PTIP promotes recurrence and metastasis of hepatocellular carcinoma by regulating epithelial-mesenchymal transition

Abstract

Hepatocellular carcinoma (HCC) is one of the most lethal tumors worldwide, which is mainly due to the high recurrence and metastasis rate after hepatectomy. In this study, we found that PTIP expression was dramatically upregulated in human HCC tissues and cell lines. High expression of PTIP was shown to be associated with aggressive clinicopathological features, including liver cirrhosis, vascular invasion and advanced stage. In addition, PTIP overexpression was independently associated with shorter survival and increased HCC recurrence in patients. Knockdown of the PTIP expression significantly inhibited invasion and metastasis in vitro and in vivo, whereas ectopic expression of PTIP significantly promoted invasion and metastasis. Mechanistically, PTIP promotes HCC progress by facilitating epithelial-mesenchymal transition (EMT). Notably, we also found that PTIP might increase miR-374a expression to promote EMT and metastasis in HCC. In summary, our study identified PTIP as a new potential prognostic indicator and therapeutic target for HCC.

Keywords: PAX interacting protein1 (PTIP); epithelial-mesenchymal transition (EMT); hepatocelluar carcinoma; metastasis; miRNA.

Figure 1. PTIP expression is up-regulated in HCC tissues and cells

(A) PTIP mRNA of human HCC cell lines was up-regulated by quantitative real-time PCR (qRT-PCR). (B) PTIP mRNA expression in 30 pairs of HCC tissues was higher than their corresponding adjacent nontumorous liver tissues (ANLTs). Expression level of PTIP was determined by qRT-PCR and normalized to GAPDH. Fold change was analyzed using the formula 2^{-(ΔΔCT[HCC/ANLT])}. (C) PTIP protein expression in HCC cell lines and normal liver cell line L02 was detected by western blot. Below was the statistical analysis of results. The representative western blot results showed that Hep3B and HCCLM3 cells had the highest PTIP expression level compared to L02 cells. (D) PTIP protein in paired HCC tissues was significantly higher than those in ANLTs. Below was the statistical analysis of results. GAPDH and β-actin was used as an internal loading control.

Figure 2. Immunohistochemistry analysis of PTIP protein expression in HCC tissues

(A) Representative IHC images of PTIP expression in ANLT. (B) Representative IHC images of PTIP in HCC. (C) The overall survival time of PTIP high expression group was significantly poorer compared to PTIP low expression group (P<0.01). (D) PTIP high expression group also had poorer disease-free survival than PTIP low expression group (P<0.001). (E, F) The association between PTIP expression and OS/DFS in non-cirrhotic versus cirrhotic patients. The results showed that high PTIP expression with cirrhotic had significantly lower OS and DFS time than low PTIP expression without cirrhotic group (P<0.01)

Figure 3. PTIP promotes proliferation and invasion of HCC cells in vitro

HCC cell lines infected with PTIP expression lentivirus or PTIP inhibition lentivirus or NC were used in these studies. Effect of PTIP on proliferation of HCCLM3 and HepG2 cell lines was assessed using MTT assays and colony formation. (A, B) The results of MTT assays showed PTIP promoted cell growth. (C, D) Effect of PTIP was on colony formation of HCC cell lines. The colonies >40 um were counted and their numbers were compared. The Wound healing assay and Transwell assay were performed to analyze the effect of PTIP on the motility and invasion of HCC cell lines. The percentage of wound healing or cells passing through the transwell membranes of each well was calculated and is compared in the diagrams. (E, F) The results of Wound healing assay showed that PTIP promoted HCC cells migration. (G, H) The results of Transwell assay showed that PTIP promoted invasion. Representative images of above diagrams are supplied in Supplementary Figures 1, 2, 3 and 4. *P<0.05; **P<0.01; ***P<0.001.

Figure 4. PTIP promotes metastasis of HCC in vivo

The orthotopic HCC tumors mouse model was constructed by using HCCLM3^NC, HCCLM3^shPTIP, HepG2^NC and HepG2^PTIP. Cells (2 × 10⁶) were injected subcutaneously into the left upper flank regions of nude mouse (3-4 weeks of age, male, BALB/c). After one month, the subcutaneous tumor tissues were obtained and cut into commensurate fragments of approximately 1 mm³. Then, each fragment was implanted into the liver (four mice for each group). (A) The results showed that tumor volumes of HCCLM3^NC group was significantly larger than that of HCCLM3^shPTIP group(1.41 ± 0.27cm³ versus 0.25 ± 0.05 cm³, P =0.02). (B) The tumor volumes of HepG2^NC group was significantly smaller than that of HepG2^PTIP group (0.18 ± 0.04cm³ versus 1.41 ± 0.38 cm³, P =0.02). (C, D) Representative pictures of lung metastasis; metastatic nodules proportion or lungs was calculated and compared. (C) The rate of lung metastasis in HCCLM3^NC group was higher than that of HCCLM3^shPTIP group. (D) The rate of lung metastasis in HepG2^NC group was lower than that of HepG2^PTIP group. Original magnification: left, 100 ×; right, 400 ×.*P<0.05; **P<0.01 based on the Students test. Error bars, standard deviation.

Figure 5. PTIP induces epithelial-mesenchymal transition (EMT) through miR-374a in hepatocellular carcinoma

(A) Representative images of cytoskeleton. (B) Western blot analyzed PTIP and EMT markers expression in HCC cells with PTIP overexpression or knockdown, and their control cells. (C) PTIP enhanced metalloproteinase (MMP-2 and MMP-9) expression in HCC cells through gelatin zymography assay. (D) The expression of MMP-2 and MMP-9 was examined by qRT-PCR. The results showed that PTIP promoted the expression of MMP-2 and MMP-9. (E) The potential microRNA target of PTIP was detected by quantitative real-time PCR (qRT-PCR). The results showed that the expression of miR-374a was significantly lower in HCCLM3^shPTIP cells. (F) miR-374a was detected in thirty pairs of HCC tissues and corresponding ANLT by qRT-PCR. Correlation analysis showed that PTIP was positively related with miR-374a (r=0.77, P<0.01). (G) Referred to the previous results of PTIP expression in human tissues, a sample (H103) with low PTIP expression and a sample (H36) with high PTIP expression were chosen. (H) Expression of PTIP, Snail, E-cadherin and vimentin were examined by IHC. PTIP, Snail and Vimentin showed positive correlation with miR-374a;E-cadherin showed negative correlation with miR-374a.