Oncogenic tuftelin 1 as a potential molecular-targeted for inhibiting hepatocellular carcinoma growth

Figure 1 Comparative analysis of tuftelin 1 messenger RNA between hepatocellular carcinoma and normal livers based on bioinformatics databases. A: The expression of tuftelin 1 (TUFT1) messenger RNA in the cancerous tissues of hepatocellular carcinoma (HCC) patients (n = 369, red, ^aP < 0.05) and normal liver tissues (n = 50, grey) from the Cancer Genome Atlas database; B: The expression of TUFT1 messenger RNA in the cancerous tissues of HCC patients (n = 225, dark blue, ^aP < 0.05) and normal liver tissues (n = 220, light blue) from the Oncomine database. The horizontal line represents the median of the two groups. Vertical bars indicate the range of data. LIHC: Liver hepatocellular carcinoma.

Figure 2 Tuftelin 1 expression with clinical staging of hepatocellular carcinoma. A, A1: Hepatocellular carcinoma (HCC) tissue with strongly positive staining; B, B1: Adjacent non-HCC tissue with negative staining, original magnifications of × 40 (scale bar: 50 μm) in A and B, and × 200 (scale bar: 50 μm) in A1 and B1; C, D: Tuftelin 1 (TUFT1) expression in different HCC staging; C1, D1: Low TUFT1 expression in the non-HCC tissues; C2-C5: The brown staining of TUFT1 gradually increases in the HCC tissues from stage I to IV (original magnification × 40); D2-D5: The staining of TUFT1 in the non-HCC tissues from stage I to IV (original magnification × 400). TNM: Tumor-node-metastasis.

Figure 3 Relationship between tuftelin 1 expression and prognosis of hepatocellular carcinoma. A: Kaplan-Meier analysis was performed to compare the overall survival (OS) between hepatocellular carcinoma (HCC) with high (n = 66) and low (n = 66) TUFT1; A1: The high (n = 19) and low (n = 40) tuftelin 1 (TUFT1) with OS at early HCC; A2: The high (n = 47) and low (n = 26) TUFT1 with OS at advanced HCC; B: Kaplan-Meier analysis was performed to compare the disease-free survival (DFS) between HCC with high (n = 66) and low (n = 66) TUFT1; B1: The high (n = 19) and low (n = 40) TUFT1 with DFS at early HCC; B2: The high (n = 47) and low (n = 26) TUFT1 with DFS at advanced HCC.

Figure 4 Expression, interfering of tuftelin 1 with proliferation of hepatocellular carcinoma cells. A: Expressions of tuftelin 1 (TUFT1) among different hepatocellular carcinoma (HCC) cell lines were detected by Western blotting, and the expressing levels of all HCC cells were significantly higher than that of the control LO2 cells (^bP < 0.001); A1: The relative ratio from TUFT1 to glyceraldehyde 3-phosphate dehydrogenase with the MHCC-97H cells showed the strongest TUFT1 expression, and the Hep3B cells showed the lowest TUFT1 expression; B: The MHCC-97H cells and interfering with TUFT1 short hairpin RNA (sh-RNA)1-3 (left); the TUFT1 was over-expressed after the Hep3B cells transfected with the constructed pEX-4 (pGCMV/MCS/T2A/EGFP/Neo) plasmid (Right); B1: The TUFT1 expression was significantly inhibited by the TUFT1-shRNA transfection (^bP < 0.001, left); the markedly increasing TUFT1 Level was compared with the control cells (^bP < 0.001, right); C: The TUFT1 activation interfering by TUFT1- shRNA3 significantly inhibited the proliferation of MHCC-97H cells (^aP < 0.05, ^bP < 0.001); D: the over-expression of TUFT1 significantly enhanced the proliferation of the Hep3B cells (^bP < 0.001). Data are presented as mean ± standard error of the mean of at least three independent experiments. NC: Blank control; sh-NC: Negative sh-RNA for tuftelin 1 mRNA.

Figure 5 Effects of tuftelin 1 on healing, invasion, and migration of hepatocellular carcinoma cells. A: The scratch test of the MHCC-97H cells, the cell healing ability was decreasing after the high tuftelin 1 (TUFT1) cells were transfected with the TUFT1-shRNA3 plasmid, and A1, the relative cell healing ability was significantly inhibited compared with the control or negative sh-RNA for tuftelin 1 mRNA (sh-NC) cells (^bP < 0.001); B: The scratch test of the Hep3B cells, the cell healing ability was increased after the cells were transfected with the over-expressing TUFT1 plasmid; B1, the relative cell healing ability was significantly improved compared with the control or blank control (NC) cells (^bP < 0.001); C: The migration and invasion abilities of the MHCC-97H cells were analyzed by the transwell test; C1: The histograms of the cell migration and invasion abilities among the different groups (^bP < 0.001): D: The migration and invasion abilities of the Hep3B cells were analyzed by the transwell test; D1: The histograms of the cell migration and invasion abilities among the different groups (^bP < 0.001). Data are presented as mean ± standard error of the mean of at least three independent experiments. sh-RNA: sh-RNA3 for tuftelin 1 mRNA.

Figure 6 Possible mechanism of tuftelin 1 expression in hepatocellular carcinoma cells progression. A: The MHCC-97H cells were transfected with the tuftelin 1 (TUFT1)-short hairpin RNA (sh-RNA)3 plasmid, and the apoptotic cells in the sh-TUFT1 group (A2) were increased more than those in the blank control (NC) (A) or negative sh-RNA for tuftelin 1 mRNA (sh-NC) group (A1); B: The histograms of the apoptotic cells among the different groups, the rate of the MHCC-97H cell apoptosis in the sh-TUFT1 group was significantly higher (^bP < 0.001) than those in the NC or sh-NC group. Data are presented as mean ± standard error of the mean of at least three independent experiments; C: Speculation of possible mechanisms of TUFT1 promotion of the progression of hepatocellular carcinoma (HCC). Aberrant TUFT1 activation promoted the proliferation and metastasis via anti-apoptosis of HCC cells.