Study On The Effect Of Muscarinic Acetylcholine M1 Receptor On Pyroptosis Of Prostate Cancer Cells Through Caspase-1/GSDMD Pathway

Objective: To study the relationship between muscarinic acetylcholine M1receptor(CHRM1)and pyroptosis of prostate cancer cells,and to study the possible molecular mechanism of pyroptosis induced by CHRM1 in prostate cancer cells through cell experiments.Methods :(1)The expression of CHRM1 in normal prostate and prostate cancer in TCGA and GEPIA tumor databases was detected.(2)Pirenzepine(PIN)and Bethanechol(Beth),specific inhibitor and agonist of CHRM1,were added into PC-3 and DU145 cells with different time concentration gradients,and cell viability was detected by CCK8.(3)Knock down CHRM1 in PC-3 cells and verify the efficiency by Western blot.(4)After PC-3 and DU145 were treated with inhibitors and agonists,the addition group and sh-CHRM1 group were set.The PI positive cell rate was detected by Hoechst-PI double staining and Annexin-PI flow cytometry,and the LDH release level was detected by LDH(lactate dehydrogenase)cytotoxicity detection reagent.(5)The phenomenon of pyroptosis in PC-3 and DU145 treated with the optimal concentration of PIN was detected under the transmission electron microscope.(6)Bioinformatics analysis was conducted through GEPIA and Oncomine online database to observe the expression levels of GSDMD,NLRP3 and caspase-1 genes related to the caspase-1 /GSDMD pathway in tumors,especially in prostate cancer.(7)The GSDMD in PC-3 was knocked down by plasmid transfection,and the efficiency was verified.(8)To analyze the level of pyroptosis,the si-GSDMD cell group and the control group were set,and then the PI-positive cell rate was detected by Hoechst-PI double staining and Annexin-PI flow cytometry as well as the LDH release level.And the expression level of molecular proteins related to the caspase-1 /GSDMD pathway was detected by western blot.Results :(1)CHRM1 was highly expressed in prostate cancer.(2)Inhibition of CHRM1 reduced the proliferation ability of PC-3 and DU145 cells in a time and concentration dependent manner within a certain concentration range,while activation of CHRM1 increased it in a similarly dependent manner within a certain concentration range.(3)Downregulation of CHRM1 increase the LDH release level of PC-3 and DU145 cells and the positive rate of PI in prostate cancer cells.Upregulation of CHRM1 could reduce LDH release level and PI positive rate.(4)According to transmission electron microscopy,the inhibition of CHRM1 caused the cell pyroptosis of prostate cancer cells.(5)The protein expression levels of GSDMD,NLRP3,ASC,caspase-1 and cleaved caspase-1 were significantly increased in sh-CHRM1 group and PIN group by western blot analysis.(6)Analysis of tumor database showed that the expression of GSDMD,NLRP3 and Caspase-1 genes were abnormal in various tumors,and GSDMD,NLRP3 and Caspase-1 expressions were decreased in prostate cancer patients.(7)After knocking down GSDMD,the PI-positive cell rate,LDH release level and the expression of cell pyroptosis related molecular proteins NLRP3,ASC,caspase-1 and cleaved caspase-1 were significantly decreased,and there was no significant difference in all levels after the addition of PIN.Conclusion: Downregulation of CHRM1 induces pyroptosis of prostate cancer cells by stimulating the Caspase-1/GSDMD pathway.