Claudin-9 enhances the metastatic potential of hepatocytes via Tyk2/Stat3 signaling

Abstract

Background/aims: We have previously identified a tight junction protein claudin-9 (CLDN9) as an upregulated gene in hepatocellular carcinoma (HCC) through an immunohistochemistry analysis. Here, we explore its function and clinical relevance in human HCC.

Materials and methods: Stable transfection of the hepatocyte line HL7702 with CLDN9 was confirmed by the real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence. The impact of CLDN9 on the cell invasion and migration was assessed in vitro by a transwell assay and wound-healing experiment. Western blotting was used to determine the activation state of the Tyk2 (tyrosine kinase 2)/Stat3 (signal transducer and activator of transcription 3) pathway. Moreover, we used a Tyk2-RNAi assay to silence the expression of Tyk2 in CLDN9 expressing hepatocytes; subsequently, the impact of the Tyk2/Stat3 signaling pathway on the cell invasion and migration in vitro was assessed by a transwell assay and a wound-healing experiment. Furthermore, an immunohistochemistry method was utilized to explore the expression levels of CLDN9 and p-Stat3 in the HCC tissues and histologically non-neoplastic hepatic tissues.

Results: We confirmed that the expression of CLDN9 significantly enhanced the metastatic ability of hepatocytes in vitro, and the activation of the Stat3 pathway by Tyk2 was an important mechanism by which CLDN9 promoted hepatocyte aggressiveness in HCC.

Conclusion: As an HCC proto-oncogene, CLDN9 affected the Stat3 signaling pathway via Tyk2 and ultimately enhanced the metastatic ability of hepatocytes.

Figure 1. a–d

The expression levels of CLDN9 in the HCC cell lines and tissues (a). The relative mRNA level of CLDN9 in hepatocyte lines and HCC cell lines (b). The relative protein expression of CLDN9 in hepatocyte lines and HCC cell lines (c). The protein expression of CLDN9 in hepatocyte tissues and HCC cell tissues (d). The corresponding statistical analysis of protein expression in hepatocyte lines and HCC cell lines. *represents p<0.05; **represents p<0.01; compared with the empty vector groups

Figure 2. a–d

Characterization of the stable expression levels of CLDN9 (a). Detection of CLDN9 in the HL7702 cell line by real-time PCR (b). Detection of CLDN9 expression in the HL7702 cell line by western blotting (c). Detection of CLDN9 expression in the HL7702 cell line by immunofluorescence (d). The corresponding statistical analysis of protein expression in the HL7702 cell line. **represents p<0.01; compared with the empty vector groups

Figure 3. a–d

The impact of CLDN9 on the proliferation and metastasis ability of cells in vitro (a). The growth curve of the HL7702 cell line was drawn by the CCK-8 method (b). The wound-healing assay was used to detect the migration ability of the HL7702 cell line in vitro (c). The transwell chamber method was used to detect the invasive ability of the HL7702 cell line in vitro (d). The corresponding statistical analysis of invasive cells. **represents p<0.01 compared with the empty vector groups

Figure 4. a, b

The impact of CLDN9 on the Tyk2/stat3 signaling pathway (a). Western blotting was used to detect the activation of the Stat3 signaling pathway in the HL7702 cell line (b). The corresponding statistical analysis of the activation status of various Stat3 pathway components. **represents p<0.01 compared with the empty vector groups

Figure 5. a–f

RNAi was used to silence the Tyk2 expression in CLDN9-expressing cells (a). Real-time PCR was used to examine the effects of silencing Tyk2 in the HL7702 cell line (b). Western blotting was used to examine the effects of silencing Tyk2 and the activation of the Stat3 signaling pathway in the HL7702 cell line (c). The corresponding statistical analysis of Stat3 signaling pathway activation (d). The transwell chamber method was used to detect the impact of Tyk2 silencing on the invasive ability of cells in vitro (e). The corresponding statistical analysis of invasive cells (f). The wound-healing assay was used to detect the migration ability of the HL7702 cell line in vitro. *represents p<0.05; **represents p<0.01 compared with the scramble group

Figure 6

Expressions of p-Stat3 in human HCC and hepatic tissue