The Role Of GPRC6A In Prostate Cancer Cell On Proliferation And Apoptosis

Objective:Prostate cancer(PCa)is a common urological malignancy in men.Androgen deprivation therapy is the main systemic treatment for patients with advanced diseases.Despite therapeutic effects,castration resistance eventually occurs in almost all patients.With the rapid development of life science technology and the improvement of medical research,biomolecules will become potential targets for PCa therapy.G-protein-coupled receptor family C group 6 member A(GPRC6A)is expressed in various tissues and organs.It binds to different endogenous ligands and can participate in a wide range of physiological functions such as metabolism and hormone regulation.The expression of GPRC6 A increased in PCa cells.Previous studies found that GPRC6 A was involved in the migration and invasion of PCa cells,but its role and mechanism in the proliferation and apoptosis of PCa cells were unclear.Therefore,in this study,we observed the role of GPRC6 A in the proliferation and apoptosis of PCa cells through overexpression and knockdown of GPRC6 A gene,and further studied the mechanism of GPRC6 A overexpression cells in order to provide new ideas and new targets for the study of PCa.Methods:Experiment 1: To observe the role of GPRC6 A in cell proliferation and apoptosis in PC3 cells with GPRC6 A knocked down and C4-2 cells with GPRC6 A overexpressed.PC3 cells with GPRC6 A knocked down were constructed,and C4-2 cells with GPRC6 A overexpressed were constructed in our previous study;cell proliferation was detected by cell counting and cell cloning;cell cycle and apoptosis were detected by flow cytometry;p-ERK and ERK expression were detected by Weatern Blot and immunofluorescence,the optimum concentration of U0126 was determined by cell count and CCK8;After subcutaneous tumorigenesis in nude mice,immunohistochemistry was used to detect Cleaved caspase3 and Weatern Blot was used to detect CyclinD1,Bcl2 and Cleaved caspase3 in tumor tissue.The effects of GPRC6 A on the growth and apoptosis of tumors were observed in vivo and in vitro.Experiment 2: The mechanism of GPRC6 A affecting cell proliferation and apoptosis in C4-2 cells overexpressing GPRC6 A was studied.The experiment was divided into three groups: C4-2 pCDH group(empty plasmid control group),C4-2 GPRC6 A group(GPRC6A overexpression group)and C4-2 GPRC6 A + U0126 group(GPRC6A overexpression cells were treated by ERK pathway inhibitor U0126).Cell proliferation and apoptosis were detected by CCK8.Cell cycle and apoptosis were detected by flow cytometry;apoptosis was detected by Tunel fluorescence staining;mitochondrial membrane potential was detected by TMRE staining.Western Blot was used to detect the expressions of Cyclin D1,P53,Bcl2,Bax,Cleaved caspase 3,Caspase 9,CHOP and Caspase 8.Results:Experiment 1: After the overexpression of GPRC6 A,cell cycle progression was significantly higher than that of the control group,proliferation was slowed down and apoptotic cells were reduced.Compared with the control group,the the expression of p-ERK was increased in C4-2 cells overexpressing of GPRC6 A.In the PC3 cells with GPRC6 A knocked down,cell cycle progression was significantly lower than that of the control group,proliferation was decelerated and apoptotic cells were increased,the expression of p-ERK was reduced.The optimum concentration of U0126 is 10μM.In the subcutaneous tumorigenesis experiment of nude mice,the tumorigenesis rate of C4-2 GPRC6 A group was higher than that of control group,and the volume and weight of tumors was larger than that of control group.The expression of CyclinD1 and Bcl 2 in tumor tissue was higher in GPRC6 A overexpression group than that in control group,and the expression of Cleaved caspase 3 was decreased in overexpression group.Experiment 2: Compared with GPRC6 A overexpressed C4-2 cells,CCK8 and flow cytometry results showed that cell proliferation and cell cycle progression slowed down after inhibition of ERK pathway.Western blot results indicated that after inhibition of ERK pathway,the expression of Cyclin D1,Bcl2 decreased,while the expression of Bax and Cleaved caspase 3 increased.Mitochondrial membrane potential increased with the high expression of GPRC6 A,and decreased after U0126 inhibited ERK pathway.The expression of Caspase 9 and Caspase 8 in mitochondrial pathway and Cleaved caspase/Pro caspase in C4-2 GPRC6 A group were significantly lower than that in C4-2 pCDH group,ERK inhibitors reversed this result.The expression of CHOP in endoplasmic reticulum pathway was increased with the inhibition of ERK pathway.Conclusion: 1.GPRC6 A can promote the proliferation and inhibit the apoptosis of PCa cells.2.GPRC6 A regulate the growth and apoptosis of PCa cells through ERK pathway.3.GPRC6 A reduce PCa cell apoptosis through ERK pathway,probably involve death receptor,mitochondria and endoplasmic reticulum pathways.In mitochondria,GPRC6 A may involve P53-Bcl2/Bax-Caspase 9-Caspase 3 pathway.