Muscarinic Acetylcholine Receptor M1 Induces Autophagy And Affects Tumor Cell Progression In Prostate Cancer

Objective The intention of this study was to investigate the influence of CHRM1 on the proliferation,metastasis and anti-apoptosis of prostate cancer.Preliminary exploration of CHRM1 indicated that it is involved in cell autophagy and thereby affects tumor cell progression and possible molecular mechanisms.Methods(1)Substitute nude mice subcutaneous tumorigenesis,Western blot,immunofluorescence and other methods to observe the expression of CHRM1 in prostate cancer tissues and cells in vitro.(2)Human prostate cancer cells PC-3,LNCaP,DU145 were cultured in vitro and treated with CHRM1 agonist carbachol(CAR)and CHRM1 specific inhibitor pirenzepine(PIN).PC-3 cells were infected with CHRM1 RNAi lentivirus,and a stably transfected strain of CHRM1 knockdown was constructed.CCK8 cell proliferation assay,plate cloning assay,wound healing assay,migration and invasion experiments,and flow cytometry detection and observation under transmission electron microscope were used to explore the proliferation,metastasis and apoptosis levels of CHRM1 in prostate cancer.(3)After stably knocking down CHRM1 or using inhibitors,qPCR and Western blot methods were used to determine CHRM1 on the expression levels of genes and proteins related to the EMT of prostate cancer cells.(4)Autophagy inhibitor 3-methyladenine(3-MA)and plasmids transfected with LC3 were added to the test to detect the level of autophagy in prostate cancer cells.(5)CHRM1 stable knockdown model,CHRM1 agonist carbachol(CAR)and CHRM1 inhibitor pirenzepine(PIN)were used,and the expression of autophagy-related markers LC3,p62 were detected through Western blot,autophagosomes were detected through transmission electron microscopy,and autophagic flow was detected through immunofluorescence.(6)Cell migration and invasion tests were performed via the addition of 3-MA,CAR,and PIN to each prostate cancer cell line to detect the migration and invasion abilities of tumor cells by CHRM1-induced autophagy.(7)After steadily knocking down CHRM1,the expression of autophagy-related genes was tested,which showed that the autophagy gene Atg5 was highly positively correlated with CHRM1.Then,a stable knockdown model of Atg5 was established.The migration and invasion test were used to detect whether CHRM1 induces autophagy-mediated metastasis in prostate cancer through Atg5.(8)After stably knocking down CHRM1 or adding an agonist,the Western blot method was used to determine the protein expression of pAMPK and p-mTOR in the AMPK / mTOR signaling pathway.Results(1)CHRM1 was expressed abundantly in prostate cancer cells.(2)CHRM1 could promote proliferation,metastasis and induces apoptosis resistance in prostate cancer cells.(3)CHRM1 promoted the epithelial to mesenchymal transformation.(4)Autophagy was activated in prostate cancer cell lines.(5)In prostate cancer cells,autophagosomes were observed via transmission electron microscopy after the activation of CHRM1.The expression of LC3 increased after the addition of CAR based on Western blot and immunofluorescence.The knockdown of CHRM1 or the addition of PIN gave the opposite results,suggesting that activation of CHRM1 can induce autophagy.(6)After the addition of the autophagy inhibitor 3-methyladenine(3-MA),CHRM1 inhibited prostate cancer cell migration and invasion.Adding 3-MA in the presence of CHRM1 agonist carbachol(CAR)can reduce CARto-cell migration levels,which is statistically significant.CHRM1 may enhance the tumor cell migration and invasion abilities by activating the autophagy levels.(7)Then,we established a CHRM1 knockdown model to detect autophagy-related genes and found that CHRM1 was highly positively correlated with Atg5.Downregulation of Atg5 inhibited the metastasis,which suggested that after activating CHRM1,the induction of autophagy to regulate the cell metastasis process is positively correlated with Atg5.(8)After knocking down CHRM1,the detection of p-AMPK,pmTOR and other protein expression suggested that CHRM1 may induce autophagy in prostate cancer through the AMPK / mTOR pathway.Conclusion In prostate cancer cells,CHRM1 could promote proliferation and metastasis and induce apoptosis resistance.CHRM1 induces autophagy-mediated metastasis in prostate cancer through the AMPK/mTOR pathway by Atg5.