The Inhibitory Effects And Mechanisms Of Blockage Of STAT3 Signal Pathway On Invasion And Metastasis Of Human Pancreatic Cancer

Objectives:To investigate the expression of signal transduction and activators of transcription-3 (STAT3) and metastasis-correlated gene in pancreatic cancer and their correlation with the clinicopathological parameters of pancreatic cancer. To investigate the effects and mechanism of blockade of STAT3 signaling pathway by JAK specific inhibitor-AG490 and STAT3 specific small interference RNA (siRNA) expression vector on the invasion and metastasis of human pancreatic cancer cells.Methods:⑴Immunohistochemistry was used to detect the expression of STAT3, phosphorylated STAT3 (p-STAT3), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in 34 cases of pancreatic cancer tissue and 10 cases of normal pancreas.⑵The invasion ability of SW1990 and CaPan-2 cells were investigated by cell invasion assay kit. Western blot and electrophoresis mobility shift assay (EMSA) were performed to detect the STAT3, p-STAT3 protein expression and STAT3 DNA-binding activity of SW1990 and CanPan-2 cells, respectively. AG490 was added into the culture media for SW1990 and CaPan-2 cells. The changes of invasion ability of SW1990 and CanPan-2 cells were investigated by cell invasion assay kit. Western blot and EMSA were performed to detect the changes of STAT3, p-STAT3 protein expression and STAT3 DNA-binding activity of SW1990 cells. Western blot and RT-PCR were performed to detect the changes of protein and mRNA expression of metastatic-correlated gene, respectively.⑶Three STAT3 siRNA expressing vectors were constructed and transiently transfected into SW1990 cells. STAT3 mRNA and protein expression were examined using RT-PCR and Western blot, respectively. The plasmid which had the most obvious gene silencing effect was choosed for stable transfection. The changes of invasion capacity in vitro and metastasis capacity in vivo of SW1990 cells before and after STAT3 silence were investigated by cell invasion assay kit and acute hematal metastasis model in nude mice. RT-PCR and Western blot were performed to detect the changes of mRNA and protein expression of metastatic-correlated gene, respectively.Results:⑴Immunohistochemistry revealed that pancreatic cancer tissues showed increased p-STAT3,MMP-2 and MMP-9 expression which compared with normal pancreas tissues. The expression level of p-STAT3,MMP-2 and MMP-9 had a significant positive relationship with clinical stage and lymphatic metastasis. Correlation between p-STAT3 and MMP-2,MMP-9 expression level was tested with Spearman rank correlation. We found a significant positive relationship between the expression of p-STAT3 and MMP-2 (r=0.583, P=0.000). We also found positive relationship between the expression of p-STAT3 and MMP-9 (r=0.410, P=0.016).⑵We found that SW1990 cells showed greater levels of invasiveness than the CaPan-2 cells. We also found that p-STAT3 protein levels were significantly higher in SW1990 cells compared to the CaPan-2 cells. Furthermore, EMSA indicated significant amount of STAT3 DNA-binding activity in SW-1990 cells, while weak binding activity was observed in CaPan-2 cells. Invasion assay in vitro indicated that SW1990 cells' invasion ability was attenuated in a dose-dependent manner when cells were treated with AG490 for 48h. In contrast, the invasion ability of CaPan-2 cells was not significantly reduced by AG490 even when used at concentration as high as 20μM. The use of AG490 not only markedly reduced the protein expression of p-STAT3 but also great reduced the STAT3 DNA-binding activity of SW1990 cells. Moreover, it was demonstrated that the protein and mRNA expression of MMP-2 and vascular endothelial growth factor (VEGF) were significantly reduced in a dose-dependent manner when SW1990 cells were treated with AG490 for 48h.⑶PCR and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.1/Neo plasmid and three STAT3 siRNA expressing vectors (pRNAT-STAT3 siRNA-I, pRNAT-STAT3 siRNA-II, pRNAT-STAT3 siRNA-Ⅲ) were successfully constructed. RT-PCR and Western blot indicated STAT3 expression in the transfected cells was inhibited significantly by three STAT3 siRNA expressing vectors at both mRNA and protein level compared with untransfected group (P<0.05). STAT3 mRNA and protein expression in pRNAT-STAT3 siRNA-II group were decreased 78.5 and 71.8% compared with untransfected group, respectively. For stable transfection, SW1990 cells were transfected with pRNAT-STAT3-RNAi-II plasmid which had the most obvious gene silencing effect. SW1990 cells were also transfected with the pRNAT-Con vector. Cells were then selected with a standard medium containing G418. Two independent transfections of pRNAT-STAT3-RNAi-II expression vector were carried out in SW1990 cells, and G418-resistant colonies were pooled to establish stable SW1990 STAT3-RNAi transfectants (SW1990-RNAi-a and SW1990-RNAi-b). We stably silenced the expression of the STAT3 and p-STAT3 by stable transfection of pRNAT-STAT3-RNAi-II plasmid in SW1990 cells, followed by reduction in invasion capacity in vitro and metastasis capacity in vivo compared to parental cells. Furthermore, silencing SW1990 cells STAT3 gene by RNAi also led to a decrease of MMP-2 and VEGF at the mRNA and protein level. Conclusions:⑴Pancreatic cancer tissues have increased p-STAT3,MMP-2 and MMP-9 expression which had a significant positive relationship with clinical stage and lymphatic metastasis. The expression of p-STAT3 have significant positive relationship with the expression of MMP-2 and MMP-9. STAT3 signal pathway promotes invasion and metastasis probably through up-regulating the expression of MMP-2,MMP-9, and consequently play a critical role in the progression of pancreatic cancer.⑵STAT3 activity have a strong relationship with the invasive ability of human pancreatic cancer cells. Blocking STAT3 activation with AG490 can inhibit the invasion ability of pancreatic cancer cells through down-regulate MMP-2 and VEGF. Blocking STAT3 signaling pathway may provide a novel strategy in preventing the invasion and metastasis of pancreatic cancer.⑶The STAT3 siRNA expression vectors are successfully constructed, which effectively inhibit STAT3 expression. Stable transfection of the STAT3 siRNA expression vector can effectively and specificly inhibit the expression of STAT3 in SW1990 cells, followed by significant reduction in invasion and metastasis capacity through down-regulate MMP-2 and VEGF. Silence of STAT3 gene with RNAi may be a useful anti-invasive therapeutic option in pancreatic cancer.