STAT3 knockdown reduces pancreatic cancer cell invasiveness and matrix metalloproteinase-7 expression in nude mice

Abstract

Aims: Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression.

Methods: A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression.

Results: STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered.

Conclusion: These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells.

Figure 1. Suppression of STAT3 mRNA and protein expression using STAT3 shRNA in pancreatic cancer SW1990 cells.

A, qRT-PCR. Stable STAT3 shRNA and control vector-transfected SW1990 cells and parental SW1990 cells were grown and RNA was isolated for qRT-PCR analysis of STAT3 mRNA expression *P<0.01 compared to SW1990 or control vector-transfected cells. B, Western immunoblot. These cells were grown for total cellular protein extraction and western immunoblot analyses of STAT3 protein expression.

Figure 2. Suppression of nude mouse xenograft growth by STAT3 knockdown.

Parental or control vector and STAT3 shRNA-transfected SW1990 cells were grown in cell culture and injected into nude mouse nail pads. Tumor volume was measured every five days up to 30 days following tumor cell inoculation.. *P<0.05 compared to the control vector or parental tumors.

Figure 3. Expression of collagen IV protein surrounding the tumor cells using immunohistochemistry.

The tumor xenografts from control vector and STAT3 shRNA-transfected cells were subjected to immunohistochemical staining of collagen IV protein. These data suggest that collagen IV expression (red arrow) is complete and continuous in STAT-3-silenced tumor (A), however the integrity of collagen IV was destroyed evidenced by incomplete and discontinuous (red arrow) staining (B).×400 magnification.

Figure 4. Suppression of tumor invasion in nude mouse xenografts by STAT3 shRNA.

These nude mouse xenografts were taken from tissues processed for HE staining. A and B, x 400 magnification, C and D, x 100 magnification. Tumor cell invasion into the microvessel is frequent in control vector tumors (marked in red arrow in A) but not obvious in STAT3-silenced tumors (B). The muscle was invaded and destroyed (black arrow) by the tumor cells (red arrow) of SW1990 cells in C, but the boundary is clear between muscle (black arrow) and tumor (red arrow) in D.

Figure 5. Suppression of microvessel density by STAT3 shRNA in nude mouse xenografts.

Tissue sections of nude mouse xenografts were stained for immunohistochemistry with the anti-PECAM-1 antibody. PECAM-1 positive microvessels in tumors were counted under a microscope and summarized. The number of microvessel was 13.00±2.36 per high power field (N = 9) in STAT3-silenced tumors compared to 16.30±2.45 (N = 10) in the parental SW1990 tumors and 15.80±2.57 (N = 10) in vector control tumors. These differences were statistically significant. *P<0.01 vs. parental SW1990 tumors, **P<0.05 vs. the control vector tumors.

Figure 6. Expression of MMP-7, MMP-9, bFGF, IL-1b, and IgT7α in nude mouse xenografts.

A, qRT-PCR. RNA was isolated from the tumor xenografts and subjected to qRT-PCR analyses. B, Western blot. Total cellular protein was extracted from the nude mouse xenografts and subjected to western blot analyses. **P<0.01; *P<0.05 compared to the control vector or parental tumors, respectively.

Figure 7. MMP-7 protein expression in pancreatic cancer cells in monolayer and in nude mouse xenografts.

Tumor cells from parental, vector control and STAT3-silenced SW1990 cells were grown in a monolayer or in nude mice. The monolayer cells and tumor xenografts were then subjected to immunocytochemistry and immunohistochemistry staining of MMP-7 protein. A, parental cells; B, STAT3-silenced cells; C, parental tumors; D, STAT3-silecnced tumors. X 400 magnification. Red arrow, tumor cells.