## 3 Discussion
To summarize, very little is known about the role of _FAM13A_ gene on tumor cellular proliferation. Only in one study, decrease in A549 cell number and proliferation was observed after FAM13 AsiRNA treatment [14]. In line with this, our results confirmed that FAM13A drives NSCLC cell proliferation, especially under hypoxia. To analyze the role of FAM13A in cancer cell metastasis, we performed wound healing and invasion assays. The wound healing results showed that FAM13A silencing had a strong negative impact on A549 and CORL-105 cell migration (Figures 3 and 4). We observed that while A549 cells responded faster to hypoxia (48 h), CORL-105 cells were overall more sensitive to hypoxia, which was reflected in more severe inhibition of migratory capacities after 72 and 96 h. In the current study we observed that hypoxia reduced cell migration, and FAM13A silencing resulted in further inhibition of cell migration both in normoxia and hypoxia. As the cell migration depends on the actin cytoskeleton we assessed the influence of FAM13A inhibition on the cytoskeleton phenotype. The phalloidin scanning revealed that FAM13A depleted cells beside different cell shapes were characterized by various actin cytoskeleton disturbances. The reorganization of F-actin filaments and presence of F-actin aggregates were observed in normoxia and enhanced in hypoxia. The structural dysregulation of the actin cytoskeleton correlate with movement of tumor cells. Based on current results we assumed that FAM13A is involved in inhibition of cell migration via altering the actin cytoskeleton. HIFs are induced rapidly upon exposure to 1% O\({}_{2}\) and the delayed effect of hypoxia on migration may reflect the time required to inhibit a downstream signaling pathway in which FAM13A may be involved. However, the results of current study are opposite to reported by [PERSON] et al. in which transient knockdown of _FAM13A_ in A549 cells caused induction of cell migration, probably due to Rho proteins activation [14]. On the other hand, the proliferation of FAM13A siRNA treatment cells was decreased, which was confirmed in this study. Some discrepancies may be due to the experimental setups. In our study we generated stable FAM13A knockdown in lung cancer cells, which allowed us to conduct experiments during longer time period. Moreover, during the migration assay, the wound was generated using inserts with a defined gap, which increases the accuracy and reproducibility of the experiments [18]. We also used cell starvation procedure before starting the experiments. Results obtained for A549 cells were confirmed by monitoring on JuLIFL system for live cells imaging. In addition, we showed that FAM13A knockdown inhibits the invasive activities of NSCLC cells. Significant decrease in cell invasion rate was observed for FAM13A shRNA A549 cells vs. controls, under both oxygen concentrations (Figure 5). The inhibition of invasive properties of A549 FAM13A depleted cells was enhanced under hypoxia as compared with normoxia. The migration and invasion of FAM13A A549 depleted cells depleted may be linked to the epithelial-mesenchymal transition (EMT). It was shown that hypoxia may induce a partial EMT phenotype in NSCLC cell lines [19]. Moreover, recent data indicated that FAM13A may regulate the Wnt signaling activity associated with EMT in A549 cells [20]. Based on the current results we assume that the hypoxia-induced FAM13A overexpression may diminish the occurrence of EMT in lung cancer cells, but further studies are needed to verify this hypothesis. The difference in effect of FAM13A knockdown on cellular processes between A549 and CORL-105 cells may be related to the cells of origin. A549 cell line was derived from primary lung carcinoma, whereas CORL-105 originated from metastasized lung adenocarcinoma (pleural effusion). We chose two different NSCLC cell lines to examine the effect of FAM13A suppression on cells from primary and metastatic site. It is possible that hypoxia promotes metastasis by modulating changes inherent only to cells in primary tumor.