| Objective:Lung cancer is the most common malignant disease in thoracic surgery, which remains a major health problem of high morbidity and mortality worldwide. Early diagnosis is difficult, as there were no specific clinical symptoms or tumor markers. Prognosis of lung cancer is still poor. The Vascular Endothelial Growth Factor (VEGF) belongs to the platelet-derived growth factor (PDGF) supergene family, is one kind of secretive plasmolysis proteins. It is investigated that the expression of VEGF becomes evidence in non-small cell lung cancer (NSCLC) tumors. VEGF induces the survival of vascular and lymphatic endothelial cells, and plays a key role in both angiogenic and metastatic potential of tumor cells. Some other studies found that the autocrine loop of VEGF is crucial for tumor survival and progression. However, there has been little work to explore the mechanism of action of VEGF autocrine. Our study is to transfect the antisense oligodeoxynucleotide (ASODN) of VEGF into NSCLC cell line A549without vascularization in vitro. Inhibition of endogenous VEGF was observed to evaluate the impact of autocrine and paracrine loops, the biological behavior of cells, and the possibility of its clinical potential value.Method:Non-small cell lung cancer cell line A549was cultured and transfected with florescence-marked VEGF antisense oligonucleotide by lipofectamine in vitro to observe the transfection efficiency. According to the different transfection methods, cells were divided into five groups. The test group was transfected with ASODN, other two oligonucleotide control groups were transfected with SODN and MODN. Last two control groups were added with lipofectamine and medium. The alteration of VEGF mRNA and protein in A549were tested respectively by RT-PCR and ELISA. The growth of A549was observed by MTT. The phases of cell cycles were detected by flow cytometry.Result:(1) More than85%cells demonstrated green florescence after transfected with ASDON in A549, while the control group only showed about5%;(2) The VEGF mRNA of test group was significantly lower than other four control groups (P<0.05) after24hours and48hours.(3) The content of VEGF protein in test group after transfecting for 24hours,48hours and72hours were all significantly decreased than other four control groups (P<0.001). The content of VEGF protein in two contrast oligonucleotide groups were also significant lower than lipofectamine and medium solution control groups (P<0.05). There was no significantly difference between lipofectamine group and medium group.(4) The survival rate of A549in test group was lower than other four control groups (P<0.05) in the1st day of transfection. After48hours, the survival rate of test group was no significantly difference with the two contrast oligonucleotide groups. It remained significantly lower than two control solution groups (P<0.01).(5) The percentage of G2phase cells of ASODN group was significant lower than other four groups.Conclusion:(1) The transfection of antisense oligonuleotide could be taken in lung cancer cells efficiently by lipofectamine. It also could be diffused efficiently between nucleus and cytoplasma.(2) The VEGF mRNA and protein expression in A549could be inhibited by VEGF antisense oligonucleotide.(3) The growth of A549can be restrained by inhibited of the endogenous VEGF without vascularization and endothelial factors involved. The growth of A549could be partially restrained in contrast oligonucleotide groups.(4) The cell cycle of lung cancer cells could be hold up on G1/S phase by inhibiting expression of VEGF.(5) The method of transfection with VEGF ASODN by lipofectamine could decrease the expression of VEGF and inhibit the survival of NSCLC cells. It may have the potential in clinical treatment.(6) There is different function on tumor survival and progression between autocirne and endocrine VEGF loops. The treatment complication may be decreased by inhibiting the signaling pathways more specifically. |
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