PAX8 and SOX13 are upregulated in stomach cancer
The TCGA stomach cancer database was first used to retrieve the expression patterns of all the PAX family transcription factors, and the expression of PAX8 was found to be significantly up-regulated in stomach cancer tissues compared to normal tissues (Figure 1A). Although the cancer-promoting role of PAX8 in most types of tumors has been widely reported, the clinical significance and molecular function of PAX8 in stomach cancer remain puzzling. In order to verify whether PAX8 is related to the pathological progression of stomach cancer, the expression pattern of PAX8 in stomach cancer tissues with different pathological progression was further explored. Interestingly, in four stomach cancer tissues with different stages, the expression of PAX8 showed an increasing trend by the pathological stages (Figure 1B).
To further explain the up-regulated expression of PAX8 mRNA in stomach cancer, we were interested in the fact that HMG-box transcription elements were found to be widely present in the promoter region of PAX8, suggesting that the increased expression of PAX8 mRNA might be related to HMG-box contained transcription factors. By analyzing the TCGA stomach cancer database, we found that the expression of HMG-box domain contained SOX13 was also up-regulated, and the SOX13 expression was positively correlated with the expression pattern of PAX8 (r = 0.6567, p<0.001) (Figure 1C).
Furthermore, qRT-PCR assay revealed that SOX13 showed consistent up-regulated expression patterns as similar as PAX8 in 36 pairs of stomach cancer tissues (Figure 1D, E). In addition, spearman correlation analysis of the mRNA lever of PAX8 and SOX13 in stomach tumor tissues suggested a strong positive correlation (r = 0.6567, p<0.001), consistent with TCGA-based results (Figure 1F).
To explore the differences of PAX8 and SOX13 expression pattern in different stomach cancer cell lines, SOX13 was found to significantly up-regulated in stomach cancer cell lines as the expression pattern of PAX8, especially in MGC803 and AGS cell lines, by comparing PAX8 and SOX13 expressed in 4 stomach cancer cell lines with GES1 cells (Figure 1G, H). However, it was noted that the mRNA and protein level of PAX8 was overexpressed in cell lines where SOX13 mRNA and protein were also significantly up-regulated, suggesting the positive correlation of SOX13 and PAX8 (Figure. 1G, H).
Upregulated SOX13 and PAX8 was associated with worse prognosis of stomach cancer patients
Immuno-histochemical staining of stomach cancer specimens was performed to explore the distribution of PAX8 and SOX13 in stomach cancer tissues. The staining section indicated that PAX8 were mainly distributed in the nucleus, as the stain result of SOX13 (Figure 2A). Furthermore, 36 clinical tissues were classified according to the staining levels of PAX8 and SOX13, and the results indicated that the protein of SOX13 and PAX8 expressed in tissues were significantly different (χ2 test, p=0.0361). In brief, 47.22% (17/36) of stomach cancer patients was accompanied with SOX13 and PAX8 overexpression, while only 22.22% (8/36) of patients showed a lower expression of PAX8 and SOX13 (Figure 2B), indicating the probability that PAX8 shared the same expression pattern as SOX13 in stomach cancer.
Furthermore, weather the level of SOX13 and PAX8 in stomach cancer was correlated with the survival of patients was explored, in order to clarify the clinical significance of SOX13 and PAX8. By comparing the survival curves, it was found that not patients with high level of PAX8 significantly resulted with worse survival compared to patients with low PAX8 expression, but also SOX13 did (Figure 2C, D). In stomach cancer patients with the same expression patterns of SOX13 and PAX8, combined low SOX13 and PAX8 expression was found to result with a better overall survival rate, but not up-regulated SOX13 and PAX8 (Figure 2E). These results suggest the clinical significance of SOX13 and PAX8 in stomach tumors, which can be used as potential biological indicators for the survival of patients with stomach cancer.
SOX13 regulates the transcription of PAX8 in stomach cancer
In order to verify that the up-regulated expression of PAX8 in stomach cancer is related to SOX13, we verified whether SOX13 can regulate PAX8 expression in stomach cancer cell lines. It was first found that different amounts of SOX13 overexpression could cause the associated increase of PAX8 mRNA and protein expression level in AGS and MGC803 cells (Figure 3 A, B). Moreover, silencing SOX13 can down-regulate PAX8 mRNA and protein expressed in AGS and MGC803 cell lines, while SOX13 overexpression can rescue the down-regulation of PAX8 to some extent caused by SOX13 knockdown. However, even overexpressed SOX13 mutants (SOX13 ins6), in which six amino acids were inserted into the HMG-box of SOX13 to deprive its ability to bind with the HMG-box DNA sequence, cannot reverse the decline in PAX8 expression (Figure 3C, D). These results confirmed that SOX13 was one of the factors regulating PAX8 expression in stomach cancer.
Since SOX13 has been proved to regulate the expression of PAX8 in stomach cancer, luciferase assay was further used to explore the combination of SOX13 with the promoter region of PAX8, in order to verify that SOX13 was a transcription factor of PAX8. Although SOX13 overexpression was found to significantly increase the expression of reporter genes containing the PAX8 promoter, SOX13 lost its ability to promote reporter gene expression, when the PAX8 promoter region was reduced by more than 600 bp on the far terminal (Figure 3E), suggesting that SOX13 may bind with the -300~-600bp regions of the PAX8 promoter to regulate PAX8 expression. Furthermore, ChIP-qRCR assay showed that SOX13 could significantly enrich the -300~-600bp region of PAX8 promoter, confirming the interaction between SOX13 and PAX8 promoter (Figure 3F).
Previous studies have shown that Aurora B and Cyclin B1, as mitotic regulators, can be regulated by PAX8 and thus affect the progression of tumor cell cycle, which promoted us to speculate whether SOX13-regulated PAX8 expression can affect the expression of Aurora B and Cyclin B1 in stomach cancer. PAX8 silencing can significantly cause the silencing of Aurora B and Cyclin B1, the expression of Aurora B and Cyclin B1 were recovered, when PAX8 was expressed in AGS and MGC803 cells, confirming that PAX8 can regulate the expression of Aurora B and Cyclin B1 in stomach cancer (Figure 3G). Moreover, wild type SOX13 expression also restores the mRNA level of Aurora B and Cyclin B1, in parallel with the upregulated expression of PAX8 to a certain extent (Figure 3G), suggesting the notion that SOX13-regulated PAX8 expression affects the expression pattern of Aurora B and Cyclin B1 in stomach cancer.
SOX13-mediated PAX8 expression promotes cellular proliferation in stomach cancer
In view of the positive effect of SOX13-regulated PAX8 expression on cellular Aurora B and Cyclin B1 expression, SOX13-regulated PAX8 expression function on cell cycle was detected. And flow cytometry assay showed that PAX8 knockdown induced G1-phase arrest in AGS and MGC803 cell lines, which could be rescued by SOX13 overexpression (Figure 4 A, B). What’s more, the overexpression of SOX13 can significantly increase the decline of EdU positive cells induced by PAX8 knockdown, no matter in AGS cells or in MGC803 cells, indicating that SOX13-mediated PAX8 expression can promote the progression of cell cycle in stomach cancer (Figure 4 C, D).
Next, whether PAX8 function on the cellular viability in stomach cancer was detected, which was one of the indicators for malignant proliferation of tumor cells. The results showed that PAX8 knockdown could directly weaken the viability of stomach cancer cells, while the up-regulation of PAX8 expression induced by SOX13 overexpression restored the cell viability (Figure 4 E). Consistently, SOX13 overexpression can significantly increase the PAX8 silencing-induced decrease of clone formation in both AGS cells and MGC803 cells, indicating that SOX13-mediated PAX8 can promote the tumorigenicity of stomach cancer cells (Figure 4 F, G). Therefore, the expression of PAX8 regulated by SOX13 can promote the progress of stomach cancer cell cycle, showing the capbilty to promote tumorigenicity of stomach cancer cells.