关键词: 乳腺癌, G蛋白偶联受体139, 细胞增殖, 细胞凋亡, 自噬, 腺苷酸活化蛋白激酶

Abstract:

Objective To discuss the effect of silencing G protein-coupled receptor 139 （GPR139） gene on the proliferation， apoptosis and autophagy of breast cancer cells， and to clarify its possible mechanism. Methods The expression of GPR139 mRNA and protein in breast cancer tissue and normal tissue was downloaded from The Cancer Genome Atlas （TCGA） and Human Protein Atlas （HPA） databases； the GPR139 short hairpin RNA （shRNA） was transfected into the human breast cancer MCF-7 and MDA-MB-231 cell lines by lentiviral interference technology， and the cells were divided into sh-NC group （infected with negative control lentivirus）， sh-GPR139 group （infected with GPR139 shRNA interference lentivirus）， and sh-NC+F05 group （after infected with negative control lentivirus， GPR139 antagonist NCRW0005-F05 was added）； methylthiazolyldiphenyl-tetrazolium bromide（MTT） method was used to detect the proliferation activities of the cells in various groups； Western blotting and real-time fluorescence quantitative PCR （RT-qPCR） methods were used to detect the expression levels of proliferation markers Ki-67， Cyclin D1， Cyclin E1， and P21 mRNA and proteins in the cells in various groups； flow cytometry was used to detect the apoptotic rates of the breast cancer cells in various groups； Western blotting method was used to detect the expression levels of adenosine monophosphate-activated protein kinase （AMPK）， phosphorylated AMPK， Unc-51 like autophagy activating kinase 1 （ULK1）， phosphorylated ULK1， microtubule-associated protein 1 light chain 3A/B （LC3A/B）， and P62 proteins in the cells in various groups； immunofluorescence method was used to detected the expression level of LC3A/B protein in the breast cancer cells in various groups. Results The TCGA and HPA database results showed that the expression level of GPR139 mRNA in human breast cancer tissue was significantly higher than that in normal tissue （P<0.001）， and the expression level of GPR139 protein was significantly higher than that in normal tissue. The MTT assay results showed that compared with sh-NC group， the proliferation activities of the breast cancer cells in sh-GPR139 group and sh-NC+F05 group were significantly decreased （P<0.01）. The Western blotting results showed that compared with sh-NC group， the expression levels of GPR139， Ki67， Cyclin D1 and Cyclin E1 proteins in the breast cancer cells in sh-GPR139 group and sh-NC+F05 group were decreased （P<0.05 or P<0.01）， and the expression level of P21 protein was increased （P<0.01）. The RT-qPCR results showed that compared with sh-NC group， the expression levels of GPR139， Ki67， Cyclin D1 and Cyclin E1 mRNA in the breast cancer cells in sh-GPR139 group and sh-NC+F05 group were decreased （P<0.05 or P<0.01）， and the expression level of P21 mRNA was increased （P<0.01）. The flow cytometry results showed that compared with sh-NC group， the apoptotic rates of the breast cancer cells in sh-GPR139 group and sh-NC+F05 group were significantly increased （P<0.01）. The immunofluorescence results showed that in MCF-7 cells and MDA-MB-231 cells， compared with sh-NC group， the expression levels of LC3A/B protein in the cells in sh-GPR139 group and sh-NC+F05 group were significantly increased （P<0.01）. The Western blotting results showed that compared with sh-NC group， the expression levels of LC3A/B， p-AMPK and p-ULK1 proteins in sh-GPR139 group and sh-NC+F05 group were significantly increased （P<0.01）， and the expression level of P62 protein was significantly decreased （P<0.05 or P<0.01）. Conclusion Silencing GPR139 gene can induce autophagy and apoptosis of breast cancer cells and inhibit cell proliferation， and its mechanism may be related to the increased phosphorylation levels of AMPK and ULK1 in the cells.

Key words: Breast cancer, G protein-coupled receptor 139, Cell proliferation, Apoptosis, Autophagy, Adenosine monophosphate-activated protein kinase