects of GPER1 and G1 on intracellular lipid peroxidation(MDA)were detected.CCK8 test detected the sensitivity changes of cells to RSL3 and Erstin under the intervention of overexpression of GPER1 and G1,and flow cytometry detected the changes of C11 BODIPY 581/591 level during the process.The methods in Chapter 3 mainly include: the key downstream enzyme SCD1 of GPER1 inhibiting ferroptosis was screened by fluorescence quantitative PCR.The regulatory effects of GPER1 on PI3K/AKT/m TOR pathway,SREBP1 and SCD1 were evaluated by WB,the changes of cell survival rate were detected by CKK8,and the changes of C11 BODIPY 581/591 level and MDA content were detected by flow cytometry.The expression correlation of GPER1,SREBP1 and SCD1 in NSCLC tissue samples was analyzed by polychromatic immunofluorescence.The methods in Chapter 4 mainly include: CCK8 assay was used to detect the sensitivity of cells to cisplatin under the intervention of expression regulation of GPER1 and G1.The nude mice subcutaneous transplanted tumor model was constructed to evaluate the effect of GPER1 on the growth of NSCLC,the effect of inhibiting GPER1 on the antitumor activity of cisplatin,and the change of ferroptosis degree of tumor body was evaluated by IHC staining.Results:The expression of GPER1 was increased in NSCLC tissue samples,and the high expression of GPER1 was associated with poor prognosis of NSCLC patients.GPER1 in NSCLC cells showed that it can reduce the cell death induced by oxidative stress,with the decrease of MDA content.Further research found that GPER1 can reduce the sensitivity of NSCLC to RSL3 and Erstin.In this process,MDA and C11 BODIPY 581/591,suggesting that GPER1 can inhibit the ferroptosis of NSCLC cells.The mechanism research found that GPER1 inhibits ferroptosis of NSCLC cells by promoting the expression of SCD1,which is realized by inducing SREBP1 activation through PI3K/AKT/m TOR pathway activated by GPER1,and further promoting the transcription of SCD1.The positive correlation between GPER1 and SCD1 expression in NSCLC tumor tissue confirmed our conclusion in cell experiment.In addition,inhibition of GPER1 can enhance the cytotoxicity of cisplatin in vitro.In vitro,inhibition of GPER1 can enhance the anti-tumor activity of cisplatin,and is achieved by enhancing cisplatin-induced ferroptosis.Conclusion:This experiment first proved that the increased expression of GPER1 in NSCLC was associated with poor prognosis.Then it was found that GPER1 could reduce ROS-induced cell death and lipid peroxidation.Further studies showed that GPER1 could inhibit ferroptosis in NSCLC cells.GPER1 induces SREBP1 activation by activating PI3K/AKT/m TOR pathway,which promotes SCD1 transcription and inhibits iron death in NSCLC cells.Inhibition of GPER1 can enhance the antitumor activity of cisplatin in vitro and in vivo.Therefore,this study clarified the specific mechanism of GPER1 inhibiting ferroptosis in NSCLC and inhibiting GPER1 enhancing the anti-tumor activity of cisplatin,providing new targets and new ideas for the treatment of NSCLC. |
