Preliminary Study On The Effect And Mechanism Of Pp GalNac-T10 On The Development And Progress Of Lung Adenocarcinoma

BackgroundN-polypeptide N-Acetylgalactosaminyltransferase 10(PP GalNac-T 10)is a polypeptide N-acetylgalactosamine transfer enzyme,which is one of the members of the family.A recent research showed that the abnormal expression of GalNac-T10 may be associated with tumor metastasis and malignant growth.It has been found that the positive rate of PP GalNac-T10 in cancer tissue is higher than adjacent tissues with lung adenocarcinoma tissue microarray;however,its cellular biological and molecular mechanism still need further study.Method1,To select the appropriate cell line for cytological experiments,qPCR and Western blot assay were used to determine the level of mRNA and protein expression of PP GalNac-T10 in different lung adenocarcinoma cell lines.2,After transfection of GalNac-T10 plasmid and the corresponding interference expression plasmid GalNac-T10,the OD changes was detected by CCK8 test to detect cell proliferation in A549 and H322 cell lines.3,To study the effects of high or low expression of GalNac-T10 on the proliferation and apoptosis of A549 and H322 cells,the changes of cell cycle and apoptosis were detected by flow cytometry.4,To study the effects of high or low expression of GalNac-T10 on the invasion of A549 and H322 cells,the number of invasive cells in each group was detected by Transwell assay after transfection.5,To determine the significant differences in gene expression due to the change of PP GalNac-T10 expression,the expression of lung adenocarcinoma cell lines was detected by gene microarray.6.To find out the potential downstream genes of PP GalNac-T10 in lung adenocarcinoma,bioinformatics data mining was performed.Results1.CCK8 experiments showed that overexpression of PP GalNac-T10 in the cells significantly inhibited the proliferation of lung adenocarcinoma cells,and inhibition of PP GalNac-T10 promoted the proliferation of lung adenocarcinoma cells to some extent.2.The results of cell cycle test showed that overexpression of PP GalNac-T10 induced the cell cycle arrest at G1 phase and inhibited cell proliferation.Cell apoptosis experiments showed that overexpression of PP GalNac-T10 promoted cell apoptosis.3.Transwell experiment showed that overexpression of pp GalNac-T10 suppressed the cell invasion of lung adenocarcinoma cells.These results indicated that PP GalNac-T10 may be a tumor suppressor gene in lung adenocarcinoma,which are consistent with the results of tissue microarray in pre experiment.4.The results of gene expression profiles showed that there were 932 differentially expressed genes in the high expression of pp GalNac-T10 treated group.In the low expression group,there were 1073 significant differences gene.The potential downstream genes of PP GalNac-T10 are AMH3 BMP2,ID1,ID3,SMAD1,SMAD9,ACVR1,DCN,FST,MAPK3,RHOA in TGF-beta pathway,and AXL,BAX,EGFR,ERBB3,FGF2,IL6,GAS6,MAPK3,NRAS,NRG1,PTEN,BCL2L11,FGFR3,NF1,PDGFA,PDGFB,PIK3CB,VEGFA,EIF4E in the EGFR pathway.5.The exploratory data analysis found that the biological processes and pathway enrichment of the differentially expressed genes were mainly related to p53 signaling pathway and FoxO signaling pathway.ConclusionOverexpression of pp GalNAc-T10 inhibited the proliferation and invasion of lung adenocarcinoma cell lines,promoted the apoptosis and arrested the cell cycle in G0/G1 phase.Therefore,our result confirmed that pp GalNAc-T10 plays a function as a tumor suppressor gene in lung adenocarcinoma.The genes AMH,BMP2,and ID1 etc.in the TGF-beta pathway,and AXL,BAX,GAS6,etc.in the EGFR pathway are mostly closely associated with pp GalNAc-T10,and potentially are regulated by pp GalNAc-T10.Furthermore,pp GalNAc-T10 is closely related to p53 pathway and FoxO pathway in lung adenocarcinoma,which is worthy of further study.