GOLPH3 is upregulated in HNSCC tissue and FaDu cells
From The Cancer Genome Atlas (TCGA) database, comparing HNSCC and adjacent normal tissues, the result showed that GOLPH3 was upregulated in tumor sites (Fig. 1a, b, *p < 0.05, **p < 0.01). In addition, we found that the prognostic survival time was significantly and negatively correlated with the expression level of GOLPH3 in cancer tissues (R = 0.211, P = 0.008, n = 158) (Fig. 1c). Then, we explore whether the expression level of GOLPH3 is consistent with tumor tissue in vitro and we investigated WSU-HN30 and FaDu cells by qPCR and western blot. As expected, the expression of GOLPH3 was significantly upregulated in FaDu cells in mRNA and protein level (Fig. 1d, e, *p < 0.05). Therefore, it suggests that the expression level of GOLPH3 was closely related to HSCC.
GOLPH3 promotes proliferation of HSCC
To explore the effect of up-regulation of GOLPH3 on the development of HSCC, we constructed a FaDu cell line that has stable overexpression and knock-down effect of GOLPH3. This two stable FaDu cell lines have been verified by qPCR and western blot (Fig. 2a, b, ***p < 0.001, **p < 0.01). The results of CCK8 and cells colony assay indicated that, compared with NC group, overexpression of GOLPH3 could significantly promote the proliferation and colony formation while GOLPH3 knockdown showed opposite effects (Fig. 2c and d - e, **p < 0.01, *p < 0.05). Taken together, the results of this part demonstrated that GOLPH3 was a necessary factor for cell proliferation process and development of HSCC.
GOLPH3 promotes migration of HSCC
To further explore the effect of GOLPH3 on migration of HSCC tumor cells, we performed the wound healing and trans-well migration assay. As expected, the results of wound closure (%) and number of cell migration showed that oeGOLPH3 group had a significant promotion on the migration of FaDu cells. Conversely, the two groups of knock-down (shGOLPH3#1 and #2) all displayed that the significant inhibition of migration ability (Fig. 3a and b, 3c and d, **p < 0.01, *p < 0.05). Taken together, the GOLPH3 expression level was positively correlated with migration ability of HSCC tumor cells.
GOLPH3 promotes HSCC tumor growth in vivo
To investigate whether the level of GOLPH3 will affect tumorigenesis in vivo, we injected nude mice with three FaDu cell lines: stable knockdown (shGOLPH3#1), overexpression (oeGOLPH3) and control (blank vector mix, CTL) respectively. Tumor was observed continuously for 15 days after injection, and the weight and volume of tumors were measured in this process. Our results showed that the weight and volume of tumors in shGOLPH3#1 group were significantly lower than that in group oeGOLPH3 and CTL (Fig. 4a - c, **p < 0.01, ***p < 0.001). The results of immunochemistry showed that the expression trend of GOLPH3 was corresponded to tumorigenic experiments (Fig. 4d). Taken together, these results indicated that GOLPH3 significantly promotes tumorigenesis of HSCC in vivo.
GOLPH3 activates the AKT/mTOR axis in HSCC in vitro
Related studies have shown that GOLPH3 can activate mTOR signaling pathway in the process of promoting tumor growth (Scott et al., 2009; Liu et al., 2018; J. H. Wang et al., 2012; Yu et al., 2020). To verify and explore the correlation between GOLPH3 and mTOR signaling pathway in HSCC, we conducted an enrichment analysis online via Metascape system based on the genes positively correlated with GOLPH3 in HNSC (Pearson-CC ≥ 0.6) and the genes were gathered from TCGA database. The results showed that GOLPH3 was significantly associated with HNSCC and mTOR signaling pathway (Fig. 5a). We further investigated the expression trend of proteins AKT1, mTOR in HNSCC, and their correlation with GOLPH3 expression respectively. The results showed that the expression of AKT1 and mTOR was significantly upregulated with the tumor progression grade of HNSCC (Fig. 5b, *p < 0.05). Besides, correlations between the expression of GOLPH3 and AKT1, mTOR were significant and positive (Fig. 5c). It showed that the expression level of GOLPH3 was positively correlated with the expression and phosphorylation level of AKT and mTOR via western blot (Fig. 5d). Taken together, our results demonstrated that GOLPH3 can activates the AKT/mTOR axis in development of HSCC.