The Experimental Study Of P73 Gene On Angiogenesis In Human Lung Adenocarcinoma

Background: Lung cancer is extremely harmful to the life and health of human beings. Its genesis is related to multiple factors. It has been proved that there is approximately 47.8% in NSCLC and 80% in SCLC with p53 gene mutation. p73 gene is a homologue of p53. p73 protein has the similary structure to p53 protein and it is located in the nucleus too. p73 gene has some kinds of variants, such as p73 a , p73 P , p73 Y , p73 5 , p73 and p73 4. A Np73 gene is a kind of shortening p73 which lack the transcriptional activation domain. p73 gene is located in human chromosome Ip36 where was found delete frequently in many human tumors. All these imply that p73 gene may be an anti-oncogene and some researches have proved it can inhibit cell growth and induce cell apoptosis.Angiogenesis is a process that form the new blood vessels from preexisting micro-vascular. The tumor can't grow beyond 2~3mm3 for lack of oxygen and nutrient without angiogenesis. With angiogenesis the tumor can obtain nutrition and the capillaries can provide a chance for the tumor metastasis. The process includes the following steps: dissolve the vascular endothelial basal membrane, endothelium cell migrate to tumor tissue, endothelium cell proliferate, form new capillary, form new basal membrane. This complex process involves the interaction of tumor cells, endothelial cell and extracellular matrix. Its regulation depends on many cytokines. Some cytokines have been separated and purified, such as VEGF, bFGF, angiogenin, TSP-1, iNOS and so on. The effect of p73 gene on angiogenesis in some human tumors has been reported. It has been proved that p73 gene was overexpression in NSCLC and nearly hasn't mutation. But the effect of p73 gene on angiogenesis in NSCLC is still unknown.Objective: To study the effect of p73 gene overexpression on angiogenesis in human lung adenocarcinoma and the difference function between the p73 variants. Then find a new way for NSCLC gene therapy.Methods: This study use A549 cell, HI299 cell, 2 kinds of lung adenocarcinoma celllines as a study object. p73oc, p73 0 and A Np73 gene were transfered into these 2 kinds of cells by liposome and the overexpresion of p73 gene cell clones were chosen by G418. The overexpression of different p73 varients cells were cultured and their effects on the cell growth curve were observed. The mRNA expression of VEGF^ bFGF and TSP-1 were detected by RT-PCR, the protein expression of VEGF^ bFGF were detected by western blot in vitro. A549 cell, A549-p73oc cell and A549-ANp73 cell were transplanted into nude mice. The effect of p73 gene on angiogenesis in transplanted tumor was studied. The volume and growth speed of transplanted tumors were observed. VEGF> bFGF and iNOS protein expression in transplanted tumors were detected by immunohistochemistry staining. MVD of transplanted tumors were counted by FVTII-RAg immunohistochemistry staining.Results: Overexpression of p73 gene cell clones were chosen successfully. The cell growth curve demonstrated that overexpression of p73cc and p73 P could inhibit the cell growth but the A Np73 could promote cell growth. Overexpression of p73a could inhibit VEGF, bFGF mRNA and protein expression and promote TSP-1 mRNA expression(p<0.05). The overexpression of p73 3 has similar function with p73a but has more obviously effect on inhibiting VEGF expression(p<0.01). Overexpression of p73ot can inhibit nude mice transplanted tumor growth, inhibit VEGF, bFGF, iNOS protein expression(p<0.05), reduced the tumor MVD(p<0.01). A Np73 gene has the contrary function of p73a in vitro and in vivo, its behavior is more like an oncogene.Conclusion: Overexpression of p73cc and p73 P can inhibit the angiogenesis in human lung adenocarcinoma. The p73 P may has more obviously effect on inhibiting angiogenesis. A Np73 gene has the contrary effect of p73cc, p73 P on angiogenesis, it seems like an oncogene. We can apply the characteristic that p73 gene nearly hasn't mutation in NSCLC to induce p73 gene overexpression in lung adenocarcinoma, especially the p73 P overexpress...