The expression of PLAGL2 in human cancers
First of all, in order to analyze the expression level of PLAGL2 in various types of cancer, the Oncomine database was applied (Figure.1A). The results indicate that PLAGL2 is significantly overexpressed in various type cancers including stomach cancer. Then, we also evaluated the expression of PLAGL2 through the TIMER database which include RNA-sep data from various malignant tumors in TCGA (Figure.1B). We can find that PLAGL2 was overexpressed in most malignant tumors. Finally, the SangerBox website was used to analyzed the expression of PLAGL2 in TCGA and GTEx (Figure.1C). We can find that PLAGL2 was obviously overexpressed in most types of cancer. The above data indicate that PLAGL2 is overexpressed in most cancer types and may play an important role in the development of various types of cancer.
Prognostic potential of PLAGL2 in cancers
Firstly, we investigated the prognostic potential of PLAGL2 in cancers through the PrognoScan. The results indicated that PLAGL2 might participate in the prognosis of breast cancer and colorectal cancer. However, as shown in the results, high PLAGL2 expression is slightly related to a better prognosis in colorectal cancer (Figure.2A-2C), and the prognosis of PLAGL2 in breast cancer is not consistent (Figure.2D-2F).
So, we further explore the prognostic value of PLAGL2 through the Kaplan-Meier plotter. We can find that high PLAGL2 expression is slightly related to a poor prognosis in stomach cancer (Figure.2G-2I). However, high PLAGL2 expression is probably related to a better prognosis in ovarian (Figure.2J-2L) and breast (Figure.2M-2O) cancer. The results above indicated that PLAGL2 might be a prognostic biomarker in stomach cancer. Then we further explored the association between the expression of PLAGL2 and the clinical characteristics of STAD through the Kaplan-Meier plotter (Table 1). We can find that high expression of PLAGL2 related to both poorer OS and PFS in female, male, HER2 negative and HER2 positive in STAD. Specifically, high expression of PLAGL2 related to both poorer OS and PFS in STAD patients belonging to stages 2, stage N1+2+3, stage N1 and stage M0.
Relationship between PLAGL2 expression and immune and molecular subtypes in human cancers
Next, we further analyzed the relationship between PLAGL2 expression and immune and molecular subtypes in human cancers through the TISIDB website. We can find that there was a clearly relationship between PLAGL2 and different subtypes of UCEC, BRCA, CESC, COAD, HNSC, KIRC, KIRP, LGG, LUSC, TGCT and LIHC (Figure.3). In addition, the expression of PLAGL2 is also related to different cancer molecular subtypes in various cancers (Figure.3S). We may deduce from the aforementioned findings that PLAGL2 may play an important role in the immunological and molecular subtypes of many malignancies.
Relationship between PLAGL2 expression and immune checkpoint (ICP) genes in human cancers
Immune cell infiltration and immunotherapy have both been shown to be significantly influenced by ICP genes. The relationship between PLAGL2 expression and ICP genes in human malignancies was then investigated (Figure.4). The results indicated that PLAGL2 expression is associated to immune checkpoint genes in a range of malignancies.
Relationship between PLAGL2 and immune cell infiltration in human cancers
We further investigated the possible association between PLAGL2 and immune cell infiltration, we can find that there is a substantial correlation in numerous cancer types (Figure.5). PLAGL2 expression is related to dendritic cells in 19 cancers, macrophages in 14 cancers, neutrophils in 23 cancers, CD8+ T cells in 14 cancers, and B cells in 20 cancers. In 15 cancers, there is a strong correlation between CD4+ T cells. Then, we also explored the relationship between the expression of PLAGL2 and different immune marker genes in STAD (Table 2). We can find that there exist a significantly relationship between the expression level of PLAGL2 and most immune markers in various immune cells in STAD.
The regulation network of PLAGL2 in stomach cancer
Previous results in this study we have demonstrated that PLAGL2 was overexpressed in STAD, and predicted a poor prognosis. So, we further explored the regulation network of PLAGL2 in stomach cancer. Firstly, As shown in the volcano map (Figure.6A-6C), we explored the co-expression genes of PLAGL2 in STAD through LinkedOmics. The differentially expressed genes associated to PLAGL2 are mostly involved in cell cycle control, according to the results of GO term analysis (Figure.6E-6G). KEGG pathway analysis revealed the enrichment of cell cycle, Staphylococcus aureus infection, basic transcription factors, complement and coagulation cascade(Figure.6H).
Prognostic potential of PLAGL2 co-expression genes in stomach cancers
Then, we further evaluated the prognostic potential of the genes positive co-expression with PLAGL2 in STAD through Kaplan-Meier plotter (Figure.7, Figure.7S). The results showed that among the top 40 genes co-expressed with PLAGL2 in STAD, most of the genes were related to the prognosis of STAD.
Relationship between PLAGL2 and different clinical subgroups in STAD.
Next, the expression of PLAGL2 in STAD with different clinical characteristics was explored through UALCAN database. The results indicated that there existed an significantly difference between the expression of PLAGL2 in different STAD patients' gender (Figure.8A), age(Figure.8B), tumor grade(Figure.8C), lymph node metastasis status(Figure.8D), cancer stage(Figure.8E), and Helicobacter pylori infection status(Figure.8F), indicating that PLAGL2 may act as an important oncogene in the progress of STAD.
The function of PLAGL2 in STAD cells
Finally, to verify the association between PLAGL2 and STAD clinicopathological characteristics, we performed Western blot and IHC to detect PLAGL2 expression in 57 paraffin-embedded STAD specimens(Figure.9A-9C). we can find that the PLAGL2 was overexpressed in STAD tissues. More importantly, our findings reveal that the PLAGL2 is associated to lymph node metastasis and tumor size in STAD(Table 3). Then, we used a lentivirus-based system to establish stable PLAGL2 knockdown 7901 and MKN-45 cell lines (Figure.9D-9F). Next, the results of CCK8 test(Figure.9G) and the colony formation test(Figure.9H-9I) revealed that PLAGL2 could promote the proliferation ability of STAD cells. Finally, the results of the xenograft subcutaneous transplantation model showed that PLAGL2 could promote the growth of STAD cells in vivo (Figure.9J-9L). In summary, the data above supports the conclusion that PLAGL2 is an oncogene in STAD and promotes the proliferation of STAD cells in vitro and in vivo.