The Effect Of Glypican6 Gene On Proliferation Of Lung Adenocarcinoma Cells And The Preliminary Study For Its Mechanism

[Objectives]The morbidity and mortality rate of lung cancer ranks the first in the world.NSCLC accounts for 85%of lung cancer,among which adenocarcinoma is a very important type,prone to early metastasis.Most lung cancer patients are diagnosed at an advanced stage with a 5-year survival rate of only 15%.Therefore,it is of great significance to screen out lung cancer markers or lung cancer-related genes that are simple,sensitive and specific and helpful for the early diagnosis of lung cancer.Some scholars found that GPC6 may be a new oncogenic gene based on high variation range and small overlap region in the analysis of whole genome of NSCLC.Therefore,we collected NSCLC(Adenocarcinoma)clinical samples to detect the expression of GPC6 gene in tumor tissues and adjacent tissues.The effect of interfering GPC6 gene on the proliferation of lung cancer cells was studied in vitro.The results of whole gene expression profile chip experiment were analyzed by IPA.Combined with the verify results of several key genes by WB,the possible mechanism of GPC6 gene affecting the proliferation of lung cancer cells was preliminarily discussed.[Materials and Methods](1)The protein expression of GPC6 gene in tumor and adjacent tissues of 10 patients with lung adenocarcinoma was detected by immunohistochemical.The mRNA expression of GPC6 gene in lung adenocarcinoma cell lines was detected by RT-qPCR.(2)Interference targets were designed with GPC6 gene as template,RNA interference lentivirus vector was constructed and packed to infect human A549 and H1299 cells to knockdown GPC6 gene.The GPC6 gene knockdown efficiency on mRNA level was detected by RT-qPCR.The protein expression of GPC6 gene was detected by WB after knockdown to verify the effect of target interference.The proliferation and apoptosis of A549 and H1299 cells were detected by Celigo,MTT,FACS and caspase3/7 detection.(3)RNA-interfering lentivirus infected human A549 cells to knockdown GPC6 gene.GPC6 gene information in A549 cells was analyzed by gene chip experiment.The classic pathway analysis,upstream regulation analysis,disease and function analysis,regulatory effect analysis and interaction network analysis of GPC6 gene were conducted by IP A,and gene network diagram was further analyzed and constructed.(4)According to the results of the whole gene expression profile chip experiment,WB was used to verify for STAT3,MET,CEBPB,PTGS2(COX2),JUN,FOS,CXCL8(IL8),SHC1 and other genes.[Results](1)GPC6 gene was differentially expressed in tumor tissues and adjacent tissues of lung adenocarcinoma patients(high expression in tumor tissues,low expression in adjacent tissues)(P<0.05).The mRNA expression of GPC6 gene was found in human 95-D,A549,H1299 and H1975 cells,with lower expression in 95-D cells and higher expression in A549,H1299 and H1975 cells.(2)Celigo and MTT detection found that the proliferation rate of human A549 and H1299 cells after GPC6 gene knockdown was significantly inhibited(allP<0.01).FACS and caspase3/7 assays showed that the apoptotic cells of human A549 and H1299 cells after GPC6 gene knockdown were significantly increased(all P<0.01),and the caspase3/7 activity was significantly increased(all P<0.01).(3)The results of whole gene expression profile chip experiment showed that the number of significantly up-regulated genes was 876,the number of significantly down-regulated genes was 935 after knockdown of GPC6 gene in human A549 cells,and the FC value was-1.2340816(P=0.008695449).The Common lung cancer driver genes,such as EGFR,BRAF,MET and RET were not significantly up-regulated,while ALK,ROS1,HER2 and KRAS were not significantly down-regulated.REL,RELA and RELB genes with NF-kB activity were down-regulated.IPA results suggest that GPC6 gene may affect the proliferation of tumor cells by regulating STAT3,MET,CEBPB,PTGS2(COX2),JUN,FOS,CXCL8(IL8),SHC1,RAP1A,and IL1R1 in HGF and IL6 signaling pathways in human A549 cells.(4)The WB verification results for downstream genes showed that STAT3 protein expression was up-regulated by 26%,MET protein expression was down-regulated by 1%,CEBPB protein expression was down-regulated by 67%,PTGS2(COX2)protein expression was significantly down-regulated,JUN protein expression was up-regulated by 205%,FOS protein expression was down-regulated by 46%,CXCL8(IL8)protein expression was down-regulated by 49%,and SHC1 protein expression was up-regulated by 87%.[Conclusions]GPC6 gene was differentially expressed in tumor tissues and adjacent tissues of lung adenocarcinoma patients.Interfering with GPC6 gene can inhibit the proliferation of human A549 and H1299 cells.GPC6 gene plays the above role in human A549 cells by regulating PTGS2(COX2),CEBPB,CXCL8(IL8),STAT3,FOS,SHC1 and other downstream genes.The high expression of STAT3 gene after knockdown of GPC6 gene was an important reason for the inhibition of proliferation of A549 cells,which may be caused by the STAT3/NF-kB/IL8 axis and CEBPB/NF-kB/IL8 axis.