Effect Of GPC1 Expression On Proliferation And Apoptosis Of A549 Lung Cancer Cells

Background:Lung cancer is one of the leading causes of cancer-related deaths,and the proportion of non-small cell lung cancer（NSCLC）can be as high as 85%.In the past two decades,significant progress has been made in the treatment of NSCLC,forming a multidisciplinary comprehensive treatment model of surgery,radiation therapy,chemotherapy,molecular targeted therapy,and immunotherapy.With the deepening of people’s understanding of the physiological and pathological mechanism and the progress of related technologies,these treatment methods have been significantly improved,which has also brought clinical benefits to NSCLC patients.However,due to the existence of radiation resistance,chemotherapy,targeted drug resistance and other problems,the overall survival is still low,and metastasis and relapse become very challenging clinical issues.Therefore,there is an urgent need to find new targets to improve the radiosensitivity of NSCLC and solve the problems of targeted drug resistance,so as to seek greater clinical benefits to improve the prognosis of NSCLC.GPC1 is a member of the Glypican family of heparan sulfate proteoglycans（HSPG）.It binds to the cell surface of the plasma membrane through glycosylphosphatidylinositol（GPI）bonds and is widely present in the cell membrane and extracellular matrix.GPC1 is overexpressed in a variety of malignant tumors such as pancreatic cancer,breast cancer,esophageal cancer,cervical cancer,and glioma,and plays an important role in tumorigenesis and progression.At the same time,we also detected the high expression of GPC1 in NSCLC tissues and serum exosomes,which is related to the degree of malignancy and prognosis,and the expression of GPC1 in serum exosome significantly decreased after radiotherapy and chemotherapy,indicating that GPC1 is expected to become a tumor marker and therapeutic target of NSCLC.Cell pathways such as cell cycle and apoptosis are the main oncogenic signaling pathways,but few studies have explored the correlation between radiation and GPC1 at the cell level,as well as the pathway characteristics,mode and mechanism of GPC1 on NSCLC.Objective:To study the effects of GPC1 on the proliferation,apoptosis and cycle progression of A549 cells,and to further understand the pathway and mode of action of GPC1 in NSCLC,so as to provide a theoretical basis for GPC1 as an important reference target for lung cancer to develop new drugs and new therapeutic models.Methods:A549 cells were irradiated with 6MV X-ray at different doses（0.5Gy,1Gy,1.5Gy,2Gy）.The expression of GPC1 protein in A549 cells was detected 12 hours after irradiation,and the changes of GPC1 expression in A549 cells after irradiation were observed.Then,through the pre-experiment of GPC1 interference vector,the constructed GPC1 shRNA1-4 expression plasmids were transfected into four groups of A549 cells respectively.The expression level of GPC1 mRNA in the four groups of A549 cells was detected by Real time PCR,and the group of GPC1-shRNA with the best silencing efficiency was selected;The screened GPC1-shRNA was transfected into A549 cells to silence the expression of GPC1.At the same time,normal control group and blank vector group were set up.The expression levels of GPC1 mRNA and GPC1 protein in control group,empty vector group and GPC1 silent group were detected by Real-time PCR and Western blotting to verify the efficiency of gene knockout;CCK8 experiment was used to observe the effect of silencing GPC1 on the proliferation of A549 cells.Flow cytometry was used to detect the effects of silenced GPC1 on A549 cell cycle and apoptosis.Results:1.Compared with the normal control group,the GPC1 protein expression level of A549 cells in groups receiving different irradiation doses（0.5Gy,1Gy,1.5Gy,2Gy）was significantly reduced（P<0.05）.2.Real time PCR results showed that among the four groups of A549 cells transfected with the GPC1 shRNA1-4 interference vector,the GPC1 shRNA group 3cells had the lowest GPC1 mRNA expression level and the highest silencing efficiency for GPC1.3.Compared with the normal control group and the empty vector group,the expression levels of GPC-1 mRNA and GPC1 protein in A549 cells of the GPC1silencing group transfected with the GPC1 interference vector were significantly reduced（P<0.05）.4.Compared with the normal control group and the empty vector group,the OD value of A549 cells in the GPC1 silent group transfected with the GPC1 interference vector decreased,and the proliferation was significantly inhibited（P<0.05）.5.Compared with the normal control group and the empty vector group,the apoptosis rate of A549 cells in the GPC1 silencing group transfected with the GPC1interference vector was significantly increased（P<0.05）.6.Compared with the normal control group and the empty vector group,the percentage of G₂/M phase of A549 cells in the GPC1 silencing group transfected with the GPC1 interference vector increased by 16.01%（P<0.05）,and the cell blockage was in the G₂/M phase.Conclusion:X-ray irradiation can inhibit the expression of GPC1 in A549 cells;Silencing GPC1 expression can promote A549 cell apoptosis,inhibit A549 cell proliferation,and cause A549 cell G2/M phase arrest...