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  • br Fig a CDC expression in AGS


    (Fig. 4a). CDC27 expression in AGS and NCI-N87 cells was then knocked down by shRNA. MTT and colony formation assays showed that deletion of CDC27 promoted cell growth and colony-forming abilities compared to those in control cells (Fig. 4b-e). CDC27 is a component of the Erastin promoting complex/cyclo-some (APC/C) and a cell cycle-regulated E3 ubiquitin ligase, which can control progression through mitosis and the G1 phase of the cell cycle [26]; hence, the expression of Cyclin B1 was examined. As shown in Fig. 4f, Cyclin B1 expression was upregulated by CDC27 deletion in both cell lines. Taken together, these results sug-gest that CDC27 is a tumor suppressor that is inactivated upon mutation.
    FLG (Filaggrin), which was reported as a TCGA pan-cancer driver gene, was mutated in one single cell and was supported by reads of mutant allele in another one tumor cell and the tumor tissue (Fig. 1c, Table 2). FLG expression was also detected in gastric cancer cell lines by western blot and higher FLG expression was found in MGC803 and SGC7901 cell lines compared with that in other cell lines (Fig. 5a). Immunofluorescence experiments revealed that FLG was localized in the cytoplasm of all the GC cell lines (Fig. 5b). FLG expression was then knocked down in SGC7901 cells 
    by shRNA (Fig. 5c). MTT and colony formation assay showed that deletion of FLG promoted cell growth and colony-forming abilities compared with those in control cells (Fig. 5d, e). Our data showed that FLG knockdown resulted in up-regulated expression of IL8, which induced inflammation. These results suggest that knock-down of FLG might promote cell growth through induction of inflammation, which is an inducer of GC.
    4. Discussion
    Until date, the intra-individual variations of GC have remained unknown. This study presents the mutation landscape of GC at the single-cell level by whole-exome single-cell sequencing for the first time and demonstrates that 24 somatic significant mutated genes (SMGs) and highlighted CDC27 and FLG might alter protein conformation and potentially contribute to promoting GC progression by inducing abnormal cell growth.
    In recent studies on cancer at the single-cell level, researchers discovered that genes might mutate at a low frequency of the pop-ulation level but at a high frequency of the single-cell level, indicat-
    Number of clones
    Fig. 5. Functional characterization of FLG. (a) Expression of FLG in GC cells detected by western blot. (b) Localization of FLG in GC cells detected by immunofluorescence. (c) The effect of FLG knockdown the expression of IL8 in SGC7901 cells. (d) The effect of FLG knockdown on cancer cell proliferation was detected by an MTT assay in SGC7901 cells and e a colony formation assay in SGC7901 cells.
    ing the existence of intra-tumoral and inter-tumoral heterogeneity [5]. Analogously, we compared single cells with the corresponding tissue and 54 additional paired tissues, and found 4 groups of mutated genes with different mutation frequencies between the single-cell level and population level: high at both levels; high at the single-cell level but low among tissues, implying the driver event in an individual but not prevalent in the population; high at the population level but low among tumor cells; and low at both levels. The roles of these four groups of genes in GC were unclear and further investigations are necessary. In colon cancer, people have found a few driver events with high mutation frequencies both at single-cell and population level, such as TP53 and APC, but also found SLC12A5, which frequently mutated among cancer cells but not population, was also potential driver [5]. In our study, we also found five genes with high mutation frequency at the single-cell level but absent in the corresponding tissue, which were also frequently mutated at the population level and might play an important role in single GC cells. As expected, CDC27 and FLG pro-mote GC progression through affecting cell growth by fingerprint-ing and functional characterization. Of these, CDC27 was reported as a TCGA pan-cancer driver gene that is recurrently mutated in prostate cancer [27] and colon cancer single cells [5]. Low CDC27 and high securin expression was reported in breast cancer patients and significantly correlated with clinical outcomes [28]. In present study, we focus to analysis of gene mutation correlated with intra-tumoral heterogeneity in GC. The protein structure determines the function of protein, hence, we used PFSC to predict the protein folding. Following gene mutation, it is not only the amino acid sequence changed, but also protein three-dimensional structure has been transformed. Future experiments in vivo, including ani-mal models, would more definitely address the functional signifi-cance of these two genes in GC.