Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

Abstract

Aim: To explore the effect of silencing of signal transducer and activator of transcription 3 (STAT3) expression by RNA interference (RNAi) on growth of human hepatocellular carcinoma (HCC) in tumor-bearing nude mice in vivo.

Methods: To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP (pSH1-siRNA-STAT3) and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSH1-siRNA-STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis of tumor cells.

Results: The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups (P < 0.01). The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased (P < 0.01). Most of the tumor tissue cells in the treated group developed apoptosis that was detected by TUNEL assay.

Conclusion: Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

Figure 1

Growth curves of SMMC7721 tumor in nude mice in different treated groups. ^aP < 0.01 vs pH1Si-Scramble group.

Figure 2

Comparison of tumor volume in nude mice in different treated groups. A: Mock-treated group; B: pSH1Si-Scramble group; C: pSH1Si-STAT3 group. Black arrow points to tumor tissue.

Figure 3

Tumor appearances in different treated groups. A: Mock-treated group; B: pSH1Si-Scramble group; C: pSH1Si-STAT3 group.

Figure 4

RT-PCR analysis of mRNA for STAT3 and related genes in tumor tissues of different treated groups. M: Mock-treated group; N: pSH1Si-Scramble group; T: pSH1Si-STAT3 group. ^aP < 0.01 vs pSH1Si-Scramble group.

Figure 5

Western blotting analysis of STAT3 and related genes protein in tumor tissues in nude mice in different treated groups. ^aP < 0.01 vs pSH1Si-Scramble group.

Figure 6

Detection of p-STAT3 protein expression by immunohistochemical assay of tumor tissues in nude mice in different treated groups (streptavidin biotin complex, × 400). A: Mock-treated group; B: pSH1Si-Scramble group; C: pSH1Si-STAT3 group. Black arrows point to positive cells, which were stained brown in the nucleus, using antibodies to p-STAT3.

Figure 7

Tumor tissues in nude mice in different treated groups (HE, × 400). A: Mock-treated group; B: pSH1Si-Scramble group; C: pSH1Si-STAT3 group. Black arrow points to positive cells, which had karyopyknosis and red staining of the cytoplasm.

Figure 8

Detection of apoptosis with TUNEL assay in tumor tissues of tumor-bearing nude mice in different treated groups (× 400). A: Mock-treated group; B: pSH1Si-Scramble group; C: pSH1Si-STAT3 group, black arrow points to positive cells, in which the nuclei were stained brown.