Reduction in the hepatocellular carcinoma antioncogene SLC39A14 during the malignant progression of hepatocellular carcinoma

Figure 1 Schematic representation of the study protocol. HCC: Hepatocellular carcinoma; DEN: Diethylnitrosamine; CCl₄: Carbon tetrachloride; qRT-PCR: Quantitative reverse transcriptase PCR; CCK-8: Cell counting kit-8; H&E: Hematoxylin and eosin.

Figure 2 SLC39A14 expression in clinical hepatocellular carcinoma samples. A: Differences in SLC39A14 expression between cancer tissues and paracancerous tissues from hepatocellular carcinoma (HCC) patients were detected by western blotting (n = 13); B: Differences in SLC39A14 mRNA expression in the cancerous and paracancerous tissues of HCC patients were detected by quantitative reverse transcriptase PCR (n = 3). ^aP < 0.01. ^bP < 0.0001.

Figure 3 SLC39A14 expression and survival analysis in The Cancer Genome Atlas cohorting. A: SLC39A14 expression in cancer patients (T) and normal individuals (N) (P < 0.05); B: Changes in SLC39A14 expression across cancer stages; C: Overall survival in male patients with high and low SLC39A14 expression (P = 0.017); D: Overall survival in female patients with high and low SLC39A14 expression (P = 0.2).

Figure 4 In vivo experimental workflow. Diethylnitrosamine and carbon tetrachloride-induced hepatocellular carcinoma in C57BL/6J mice. DEN: Diethylnitrosamine; CCl₄: Carbon tetrachloride.

Figure 5 Evaluation of hepatocellular carcinoma model induction efficacy. A: Appearance and hemotoxylin and eosin staining of liver lobules in the control and model groups. Abnormal mitosis of hepatocellular carcinoma cells is indicated by black arrows; magnification, 200 ×; B: Liver body ratios for the control and model mice; C: Alanine aminotransferase for the control and model mice; D: Aspartate aminotransferase for the control and model mice. H&E: Hemotoxylin and eosin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase. ^aP < 0.001, ^bP < 0.01.

Figure 6 Changes in SLC39A14 expression during hepatocellular carcinoma progression. A batch of mice was collected every 2 weeks during the induction process, and western blotting was performed to detect the changes in ZIP14 protein expression during the development of hepatocellular carcinoma (n = 8). ^aP < 0.05, ^bP < 0.01, ^cP < 0.0001.

Figure 7 Checking transfection efficiency in Huh7 cells. A: ZIP14 expression after overexpression plasmid transfection; B: ZIP14 expression after siRNA transfection. ^aP < 0.05, ^bP <0.01.

Figure 8 SLC39A14 in cell proliferation. A: Cell counting kit-8 (CCK-8) assay in Huh7 cells after SLC39A14 overexpression; B: CCK-8 assay in Huh7 cells after SLC39A14 knockdown; C: Colony formation experiment in Huh7 cells after SLC39A14 overexpression; D: Colony formation experiment in Huh7 cells after SLC39A14 knockdown. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 9 SLC39A14 in cell apoptosis. A: Apoptosis detection by flow cytometry after SLC39A14 overexpression; B: Apoptosis detection by flow cytometry after SLC39A14 knockdown. ^aP < 0.05, ^bP < 0.01.

Figure 10  Alterations in the BCL-2/BAX/caspase-3 pathway. A: Pathway proteins BCL2/BAX expression after overexpression plasmid transfection; B: Pathway proteins cleaved caspase 3/caspase 3 expression after overexpression plasmid transfection; C: BCL2/BAX expression after siRNA transfection; D: Cleaved caspase 3/caspase 3 expression after siRNA transfection. ^aP < 0.01, ^bP < 0.05.

Figure 11 SLC39A14 in cell metastasis. A: Cell metastasis detection by scratch assay after SLC39A14 overexpression; B: Cell metastasis detection by Transwell assay after SLC39A14 overexpression; C: Cell metastasis detection by scratch assay after SLC39A14 knockdown; D: Cell metastasis detection by Transwell assay after SLC39A14 knockdown. ^aP < 0.05, ^bP < 0.01.

Figure 12  Alterations in epithelial-mesenchymal transition. A: Epithelial–mesenchymal transition (EMT)-related protein E-cadherin expression after overexpression plasmid transfection; B: EMT-related protein N-cadherin expression after overexpression plasmid transfection; C: EMT-related protein vimentin expression after overexpression plasmid transfection; D: E-cadherin expression after siRNA transfection; E: N-cadherin expression after siRNA transfection; F: Vimentin expression after siRNA transfection. EMT: Epithelial–mesenchymal transition. ^aP < 0.05, ^bP < 0.01.

Figure 13 SLC39A14 is regulated by the transcription factor STAT3. A: Bioinformatics analysis identified STAT3 as one of potential transcription factor driving SLC39A14 expression; B and C: Binding sites for STAT3 in the promoter region of SLC39A14 were identified according to the JASPAR website; D: Pearson’s correlational analysis was performed to explore the correlation between the mRNA levels of STAT3 and SLC39A14 in hepatocellular carcinoma patients in The Cancer Genome Atlas dataset (r = 0.41, P < 0.0001).