GALNT10 knockdown in A549 cell line and over-expressing in the H322 cell line

Aberrant mucin-type O-glycosylation affects many cellular properties and is associated with many cancers. N-acetylgalactosaminyltransferase 10 (GALNT10) is a GalNAc-transferase that initiates protein O-glycosylation. The aim of this study was to explore the role of GALNT10 in lung adenocarcinoma. Immunohistochemistry was performed to study the expression of GALNT10 in an lung adenocarcinoma tissue microarray. We found that GALNT10 is frequently down-regulated in lung adenocarcinoma tissues and is associated with pathological classification and TNM stage. Additionally, we demonstrated that knockdown of GALNT10 with small interference (si) RNA promoted cell proliferation, cell colony formation and cell invasion capacity in A549 cells by using CCK8 assay, flow cytometry, cell colony formation, and transwell invasion assay.Moreover, whole genome microarray analysis showed that 287 genes were up-regulated and 137 were down-regulated in A549 cells upon GALNT10 knockdown. Functional enrichment analysis reveal that GALNT10 knockdown in A549 cells leads to differential gene expression in pathways, including TNF signaling pathway. These findings suggest that down-regulation of GALNT10 plays an important role in the cell proliferation and invasive behavior of lung adenocarcinoma via modifying O-glycosylation and activity of TNF pathway. In addition, we suggest that GALNT10 may be a tumor suppressor gene in lung adenocarcinoma and that targeting GALNT10 could be a promising approach for lung adenocarcinoma therapy. Gene expression analysis was carried out as previously described (Wang et al., 2014; Huang et al., 2015). Briefly, total RNA from control and GALNT10 siRNA knockdown A549s cells were extracted in triplicates using GeneJET RNA Purification kit (Thermo Scientific) following the manufacturer’s protocol and quantified by NanoDrop spectrophotometer. The quality of the RNA samples was examined by capillary electrophoresis using the Agilent 2100 Bioanalyzer (Agilent). Fluorescently labeled cDNA targets were generated from 10 μg of total RNA from each corresponding A549s cell clone following user’s manual. Agilent Gene Expression Hybridization Kit was used for hybridization according to the manufacturer’s instruction. Array hybridization, washing, scanning, data extraction and analyses were performed as previously described (Keita et al., 2013). Furthermore, we investigated the function GALNT10 of by over-expressing GalNAc-T10 in the H322 cell line and by down-regulating GalNAc-T10 expression by shRNA interference in the A549 cell line, and by evaluating proliferation, invasion and apoptosis in the corresponding cells. These in vitro experiments showed that increased gene expression of GalNAc-T10 could inhibit the growth, invasion and promote H322 cell apoptosis. On the contrary, reduced expression of the GalNAc-T10 gene can promote the growth, invasion, and inhibit A549 cell apoptosis.