Decreased STAT4 indicates poor prognosis and enhanced cell proliferation in hepatocellular carcinoma

Abstract

Aim: To investigate the role of signal transduction and activation of transcription 4 (STAT4) in the development and progression of human hepatocellular carcinoma (HCC).

Methods: Recent genetic investigations have identified that a genetic variant of STAT4 is associated with hepatitis B virus (HBV)-related HCC. The level of STAT4 in 90 HCC patients was examined via Western blot and immunohistochemical analyses. The correlation between STAT4 expression and the clinicopathological characteristics of the patients was analyzed. The level of STAT4 expression in the HCC liver tissues was significantly lower than that in the non-HCC liver tissues and correlated with tumor size, histological grade of HCC and serum hepatitis B surface antigen level in HCC patients. The data were statistically analyzed using SPSS. Furthermore, siRNA oligos targeting STAT4 were employed to investigate the influence of STAT4 RNA interference on HCC cell physiology. Based on Cell Counting Kit-8 and flow cytometric assays, we found that depletion of STAT4 expression significantly enhanced the proliferation of L02 cells.

Results: STAT4 protein expression was significantly lower in HCC tissues than in normal liver tissues. Immunohistochemistry followed by statistical analysis revealed that the expression of STAT4 negatively correlated with Ki67 expression (r = 0.851; P < 0.05) and positively correlated with maximal tumor size (P < 0.05), HBV (P = 0.012) and histological grade (P < 0.05). Kaplan-Meier analysis revealed significant differences in the survival curves between HCC patients expressing low and high levels of STAT4 and Ki67 (P < 0.05). Based on a multivariate Cox proportional hazard model, STAT4 expression was an independent prognostic indicator for HCC patients who underwent curative resection. In vitro, following the release of L02 cell lines from serum starvation, the expression of STAT4 was downregulated, and transfection of L02 cells with siRNA targeting STAT4 inhibited cell proliferation.

Conclusion: Our data indicate that STAT4 may inhibit HCC development by modulating HCC cell proliferation.

Keywords: Hepatocellular carcinoma; Prognosis; Proliferation; Signal transduction and activation of transcription 4.

Figure 1

Expression of signal transducer and activator of transcription 4 in human hepatocellular carcinoma tissues. A, B: Expression of STAT4 in eight representative matched samples of HCC tissue (T) and adjacent noncancerous liver tissues (N) via Western blot and SigmaPlot. STAT4: Signal transducer and activator of transcription 4; HCC: Hepatocellular carcinoma.

Figure 2

Immunohistochemical analysis of signal transducer and activator of transcription 4 and Ki67 expression in hepatocellular carcinoma and adjacent noncancerous liver tissues. Paraffin-embedded tissue sections were stained with antibodies against STAT4 and Ki67 and counterstained with hematoxylin. High expression of STAT4 (E, F) and Ki67 (C, D) was detected in the HCC tissues. Low expression of STAT4 (A, B) and high expression of Ki67 (G, H) were detected in adjacent noncancerous liver tissues (SP × 400) based on immunohistochemistry. STAT4: Signal transducer and activator of transcription 4; HCC: Hepatocellular carcinoma.

Figure 3

Relationship between signal transducer and activator of transcription 4 expression and the Ki67 proliferation index in hepatocellular carcinoma. A: Scatterplot of STAT4 expression vs Ki-67 expression; the regression line indicates a correlation based on the Spearman correlation coefficient; B: Kaplan-Meier survival curves of 90 HCC patients stratified into low and high STAT4 expression show a highly significant difference between these groups (P < 0.05, log-rank test using SPSS). STAT4: Signal transducer and activator of transcription 4; HCC: Hepatocellular carcinoma.

Figure 4

Western blot analysis of signal transducer and activator of transcription 4 protein expression in hepatocellular carcinoma cells compared to normal hepatocyte (L02) cells. A: GAPDH was used as a loading control. Each experiment was repeated at least 3 times; B: Expression of STAT4 and cell cycle-related molecules in proliferating HCC cells. HepG2 cells were synchronized via serum starvation for 72 h. Upon serum refeeding, cell lysates were prepared and analyzed via Western blot using antibodies directed against STAT4 and cyclin D1. GAPDH was used as a control for protein load and integrity. S: Serum starvation; R: Serum refeeding; C: Flow cytometric quantification of the cell cycle status in HepG2 cells. The cells were synchronized at G1 via serum starvation for 72 h; then, progression into the cell cycle was induced by adding medium containing 10% FBS for the indicated period (R4 h, R8 h, R12 h, or R24 h). STAT4: Signal transducer and activator of transcription 4; HCC: Hepatocellular carcinoma.

Figure 5

Signal transducer and activator of transcription 4 knockdown inhibited cell proliferation and the effects of altered signal transducer and activator of transcription 4 expression on the cell cycle status of L02 cells. A: Western blot analysis revealed that treatment with STAT4-siRNA markedly decreased the STAT4 and cyclin D1 levels at 48 h after siRNA transfection in L02 cells compared to transfection with negative control siRNA and mock transfection; B: siSTAT4-1 and -2 were used to knockdown STAT4 in HepG2 cells, resulting in the delay of the G1-S transition and significant arrest in the G1 phase after these cells were released from starvation. The data are presented as the mean ± SD of three experiments; C: A CCK-8 assay showed that STAT4 knockdown inhibited cell proliferation. CCK-8 reagents were added to the medium for 2 h. The absorbance was measured at each indicated time point (0, 1, 2, and 3 d). Each value was derived from three independent experiments. STAT4: Signal transducer and activator of transcription 4; HCC: Hepatocellular carcinoma.