eIF6 is overexpressed in HCC clinical samples and cell lines
We analyzed a total of 424 eIF6 expression profiles from the TCGA liver carcinoma dataset, including 50 normal tissues and 374 tumor tissues. The results showed that eIF6 expression was higher in HCC tissue than in normal liver tissue (p<0.001) (Fig. 1a). Furthermore, the expression of eIF6 was also significantly elevated in HCC tissues compared with normal liver tissues by the GEO dataset, including GSE64041, GSE14520, GSE57957, and GSE45436 (all p<0.001) (Fig. 1b-e).
To further validate the above bioinformatics findings, we evaluated the expression levels of eIF6 in 6 paired fresh HCC tissues and their adjacent normal liver tissues by western blot. As shown in Fig. 2a and b, eIF6 levels were evidently increased in fresh HCC tissues (p<0.05). Meanwhile, the relative protein level of eIF6 was significantly upregulated in 7 HCC cell lines (SMMC7721, Huh7, HepG2, BEL-7404, HB611, 97H, BEL-7405) compared with that in normal liver cells (LO2) and was much higher in BEL-7404 and 97H cells (p<0.05) (Fig. 2c). Furthermore, we also performed an IHC assay to verify the above results. The paraffin-embedded tissues were diagnosed as HCC based on HE staining before collection, and eIF6 was mainly located in the cytoplasm, as shown by IHC staining (Fig. 2d). Moreover, statistical analysis showed that the expression of eIF6 was distinctly elevated in 64.71% (44/68) of HCC tissues (Table 2), and its high expression was more than that of normal liver tissues (p<0.001) (Fig. 2e).
eIF6 expressionis positively correlated with tumor size, vascular invasion and poor prognosis in HCC patients
To further investigate the role of eIF6 in the development of HCC, we analyzed the relationship between the expression of eIF6 and the clinical characteristics of HCC patients from IHC results. The 68 HCC patients were divided into the eIF6-high expression group (n=44) and the eIF6-low expression group (n=24), and the clinical features of these patients are summarized in Table 2. The data show that the expression of eIF6 was significantly correlated with tumor size (p<0.001) (Fig. 2f) and vascular invasion (p<0.001) (Fig. 2g). In other words, patients with large tumor diameters or vascular invasion occurrences were frequently identified with eIF6 overexpression. However, eIF6 overexpression had no relationship with other clinicopathological features, including gender, age, differentiation, lymphatic metastasis, distant metastasis, HBV infection, AFP level, and liver cirrhosis occurrence. Kaplan-Meier survival curves indicated that overall survival (OS) time of the eIF6 high expression group was significantly shorter than that of the eIF6-low expression group from the TCGA public database (p=0.015) (Fig. 2h). These results indicate that the expression of eIF6 is correlated with tumor size, vascular invasion and poor prognosis in HCC patients.
eIF6 expression is a reliable clinical diagnostic biomarker for HCC
We established ROC curves to evaluate the clinical diagnostic value of eIF6 in HCC. The area under the ROC curve (AUC) from TCGA was 0.902 and indicated a high diagnostic power (Fig. 3a). Meanwhile, the AUC values from GSE64041, GSE14520, GSE57957 and GSE45436 further validated this result (Fig. 3b-e). The ROC curve of the eIF6 protein also showed authentic diagnostic value in our IHC results (AUC=0.884) (Fig. 3f).
Knockdown of eIF6inhibits the malignant progression of HCC cells in vitro
To determine the possible biological role of eIF6 in HCC cells, we constructed stable eIF6 knockdown BEL-7404 and 97H cell lines. The infection efficiency was measured by western blotting (p<0.001) (Fig. 4a, b).The results of CCK-8 assays revealed that the proliferation activity of eIF6 knockdown cells was deeply decreased compared with that of their control cells (Fig. 4c, d). The quantitative analysis from plate colony assays suggested that the number of colonies formed by eIF6 knockdown cells was markedly lower than that of the control cells (p<0.001) (Fig. 4e). Furthermore, the invasive capabilities of HCC cells were assessed by Transwell assays with Matrigel. We found that eIF6 knockdown dramatically decreased the number of invasive cells passing through the Matrigel to the underside of the membrane (p<0.001) (Fig. 4f).
We also detected the effect of eIF6 on apoptosis and the cell cycle in BEL-7404 and 97H cells by flow cytometry assay. As shown in Fig. 5a, knockdown of eIF6 increased the proportion of BEL-7404 and 97H cells in G0/G1 phase, but decreased the proportion of cells in S phase, which was similar to the proliferation assay results. For cell apoptosis, knockdown of eIF6 induced a significant increase in the total apoptosis rate in BEL-7404 cells (p<0.01) and 97H cells (p<0.001) (Fig. 5b). Accordingly, these results showed that the increase in cell cycle G0/G1-phase arrest and apoptosis might contribute to the suppression of cell proliferation after depletion of eIF6.
Knockdown of eIF6 arrested tumor growth in a nude subcutaneous xenograft model.
The effect of eIF6 on HCC cell growth was further confirmed in a nude mouse xenograft model. Cells treated with the eIF6 knockdown (BEL-7404-sheIF6) and control (BEL-7404-shNC) were injected into nude subcutaneous tissue, and the tumor size was measured every two days. BEL-7404-sheIF6 cells injected into nude mice developed smaller (p<0.05) and lighter tumor (p<0.01) with slower growth rates compared with those in the control (n=6) (Fig. 6a-c). Finally, IHC staining of Ki-67 antibody indicated that the Ki-67 index in the BEL-7404-sheIF6 groups was lower than those that in the control group (p<0.05) (Fig. 6d, e), further confirming that eIF6 knockdown inhibited HCC cell proliferation in vivo.
The potential regulatory network and functional enrichment analysis of eIF6 in HCC
To further investigate the potential signaling pathways by which eIF6 promoted tumor malignant progression, we examined 355 eIF6-associated proteins using UniHI websites (the details given in Supplementary Table S2) and then constructed an eIF6 centered network showing the top 20 proteins using GeneMANIA (Fig. 7a). Subsequently, we submitted them to the Metascape online website for pathway enrichment analysis. The results showed that the associated proteins received a high enrichment in the cell cycle (p<0.001) and mTOR signaling pathways (p=0.00338) (Fig. 7b, and the details are shown in Supplementary Table S3), which are associated with the malignant proliferation of tumor cells.
eIF6 activated mTOR-related cancer signaling pathways in HCC progression
To confirm above result of enrichment analysis, we examined the effect of eIF6 knockdown on mTOR activity by western blotting. As shown in Fig. 8a, eIF6 knockdown dramatically decreased the phosphorylated-mTOR (p-mTOR) level in HCC cell lines. At the same time, cell cycle and invasion regulators, including MYC, CCND1, CDK4, CDK6 and CTNNBL1, were downregulated after eIF6 knockdown, while the apoptosis regulator cleaved-CASP3 was upregulated (Fig. 8b). The detection of a coefficient correlation between eIF6 and the above related markers showed that eIF6 expression was positively correlated with MYC, CCND1, CDK4, CDK6 and CTNNBL1 (Fig. 8c-g), which was consistent with the western blot results. Consequently, we concluded that eIF6 activated mTOR-related multiple cancer signaling pathways to promote the malignant progression of human HCC (Fig. 8h).