PDGF-D Promotes Proliferation And Invasion Of Colorectal Cancer By Notch1/Twist1 And Notch1/MMP9 Signaling

Part Ⅰ:Platelet-derived growth factor-D promotes colorectal cancer cell migration, invasion and proliferation by regulating Notchl and MMP9Objective:Colorectal cancer(CRC) has been one of the most common cancer for decades in the worldwide and significantly associates with cancer related deaths. The pathogenesis of CRC is linked with the process of activating oncogenes and inactivating anti-oncogenes. The platelet-derived growth factor-D(PDGF-D) was confirmed to regulate migration, invasion, proliferation, apoptosis and metastasis of different cancer cells. Over-expression of PDGF-D exists in various human malignancies, for example, pancreatic cancer, prostate cancer and breast cancer. However, the expression and function of PDGF-D and its related molecular mechanism in CRC remain unclear. Thus we detected the expression of PDGF-D in CRC tissues and explored the potential mechanism, to clarify the role of PDGF-D in colorectal cancer.Methods:Immunohistochemical staining was used to detect the expression of PDGF-D in CRC tissues and adjacent nontumor tissues. The qPCR and western blot were also used to evaluate protein and mRNA levels of PDGF-D. Several colon cell lines were screened for the expression of PDGF-D by qPCR and then confirmed by western blot assay. Deregulation of PDGF-D in SW480 cell line was successfully established by lentiviral transfection, and similarly, upregulation of PDGF-D in CT116 cell line. The Flow cytometry was applied to test the apoptosis rate of transfected cells. The migration, invasion and proliferation of cancer cells were detected by cell transwell assay and CCK8 assay correspondingly.Results:53.3%(24/45) CRC tissues were positive for PDGF-D protein expression in the cytoplasm while the paired normal colorectal tissues were 11.1%(5/45), P<0.0001. The qPCR results showed that PDGF-D mRNA expression was averagely 4.6 times higher in CRC tissues than that of normal tissues, P<0.05. SW480 were chosen for its stable high expression of PDGF-D protein, and HCT116 for its relative low expression. Silencing PDGF-D in SW480 cell line inhibited migration, invasion and proliferation distinctly, with the reduced expression of Notchl and MMP9. What’s more, up-regulating PDGF-D in HCT116 showed opposite results.Conclusion:PDGF-D promotes colorectal cancer cell migration, invasion and proliferation by regulating Notchl and MMP9.These findings imply PDGF-D could be developed into a potential therapeutic target for colorectal cancer treatment.Part II:Platelet-derived growth factor-D promotes colorectal cancer cell migration, invasion and proliferation by Notch1/Twist1 and Notch1/MMP9 signalingObjective:To explore the specific molecular mechanism of PDGF-D in the tumorgenesis of colorectal cancer.Methods:After downregulated of PDGF-D in SW480 cells, the cDNA-Notchl-lentivirus were transfected into them-. When the Notch1 expression was up-regulated successfully, the cell transwell assay and CCK-8 assay were performed to detect the changes of tumor cell migration, invasion and proliferation. Furtherly, the potential downstream signaling of PDGF-D/Notchl were screened and proven by qPCR and western blot. Similarly, after upregulated of PDGF-D in HCT116 cells, the DAPT agent (a Notch 1 inhibitor) was used to down-regulate Notchl expression. Then the cell transwell assay and CCK-8 assay were performed to detect the changes of tumor cell migration, invasion and proliferation. The potential downstream signaling of PDGF-D/Notchl were also screened and proven by qPCR and western blot.Results:After downregulated of PDGF-D in SW480 cells, the Notchl expression was up-regulated successfully by cDNA-Notch1-lentivirus transfection. The migration, invasion and proliferation of cancer cells significantly enhanced and the downstream molecules such as Twistl and MMP9 were upregulated accordingly. After upregulated of PDGF-D in HCT116 cells, the Notchl expression was down-regulated successfully by DAPT. The migration, invasion and proliferation of cancer cells significantly dropped and the downstream molecules such as Twistl and MMP9 were downregulated accordingly.Conclusion:Platelet-derived growth factor-D promotes colorectal cancer cell migration, invasion and proliferation by Notch1/Twistl and Notch1/MMP9 signaling.