Study About The Mechanism Of Platelet-derived Growth Factor-B Signaling In Metastasis Of Gastric Carcinoma

PART I CLINICOPATHOLOGICAL SIGNIFICANCE OFPDGF-B AND PLATELET-DERIVED GROWTH FACTORRECPERTOR-β EXPRESSION IN GASTRIC CARCINOMAObjective: To observe the relationships between expressions ofplatelet-derived growth factor B(PDGF-B), platelet-derived growth factorreceptor β(PDGFR-β) and clinicolpathological features of patients withgastric carcinoma.Methods: A series of32gastric carcinoma cases that had undergonesurgical resection was collected. Gastric carcinoma tissues and normalgastric mucosa tissues were examined immunohistochemically usingantibodies against PDGF-B and PDGFR-β, followed by further analysisabout the correlation between PDGF-B and PDGFR-β expression and theclinicolpathological features of patients.Results: In surgical specimens, tumor cells expressed PDGF-B,positive rate was70.3%(45/64); but PDGFR-β was expressedpredominantly by tumor stromal cells, positive rate was60.9%(39/64); both of them were much higher than those of in normal gastric mucosatissues(4.7%,3/64and3.1%,2/64respectively). Expressions of PDGF-Band PDGFR-β were positively correlated with the depth of cancer invasion,lymph node metastasis and tumor-node-metastasis(TNM) stage(P<0.05);but expressions of PDGF-B and PDGFR-β were no correlated with thetumor cell differentiation(P>0.05).Conclusions: Our data indicate that expressions of PDGF-B andPDGFR-β were much higher in gastric carcinoma tissue and were correlatedwith cancer progression and lymphogenous metastasis of gastric carcinoma;suggest that detection of PDGF-B and PDGFR-β were important markers forjudgment of tumor metastasis and prognosis of gastric carcinoma. PART II CONSTRUCTION OF LENTIVIRAL VECTORPLENO-DCE-PDGF-B AND STABLE PDGF-BOVEREXPRESSION GASTRIC CARCINOMA CELL LINEObjective: To construct a lentiviral vector that stably expressplatelet-derived growth factor B (PDGF-B) and to construct stable PDGF-Boverexpression gastric carcinoma cell line by lentiviral vector transfection. Methods: The lentiviral vector pLeno-DCE-PDGF-B was constructedand transfected into293T cells. The supernatant containing the lentivirusparticles was harvested to determine the virus titer and high titer lentivirusparticles was gathered. Then PDGF-B lentiviral vector was transfected intoSGC7901and BGC823gastric carcinoma cells for construction of stablePDGF-B overexpression gastric carcinoma cell lines. Western-blot andimmunofluorescence were used for evaluation of the construction of stablePDGF-B overexpression gastric carcinoma cell lines.Results: The lentiviral vector was correctly constructed and verified bysequencing. High titer PDGF-B lentiviral vector was acquired successfully.After transfection, two gastric carcinoma cell line SGC7901and BGC823were green color by detection of fluorescence microscope; and theexpression of PDGF-B protein in transfected SGC7901and BGC823gastric carcinoma cells were much higher than that of in normal SGC7901and BGC823gastric carcinoma cells(P<0.05).Conclusion: We have successfully constructed PDGF-B lentiviralvector and acquired high titer PDGF-B lentiviral vector. Lentiviral vectortransfection was an effective method for construction of stable PDGF-Boverexpression gastric carcinoma cell lines. PART III THE INFLUENCE ON GROWTH ANDINVASION OF GASTRIC CARCINOMA CELL CAUSEDBY PDGF-B OVEREXPRESSION AND ITS MECHANISMObjective: To observe the influence on growth and invasion of gastriccarcinoma cell caused by PDGF-B overexpression and to approach themechanism about how PDGF-B to affect the growth and invasion of gastriccarcinoma cells.Methods: Stable PDGF-B overexpression gastric carcinoma cell lineswere constructed. MTT assay was used for detection of cell growth andtranswell invasion assay was used for detection of invasion for both PDGF-Boverexpression and normal SGC7901and BGC823gastric carcinoma cells.Western-blot was used for detection of expression of AKT-1and E-cadherinbefore and after transfection to approach the mechanism.Results: Growth curve of PDGF-B overexpression SGC7901andBGC823gastric carcinoma cells were much higher than those of normalSGC7901and BGC823gastric carcinoma cells(P<0.05). Invasion cellnumber of PDGF-B overexpression SGC7901and BGC823gastriccarcinoma cells(65.4±2.4and70.2±3.2, respectively) were much higher thanthose of normal SGC7901and BGC823gastric carcinoma cells(21.6±1.6and30.2±2.2, respectively)(P<0.05). Expression of AKT-1in PDGF-B overexpression SGC7901and BGC823gastric carcinoma cells were muchhigher than normal SGC7901and BGC823gastric carcinoma cells(P<0.05).Expression of E-cadherin were no difference between PDGF-Boverexpression and normal SGC7901and BGC823gastric carcinomacells(P>0.05).Conclusions: Overexpression of PDGF-B increases the growth andinvasion of gastric carcinoma cells so as to promote tumor metastasis.Activation of PI3K/AKT signal pathway by PDGF-B autocrine might leadthe increases of the growth and invasion of gastric carcinoma cells. PART Ⅳ THE RELATIONSHIP BETWEEN PDGF-BSIGNALING, EPITHELIAL–MESENCHYMALTRANSIYION AND METASTASIS OF GASTRICCARCINOMA AND ITS MECHANISMObjective: To approach the relationships between PDGF-B signaling，epithelial–mesenchymal transition(EMT) and metastasis of gastriccarcinoma and to investigate the mechanism of PDGF-B signaling inmetastasis of gastric carcinoma. Methods: Expressions of AKT-1, ERK-1, E-cadherin and N-cadherinprotein in SGC7901and BGC823gastric carcinoma cells before and afterPDGF-B transfection were detected by western-blot. Cell coculture wasused for activation of PDGF-B signaling. RT-PCR was used for detection ofmRNA of AKT-1, ERK-1, E-cadherin and N-cadherin in coculture PDGF-Boverexpression SGC7901and BGC823gastric carcinoma cells and incoculture normal SGC7901and BGC823gastric carcinoma cells, alsowestern-blot was used for detection of protein of AKT-1, ERK-1, E-cadherinand N-cadherin in coculture PDGF-B overexpression SGC7901andBGC823gastric carcinoma cells and in coculture normal SGC7901andBGC823gastric carcinoma cells.Results: There were no differences between expressions of E-cadherin,N-cadherin and ERK-1protein in SGC7901and BGC823gastric carcinomacells before and after PDGF-B overexpression(P>0.05); the expressions ofAKT-1protein in PDGF-B overexpression SGC7901and BGC823gastriccarcinoma cells were much higher than those of in normal SGC7901andBGC823gastric carcinoma cells(P<0.05). The expressions of mRNA andprotein of AKT-1and ERK-1in cocultured PDGF-B overexpressionSGC7901and BGC823gastric carcinoma cells were much higher than thoseof in cocultured normal SGC7901and BGC823gastric carcinomacells(P<0.05). Expressions of mRNA and protein of EMT-related E-cadherinin coculture PDGF-B overexpression SGC7901and BGC823gastric carcinoma cells were much lower than those of in cocultured normalSGC7901and BGC823gastric carcinoma cells(P<0.05), and expressions ofmRNA and protein of ETM-related N-cadherin in cocultured PDGF-Boverexpression SGC7901and BGC823gastric carcinoma cells were muchhigher than those of in cocultured normal SGC7901and BGC823gastriccarcinoma cells(P<0.05). There were no differences between expressions ofAKT-1, ERK-1, E-cadherin and N-cadherin protein in normal SGC7901and BGC823gastric carcinoma cells before and after coculture(P>0.05).There were no differences between expressions of AKT-1protein inPDGF-B overexpression SGC7901and BGC823gastric carcinoma cellsbefore and after coculture(P>0.05); there were apparently differencesbetween expressions E-cadherin, N-cadherin and ERK-1protein inPDGF-B overexpression SGC7901and BGC823gastric carcinoma cellsbefore and after coculture(P<0.05).Conclusions: PDGF-B signaling might not induce EMT in gastriccarcinoma cells through autocrine. PDGF-B signaling might induce EMT ingastric carcinoma cells through paracrine and joint action with tumorstromal cells so as to promote metastasis of gastric carcinoma. BothMAPK/ERK signal pathway(a downstream targets of PDGF signalingpathway) and tumor microenvironment might participate in the process ofEMT. Consequently, PDGF-B signaling might be an effective therapy targetfor gastric carcinoma.