Glycolysis-related gene dihydrolipoamide acetyltransferase promotes poor prognosis in hepatocellular carcinoma through the Wnt/β-catenin and PI3K/Akt signaling pathways

Figures

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  Figure 1. HCC has increased DLAT expression. (A) Pan-cancer DLAT expression; (B) unpaired HCC tissues (n=374) and paracancerous tissues (n=50) in TCGA database both have high levels of DLAT expression; (C) paired HCC tissues (n=50) and paracancerous tissues (n=50) in TCGA database both have high levels of DLAT expression; and (D) the ROC analysis of DLAT revealed that (E) the HCC tissues in the HCCDB had considerably higher mRNA levels than the nearby normal tissues. *P<0.05, **P<0.01, ***P<0.001 indicate that the difference is statistically significant. DLAT, dihydrolipoamide acetyltransferase; TPM, transcripts per million; ns, no significant; TPR, true positive rate; FPR, false positive rate; AUC, area under the curve; CI, confidence interval; STD, standard deviation; IQR, interquartile range; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; ROC, receiver operating characteristic; HCCDB, Hepatocellular Carcinoma Database; mRNA, messenger ribonucleic acid.

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  Figure 2. DLAT and the prognosis of HCC patients in the Kaplan-Meier-plotter database. (A) Correlation of DLAT with OS, DSS, and PFI in HCC patients; (B) correlation of DLAT with AFP ≤400 ng/mL, G1, G2, G3, M0, N0, Stage I, T1, T2, Child-Pugh grade A, and the prognosis of HCC patients in different subgroups with or without vascular invasion. DLAT, dihydrolipoamide acetyltransferase; HR, hazard ratio; AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; OS, overall survival; DSS, disease‐specific survival; PFI, progression-free interval.

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  Figure 3. A calibration plot and nomogram. (A) A nomogram for HCC patients that predicts the survival probability of HCC patients at 1, 3, and 5 years; (B) a calibration plot of that nomogram. DLAT, dihydrolipoamide acetyltransferase; AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma.

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  Figure 4. GeneMania was used to create the gene-gene interaction network of DLAT, and STRING was used to create the PPI network. DLAT, dihydrolipoamide acetyltransferase; STRING, functional protein association networks; PPI, protein-protein interaction.

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  Figure 5. The DLAT screening of co-expressed genes. Heatmaps of the top 50 genes favorably and negatively linked with DLAT in human colorectal cancer in (A) and (B), respectively. ***P<0.001 indicate that the difference is statistically significant. DLAT, dihydrolipoamide acetyltransferase; TPM, transcripts per million.

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  Figure 6. Correlation between DLAT expression and immune infiltration level. (A) The relationship between DLAT expression and the levels of immunological infiltration; (B) the expression of DLAT was closely linked to immune cell infiltration in HCC, and (C) CTLA-4, CD274, and PDCD1 sex scatterplots. *P<0.05, **P<0.01, ***P<0.001 indicate that the difference is statistically significant. DLAT, dihydrolipoamide acetyltransferase; TPM, transcripts per million; CD, cluster of differentiation; Tcm, Central Memory T cell; Th2, T helper 2; aDC, activated dendritic cells; iDC, immature dendritic cell; PDC, pyruvate dehydrogenase complex; PDCD1, programmed cell death protein 1; HCC, hepatocellular carcinoma.

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  Figure 7. Correlation between DLAT and molecular-targeted drug therapy molecules in TCGA database. TPM, transcripts per million; DLAT, dihydrolipoamide acetyltransferase; TCGA, The Cancer Genome Atlas.

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  Figure 8. Six statistically significant probable relevant paths. The biological process gene set from MSigDB known as Gene Ear Biology was used. There were 1,600 different combinations of a random sample. NOM-p stands for nominal P value, FDR-q for false discovery rate, and NES for normalized enrichment score.

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  Figure 9. Liver cancer tissues have elevated DLAT expression. (A) 25 liver cancer tissues were examined using fluorescence quantitative PCR to identify the expression of DLAT; (B) a DLAT knockdown liver cancer cell line was created. **P<0.01. DLAT, dihydrolipoamide acetyltransferase; shDLAT, short hairpin DLAT; PCR, polymerase chain reaction.

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  Figure 10. The proliferation and invasion of hepatoma cells were significantly reduced when DLAT was knocked down. (A,B) The Transwell and CCK-8 results both showed that DLAT knockdown affected the capacity of SMMC-7721 and HepG2 liver cancer cells to proliferate. (C,D) The Transwell results also showed that DLAT knockdown affected the ability of SMMC-7721 and HepG2 liver cancer cells to proliferate (crystal violet staining, 200×). *P<0.05, **P<0.01. OD, optical density; shDLAT, short hairpin DLAT; DLAT, dihydrolipoamide acetyltransferase.

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  Figure 11. The knockdown of DLAT significantly inhibited the Wnt/β-catenin and PI3K/Akt signaling pathways in the liver cancer cells. (A) Western blot detection showed the effect of DLAT knockdown on the expression of β-catenin, PI3K, and Akt; (B) grayscale analysis and statistics of catenin protein bands on the expression of β-catenin; (C) grayscale analysis and statistics of PI3K and Akt protein bands. **P<0.01. DLAT, dihydrolipoamide acetyltransferase; p-PI3K, phosphorylated phosphoinositol 3 kinase; T-PI3K, total phosphatidylinositol-4,5-bisphosphate 3-kinas; p-Akt, Phosphorylated protein kinase B; T-Akt, total protein kinase B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; shDLAT, short hairpin DLAT; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinas; Akt, protein kinase B.