GALNT3 Regulates Non-small-cell Lung Carcinoma Proliferation And Migration Via MUC1

Objective:GALNT3 is a member of the N-acetylgalactosamine transferase family,which functions as glycosyltransferase and catalyzes the first step of O-glycan synthesis,transferring the N-acetylgalactosyl group on UDP-GalNAc to serine or threonine residues in specific sequences of polypeptide chains.Abnormal O-glycation is often associated with tumor cell surface.GALNT3 is involved in the occurrence and development of various tumors,and has been reported to promote cancer in pancreatic cancer,ovarian cancer,colorectal cancer,thyroid cancer,renal cell carcinoma,gallbladder carcinoma and intrahepatic bile duct carcinoma.The current known reports in non-small cell lung cancer are unclear.MUC1 encodes a protein that is a member of the mucin family.Mucins are a class of highly O-glycosylated proteins.An important role of mucins is to form a mucus barrier on the surface of the epithelium,which has a protective function.It also plays a role in intracellular signaling.The N-terminal alpha subunit plays a role in cell adhesion and the C-terminal beta subunit is involved in cell signaling.This mucin overexpression and glycosylation changes have been linked to cancer.Methods:1.The expression level of GALNT3 in non-small cell lung cancer was analyzed through some biological information databases.2.H1299 and A549 cells were inoculated in a medium containing 10%fetal bovine serum(FBS)and cultured in a carbon dioxide incubator at 5%concentration at 37℃.3.Plasmid transfection or interference with GALNT3 expression required the setting of control group.The reagent Lipofectamine3000 was used.One day later,cells were counted and functional tests were performed.Two days later,total proteins were extracted and related proteins were verified by western blotting.4.Immunohistochemical staining was used to detect the expression of GALNT3 in lung cancer tissues and its localization in cells.GALNT3 rabbit polyclonal antibody was added to the tissue sections and incubated overnight at 4℃.Incubate the secondary antibody the next day.DAB staining and hematoxylin staining were used to stain the cells and observed under microscope.5.Western blot assay.Proteins with different molecular weights are separated by electrophoresis,and the proteins on the glue are transferred to the polyvinylidene fluoride membrane.Soak the film in 5%skim milk and seal it for 2 hours.Incubate the primary antibody and place it in a shaker in a 4℃ refrigerator overnight.On the second day,the second antibody was incubated at 30℃ for 2 hours.Proteins were visualized using enhanced chemiluminescence.Each strip was quantitatively evaluated using ImageJ software.6.CCK-8 cell proliferation was measured by adding 10 microliters of CCK-8 solution to each of the 96-well plates.The 96-well plate was wrapped in tin foil and put back into the incubator.Two hours later,the color value of each well was measured with an enzyme marker with a wavelength of 450nm.5 secondary holes were provided for each treatment factor.A 96-well plate was taken out at the same time every day for a total of 5 days.7.Colony formation test:six well plates were taken out and washed with PBS for 3 times.Set with pre-cooled methanol for 15 minutes.Wash with PBS 3 times.Dye crystal violet solution for half an hour.Rinse with running water and count the colonies after drying.8.Transwell experiment:600 μL culture medium with 20%serum concentration was added into each well of the lower chamber of the 24-well plate,then into the Transwell chamber,and 200 μl cell suspension was added into the upper chamber.After about a day of culture,the transwell chamber was taken out and fixed and stained in a manner similar to that of the colony,photographed and counted under a microscope.9.Immunofluorescence:An appropriate number of cells were inoculated on a 24-well plate.The next day was fixed with 4%paraformaldehyde for 15 minutes,then treated with 0.1%TritonX-100 for 10 minutes.Seal it with 3%BSA for 2 hours.Incubate primary antibody and incubate overnight at 4℃.The next day,the primary antibody was recovered and washed with phosphate buffered saline(PBS)for 3 times.Incubate with the secondary antibody at 37℃ for 2 hours(this step begins the operation in the dark).The nuclei were stained with 4’,6-diaminyl-2-phenylindole(DAPI)for 10 min.Finally,confocal microscopy was used to obtain random cell images.10.Immunocoprecipitation(Co-IP):After cell lysis,the cells were centrifuged for 15 minutes at 12,000 revolutions per minute in a high speed centrifuge precooled at 4℃.60μL protein A/G beads were added to the supernatant and rotated for at least 2h.The mixture is then centrifuged for five minutes.The supernatant after centrifugation is divided into two parts.The target antibody is added to one part and anti-mouse/rabbit IgG is added to the other.Place the mixture in a rotating oscillator overnight at 4℃.On the second day,25 μ L agarose A/G magnetic beads were added to each tube and incubated at 4℃ for 6 hours.Cell lysates were washed,2XLoadingBuffer was added,and heated in boiling water for 10 minutes.Finally,western blotting was performed.11.Database:The UALCAN database was used to analyze the expression of GALNT3 in normal tissues and lung cancer tissues and at different stages of lung cancer.GEPIA online database also analyzed the expression of GALNT3 and obtained the survival curve of patients.Check the binding of GALNT3 with other proteins in String database.Results:1.The results of UALCAN and GEPIA databases showed that the expression of GALNT3 in normal tissues was less than that in lung cancer tissues,and its expression was negatively correlated with patient survival.UALCAN database showed that the expression of GALNT3 in all stages of lung cancer was higher than that in paracancer tissues.Immunohistochemical staining showed that the expression of GALNT3 in non-small cell lung cancer was higher than that in normal alveolar or bronchial tissues and was more expressed in cytoplasm.Clinicopathologic factor analysis showed that GALNT3 was positively correlated with TNM stage,lymph node metastasis and tumor size.Therefore,GALNT3 is associated with poor prognosis in patients with non-small cell lung cancer.2.In H1299 and A549 cells transfected with GALNT3 overexpression plasmid or siGALNT3,colony formation assay and CCK-8 proliferation assay were performed.The results showed that the increased expression of GALNT3 promoted the proliferation of H1299 and A549 cells,while the decreased expression of GALNT3 inhibited the proliferation of H1299 and A549 cells.Western blotting showed that overexpression of GALNT3 could increase the expression of proliferation-related proteins3.The expression of GALNT3 was also changed in H1299 and A549.Cell migration experiment showed that transfection of GALNT3 overexpressed plasmid could promote the migration of the two types of cells,while transfection of siGALNT3 had the opposite result.Western blotting also confirmed the results at the protein level。4.Western blot results showed that GALNT3 could positively regulate MAPK signaling pathway.5.We incubated with MAPK signaling pathway inhibitor PD98059 in H1299 and A549 cells transfected with GALNT3 overexpression plasmid.Combined with functional experiments,we concluded that PD98059 could reverse the effect of GALNT3 overexpression on MAPK signaling pathway.6.The interaction between GALNT3 and MUC1 was demonstrated by immunocoprecipitation and immunofluorescence imaging.GALNT3 was found in A549 and H1299 cells to regulate MUC1 at the protein level,while MUC1 could not regulate GALNT3 expression at the protein level.Subsequent western blotting showed that increased Erk phosphorylation levels were offset by inhibition of MUC1.The increase in Mek level was offset by the interference of MUC1 due to transfection of Galnt3-expressing plasmid.Subsequent proliferation and migration experiments of CCK-8 verified that silencing MUC1 could reverse the promoting effect of transfection of GALNT3 on cell proliferation and migration.Overall,these results suggest that GALNT3 can promote the proliferation and migration of non-small cell lung cancer by regulating the MAPK signaling pathway through MUC1.Conclusion:1.The high expression of GALNT3 in non-small cell lung cancer cells is positively correlated with lymph node metastasis,TNM staging and tumor size.2.GALNT3 promotes proliferation and migration of non-small cell lung cancer cells.3.GALNT3 positively regulates the MAPK signaling pathway.4.GALNT3 binds to MUC1 and regulates its expression.5.GALNT3 regulate the proliferation and migration of non-small cell lung cancer cells through MUC1.