YTHDC1 is widely expressed in a variety of tumors
Based on the TCGA database, we recognize that the expression of YTHDC1 exists in all 33 tumors (FPKM > 2), of which the highest expression was in LAML and THYM (Fig. 1A). Gene differential analysis showed that YTHDC1 was differentially expressed in 9 tumors, of which 5 tumors (CHOL, COAD, ESCA, LIHC and STAD) were significantly up-regulated and 4 tumors (BRCA, KICH, UCEC, THCA) were significantly down-regulated, suggesting its potential function in tumorigenesis (Fig. 1B). Furthermore, we analyzed the correlation between YTHDC1 expression and tumor stage. It was found that YTHDC1 expression was up-regulated in advanced liver cancer and down-regulated in five tumors (BLCA, KIRC, LUSC, SKCM and THCA) (Fig. 1C). In recent years, protein post-translational modifications (PTMs) has been found to play an important role in tumorigenesis and development, and phosphorylation modification is the most extensive PTMs in vivo. Based on CPTAC database, we found that the phosphorylation of YTHDC1 was abnormally modified in a variety of tumors (Fig. 1D) . In conclusion, the above results suggest that YTHDC1 may be involved in the development of a variety of tumors.
YTHDC1 is associated with multiple tumor prognosis
Univariate Cox regression analysis showed that high expression of YTHDC1 was associated with better prognosis in patients with four tumors (BLCA, KIRC, LGG and SKCM) (Fig. 2A). KM survival analysis also suggested that the high expression of YTHDC1 in BLCA KIRC CESC and LGG was associated with better overall survival (Fig. 2B). These results also suggest that YTHDC1 may be a potential prognostic marker for some tumors.
Prediction of YTHDC1 related functions by gene enrichment analysis
We performed gene enrichment analysis (GSEA) to predict the potential function of YTHDC1 in all the above tumors (Fig. 3A). Interestingly, YTHDC1 is associated with NK cell-mediated cytokines in BRCA. In LUSC and MESO, it is closely related to toll like receptor signal. In PRAD, YTHDC1 is associated with cytokine transmission. Among the tumors identified above which YTHDC1 may play an important role, YTHDC1 is related to calcium signal transduction in LIHC and fatty acid metabolism signal in BLCA which are closely related to tumor immune regulation [15, 16]. Therefore, we speculate that YTHDC1 may be involved in the shaping of tumor immune microenvironment.
Correlation analysis between YTHDC1 and immune marker
Using TISIDB database, we found that YTHDC1 was closely related to a variety of immune markers . MHC complex is an important signal for the body to initiate immune response. In the process of tumorigenesis and development, the abnormal expression of HLA-A and HLA-B signals promotes immunosuppression . We found that YTHDC1 was negatively correlated with MHC complex expression in a variety of tumors including BLCA, BRCA, LIHC and etc. (Fig. 4A). Interestingly, the expression of YTHDC1 is closely related to the expression of a variety of inflammatory activators and immunosuppressive molecules (Fig. 4B and 4C). This seemingly contradictory result may be attributed to the synergistic formation of pro-inflammatory and anti-inflammatory microenvironment in the tumor and YTHDC1 is involved in promoting the formation of tumor inflammatory microenvironment, which further supports the role of YTHDC1 in tumor immune microenvironment .
Correlation analysis between YTHDC1 and tumor microenvironment
Tumor growth in the body is inseparable from the support of tumor matrix and immune microenvironment . Therefore, we performed correlation analysis between YTHDC1 and tumor microenvironment. The results showed that YTHDC1 are closely associated with matrix scores of eight tumors (ACC, BLCA, GBM, LGG, LUSC, SARC, TGCT, UCS) (Fig. 5A) and immune scores of ACC, BLCA, GBM, KIRP, PCPG, SARC, THYM and UCS (Fig. 5B). These results also support the aforementioned results from another perspective.
YTHDC1 is closely related to tumor mutation burden, microsatellite instability and macrophages in a variety of tumors
Tumor mutation burden (TMB) and microsatellite instability (MSI) have been recognized to play an important role in tumor immune signal transduction and microenvironment formation [20, 21]. The analysis results showed that YTHDC1 are positively associated with TMI in LAML, READ and LIHC, and negatively associated with TMI in BRCA and THCA (Fig. 6A). YTHDC1 was positively correlated with MSI in eight tumors (LUAD, LUSC, LGG, ACC, STAD, READ, SARC and COAD) and negatively correlated in DLBC (Fig. 6B). Tumor associated macrophages (TAMs) are not only involved in the formation of tumor immunosuppressive microenvironment, but also directly involved in tumor growth and metastasis. Our results showed that YTHDC1 are closely associated with TAMs infiltration in multiple tumors such as BRCA and LIHC (Fig. 6C). These results further support the role of YTHDC1 in tumor immune microenvironment.
Independent data sets and clinical samples were used to verify the expression of YTHDC1 in hepatocellular carcinoma
Furthermore, we implemented bioinformatics analysis and experiments to partially verify the above results. Previous results have suggested that YTHDC1 may play an important role in a variety of tumors. YTHDC1 is not only abnormally expressed in liver cancer, but also related to the formation of immune microenvironment of liver cancer. In the early stage, we mainly engaged in liver cancer related research. Therefore, we focused on the role of YTHDC1 in liver cancer. First we analyzed the expression of YTHDC1 in hepatocellular carcinoma in two independent datasets (ICGC-JP and CPTAC-LIHC datasets) which YTHDC1 is high expression in LIHC (Fig. 7A and 7B). We also collected 20 pairs of clinical samples for RT-PCR which the results showed that the mRNA of YTHDC1 was highly expressed in hepatocellular carcinoma (Fig. 7C). Western blot and IHC experiments showed that the results showed that the protein of YTHDC1 was highly expressed in hepatocellular carcinoma (Fig. 7D and 7E).
YTHDC1 promotes liver cancer proliferation and macrophage recruitment
To verify the function of YTHDC1 in hepatocellular carcinoma, we used siRNA to intervene in Huh-7 cells (Fig. 8A and 8B). CCK8 experiment showed that the intervention of YTHDC1 damaged the growth ability of Huh-7 cells (Fig. 8C). Co-culturing Huh-7 and THP-1 cells, after inhibiting YTHDC1, the ability of Huh-7 cells to recruit macrophages decreased (Fig. 8D). These results support the malignant function of YTHDC1 in hepatocellular carcinoma to a certain extent.