1. Elaiophylin is a novel autophagy inhibitor in the ESCC cells
To identify whether Elaiophylin play as an autophagy inhibitor like it does in ovarian cancer and multiple myeloma with mutant TP53, we treated ESCC cells with different concentrations. And CQ as a positive control. The western blot assay results indicated that the ratio of LC3II/LC3I was increased after exposed to Elaiophylin. And the expression level of p62 was upregulated at the protein level (Fig. 1. A). Statistical analysis of A (Fig. 1. B). We further transfected GFP-LC3 fusion protein to ESCC cells, then exposed to Elaiophylin with different concentrations. The results showed that the GFP-LC3 puncta was increased in a dose-dependent manner after Elaiophylin treated (Fig. 1. C, E). The statistical analysis of C (Fig. 1. D, F). To further verify whether Elaiophylin can inhibit the autophagy at the late stage, we transfected mCherry-GFP-LC3 plasmid to ESCC cells. Since EGFP fluorescence is quenched by acid protease in the lysosome, thus the puncta of yellow was represented of autophagosome, whereas the red was represented of autolysosome (Fig. 1. G, H). Statistical analysis of G (Fig. 1. I). The results indicated that Elaiophylin could inhibit autophagy flux at the late stage in the ESCC cells.
2. Elaiophylin exerts anti-proliferative, anti-migrative, and pro-apoptotic effects on ESCC cells
To assess the other potential function of Elaiophylin on ESCC cells, we detected the cell viability, proliferation, migration and apoptosis rate of the cells and confirmed its effect by measuring the protein expression level of E-cadherin, Bax and Bcl-2 after dealing with Elaiophylin. Utilizing the CCK8 assay, we observed that the cell viability was decreased by Elaiophylin in a dose-dependent manner in both two ESCC cell lines when compared with the control. (Fig. 2. A, B). As cancer metastasis contributed to poor prognosis of ESCC patients, transwell migration assay was also carried out to determine the effects of Elaiophylin on the ability of migration of ESCC cells. After staining with crystal violet, the inhibition of ESCC cell migration was evident, as less cell numbers were observed after treating with Elaiophylin compared to the control. (Fig. 2. C). Statistical analysis results of C (Fig. 2. D). In addition, the results of immunofluorescent staining for Ki67 showed that the Ki67 positive cells was decreased by Elaiophylin, which indicated that the proliferation rate of ESCC cells was attenuated (Fig. 2. E, G). Statistical analysis results of E, G (Fig. 2. F, H). Notably, flow cytometry assay established increased apoptosis rates in ESCC cells by treating with Elaiophylin (Fig. 2. I). Statistical analysis results of I (Fig. 2. J). Moreover, western blot assay analysis showed increased protein expression of E-cadherin, Bax by Elaiophylin, whereas the Bcl-2 expression level was decreased (Fig. 2. K, L, M).
3. RNA-seq results indicated that Elaiophylin could inhibit the activation of PI3K/AKT signaling pathway
To elucidate the mechanism and target of Elaiophylin, we conducted RNA-seq analysis (GSE171167) to identify the genes that were transcriptionally affected by Elaiophylin. In RNA-seq, the correlation of expression levels between samples is an important indicator to test the reliability of the experiment and whether the sample selection is reasonable. The closer the correlation coefficient is to 1, the higher the similarity of the expression patterns between samples. The Encode plan recommends that the square of Pearson’s correlation coefficients of the two biological replicates in each group of the two cell lines are both greater than 0.97, indicating that the sample has good reproducibility and the sequencing results are credible (Fig. 3. A, F). Then the differently expressed genes were screened, and the screening criterion was |log2(FoldChange)|༞1&padj༜0.05. The volcano chart shows the distribution of differential genes for each comparison combination (Fig. 3. B, G). Red dots indicate genes that are up-regulated, and green dots indicate genes that are down-regulated. The results showed that there were 360 up-regulated genes and 891 down-regulated genes in Eca109 cells (Fig. 3. B). And there are 126 up-regulated genes and 435 down-regulated genes in KYSE450 cells (Fig. 3. G). Then cluster all differentially expressed genes. The differential genes of all comparison groups are combined as the differential gene set. We use mainstream hierarchical clustering to perform cluster analysis on the FPKM value of genes, and perform uniform processing on the rows. Shown in the form of a heat map. Genes with similar expression patterns in the heat map will be grouped together. The color in each grid is the value obtained after the row of expression data is normalized. Therefore, the colors in the heat map can only be compared horizontally, not vertically. The results showed that in the two cell lines, the expression levels of differentially expressed genes in different samples were significantly different (Fig. 3. C, H). Finally, we used clusterProfiler software to perform GO (Gene Oncology) function enrichment analysis for differential genes, and KEGG (Kyoto Encyclopedia of Genes and Genomics) function enrichment analysis. GO is a comprehensive database describing gene function, divided into three parts: biological process and cell composition, and molecular function. We mainly focus on the enrichment analysis of biological processes. Figure 3D mainly shows the results of the enrichment analysis of the biological process of Eca109 cells. The abscissa in the figure represents the ratio of the number of differential genes annotated to GO Term to the total number of differential genes, the ordinate is GO Term, and the size of dot represents the annotation to GO the number of genes on Term, the color from red to purple represents the significance of enrichment. The results showed that in the two kinds of cells, Elaiophylin mainly affected the binding of intercellular cadherin molecules and intercellular adhesion molecules (Fig. 3. D, I). KEGG enrichment results show that the abscissa is the ratio of the number of differential genes, the ordinate is the KEGG pathway, the size of the dot represents the number of genes annotated to the KEGG pathway, and the color from red to purple represents enrichment. The results show that Elaiophylin mainly affects the PI3K-Akt signaling pathway in the two cell lines (Fig. 3. E, J).
4. Elaiophylin might inhibit the expression of EIF4B via PI3K/AKT signaling pathway
In order to analyze the genes that were downregulated by Elaiophylin both in Eca109 and KYSE450 cells, we firstly found the common DEGs by Venn (Fig. 4. A). Then we annotated the downregulation DEGs to GO (Fig. 4. B) and KEGG (Fig. 4. C) analysis, as predicted, the results showed that the genes were mostly enrichment in the PI3K-Akt signaling pathway. Hence, we verified the genes enrichment in PI3K-Akt pathway by RT-qPCR assay. Noteworthy is that the expression of COL4A1, EIF4B, ITGA5 and ITGB1 was blocked at the mRNA level both in the Eca109 and KYSE450 cells (Fig. 4. D, E). Since EIF4B was previously reported to play an essential role in cell proliferation and survival, especially in cancer cells, but has not been explored in esophageal cancer, we therefore checked the EIF4B protein expression. Furthermore, we also assessed the PI3K, p-PI3K, AKT, p-AKT protein expression level. Finally, we observed that the EIF4B, p-PI3K, AKT, p-AKT protein expression level was downregulated by Elaiophylin, which further confirmed that the activity of PI3K-AKT signaling pathway was inhibited (Fig. 4. F). Taken together, from what discussed before, we speculated that Elaiophylin might target to EIF4B to regulate the proliferation, migration and apoptosis of ESCC cells.
5. Knockdown of EIF4B suppresses the proliferation, migration of ESCC cells and promotes apoptosis
To further confirm the function of EIF4B in ESCC cells, we continue knocked down the EIF4B in ESCC cells to test if silencing EIF4B affect its proliferation, migration and apoptosis. All cells were divided into three groups, scramble, shEIF4B and shEIF4B + EIF4B-OE. First of all, western blot assay was employed to detect the EIF4B expression across all groups, the results demonstrated that the EIF4B was effectively knocked down after transfection of shEIF4B, and was reversed by co-transfection with EIF4B-OE (Fig. 5. A, B). Transwell assay showed that the migratory ability was attenuated when silencing the EIF4B, and can be reversed by co-transfection with EIF4B-OE (Fig. 5. C, D). And efficient shRNA-directed knockdown of EIF4B expression could inhibit the proliferation of Eca109 and KYSE450 cells, as the percentage of ki67 positive cells was decreased in the shEIF4B group compared to scramble and shEIF4B + EIF4B-OE group (Fig. 5. E, F, G, H). Using Annexin V-FITC/PI assay, we also observed that effective knockdown of EIF4B could accelerate the apoptosis of ESCC cells compared to the scramble and shEIF4B + EIF4B-OE group (Fig. 5. I, J). Additionally, western blot assay examined the protein expression of E-cadherin, Bax, and Bcl-2, the results showed that E-cadherin, Bax was upregulated when silencing EIF4B and Bcl-2 was downregulated, which was reversed by overexpression of EIF4B (Fig. 5. K, L). All the data suggested that knockdown of EIF4B could inhibit the proliferation and migration of ESCC cells and promotes its apoptosis. Whereas, overexpression of EIF4B on the basis of blocking the expression of EIF4B abrogated the effect of silencing of EIF4B, which indicated that the EIF4B might play an important role in ESCC progression.
6. Upregulation of eIF4B could rescued the phenotype on ESCC cells caused by Elaiophylin
To ascertain whether the Elaiophylin affects the ability of proliferation, migration and apoptosis of ESCC cells via EIF4B, we transfected eIF4B-OE plasmid to ESCC cells on the basis of treating with Elaiophylin. All cells were divided to three groups vector, Elaiophylin only and EIF4B-OE on the basis of Elaiophylin. Firstly, the EIF4B protein expression level was examined by western blot, the result indicated that it was down regulated by Elaiophylin, but was reversed by co-transfection of EIF4B-OE. (Fig. 6. A, B). The transwell migration assay showed that transfection with EIF4B-OE could blocked the migration-inhibitory effect resulted from Elaiophylin (Fig. 6. C, D). Interestingly, the IHC of ki67 of the different groups illustrated that the transfection with EIF4B-OE on the basis of Elaiophylin could enhance the proliferation of Eca109 and KYSE450 cells when compared with the Elaiophylin only group. (Fig. 6. E, F, G, H). In addition, the cell apoptosis rate was also decreased when transfection with EIF4B-OE on the basis of Elaiophylin compared to the Elaiophylin only group as detected by flow cytometry (Fig. 6. I, J). In the end, in order to confirm the effect of different groups on the proliferation, migration and apoptosis in Eca109 and KYSE450 cells, the protein level of E-cadherin, Bax, Bcl-2 was examined by western blot analysis. The result showed that the expression level of E-cadherin, Bax, Bcl-2 in the Elaiophylin group was reversed by transfection of EIF4B-OE (Fig. 6. K, L). The phenomenon indicated that eIF4B-OE could reverse the effect of the Elaiophylin on ESCC cells.