Study On The Role And Mechanism Of GABA Receptor GABRD In Breast Cancer

Objective:Breast cancer is one of the most common malignant tumors in women,and there are still some patients who have a fast progression and poor prognosis after treatment.GABA receptors are important inhibitory neurotransmitter receptors that are closely related to psychosocial disorders in patients and are involved in regulating the development of multiple cancers.However,the role and molecular mechanism of breast cancer are unclear.The aim of this experiment is to investigate the expression of GABA receptors in breast cancer and its mechanism for the development of breast cancer.Methods:1.The expression of GABA receptor in breast cancer was investigated by TCGA and other related databases,and the selected differential gene GABRD was analyzed for survival.2.Normal breast tissue and human breast cancer were selected for immunohistochemical experiments to validate the data obtained from the bioassay.3.Real-time fluorescence quantitative PCR(q PCR)experiments and Western blot experiments were conducted to detect the expression of GABRD in normal breast epithelial cells and different strains of breast cancer cells.4.The effects of GABRD on the proliferation of breast cancer cells were observed by knock-down/overexpression experiments with different expressions and counting and cloning of cells.5.Cell cycle detection was performed before and after knock-down GABRD,and cell cycle blockage was observed.6.Sequencing of breast cancer cells before and after knockdown GABRD,GSEA analysis of the results and sorting out differences in gene changes,and selection of possible pathways.The pathway of sequencing analysis is verified by a drug-added experiment with cells.Results:1.Analysis of the TCGA database,GABRD is the only GABA receptor that is upregulated in breast cancer tissue(P<0.0001)and the high expression of GABRD is associated with a decrease in Overall Survival(OS)(P=0.0306).2.Immunohistochemistry with normal breast tissue and breast cancer tissue to validate the results of bioassay.GABRD was found to be highly expressed in breast cancer tissues(P <0.0001)and can be used as an independent risk factor for breast cancer prognosis(P <0.0001).3.In vitro experiments on GABRD.The q PCR and Western blot experiments found high expression of GABRD in breast cancer cells(P<0.05)compared to normal breast epithelial cells.Selection of high-expression GABRD breast cancer cells for the knock-down experiment found that after the knock-down GABRD(P<0.0001),cell count shows slower proliferation of breast cancer cells(P<0.0001)and further validated by cloning formation experiments(P<0.0001).4.Flow detection cell cycle experiments showed that cells were blocked to phase G0/G1(P<0.05)after knock-down GABRD,affecting breast cancer cell proliferation.5.Overexpression of GABRD in breast cancer cells with low-expression GABRD(P<0.0001),cell count experiments show that breast cancer cells proliferate faster(P<0.0001)and the clone formation experiment further validates the result(P<0.05).6.Sequencing of breast cancer cells before and after knockdown GABRD and GSEA analysis of sequencing results,selecting the most meaningful interferon α pathway(si1:|NES|=1.61,FDRq-val=0.027;si2:|NES|=2.69,FDRq-val=0.000).And the sequencing results were verified by a dosing experiment.The results showed that when GABRD was knocked down and then interferon signaling pathways were blocked,breast cancer proliferation suppression was returned(P<0.0001).It is proved that the anti-proliferation effect of knock-down GABRD in breast cancer cells can be attributed to the activation of interferon α signaling pathways.Conclusion:GABRD is highly expressed in breast cancer compared with normal breast tissue and affects breast cancer prognosis as an independent risk factor.At the same time,GABRD can promote the proliferation of breast cancer cells by inhibiting interferon αsignaling pathways.