P14ARF Gene On The Growth And Invasion Of Pancreatic Cancer And Their Mechanisms

Objective p14ARF is a new cell cycle regulation gene mainly causing G1 and G2/M arrest. Since it was found that the aberrance of p14ARF gene, such as deletion and promoter hypermethaylation had a high relationship with the occurrence, development and prognosis of malignant tumor, p14ARF had been regarded as a tumor suppressor gene. P14ARF is the upstream gene of p53. It can facilitate growth arrest through the p53 pathway by hindering the down-regulation of p53 activity mediated by MDM2, through the formation of a protein complex with MDM2. This is the p14 (ARF)-MDM2-p53 cell cycle regulation system. P53 gene is an acknowledged tumor suppressor gene, which plays an important role in the development /progression/invasion and metastasis of human cancers. We first try to establish the model of pancreatic carcinoma cell line PC-3 transfected with p14ARF gene, and observe the growth inhibition effect. Meanwhile, the invasion-associated factors, such as MMP2, MMP9, VEGF, chemokines (IL-1p,IL-8 and CXCR4), vascular cell adhesion molecule-1 (VCAM-1) are analyzed from the level of gene transcription to protein expression, to clarify the mechanism of invasion and metastasis in pancreatic cancer, and to illustrate the inner regulation of these invasion-associated gene exerted by p14ARF gene. It would provide the basis of clinical gene therapy.Methods The combined plasmid pCI-neo-p14ARF containing extrinsic human wild-type p14ARF whole-length sequence of cDNA and null vector plasmid pCI-neo were transfected into pancreatic carcinoma cell line PC-3 with the lipofection reagent Fugene 6 respectively, G418 was used to screen out the corresponding positive clone of PC-3/p14 and PC-3/vec, the p14ARFmRNA and protein expression in PC-3/p14 cell were measured by the RT-PCR and Western blot methods. MTT assay was used to compare the change of growth curve of PC-3, PC-3/vec and PC-3/p14 cell. The three kinds of cell (PC-3, PC-3/vec and PC-3/p14) were injected subcutaneously to nude mice. After the injection, the mice were observed for three months for subcutaneous tumor formation. Gene expression microarray analyzed the difference of gene between PC-3 cell and PC-3/p14. The alteration of cellular migration property was evaluated by cell migration through a Matrigel model basement membrane. The expression of MMP2 and MMP9 was detected by RT-PCR and immunohistochemistry. We used RT-PCR and Western blot methods to observe the change of angiogenesis-associated factor, VEGF, in three cells, microvessel density (MVD) was also measured by immunohistochemistry with CD34 antibody. Finally, the chemokines (lL-1p,IL-8 and CXCR4) and VCAM-1 were assayed by RT-PCR.Results The p14ARF gene was deleted in PC-3 cell. The combined plasmid pCI-neo-p14ARF and ull vector plasmid pCI-neo were successfully transfected into pancreatic carcinoma cell line PC-3 by lipofection, the positive clone were screened out by G418. The mRNA transcription and protein translation of p14ARF gene in PC-3/p14 cell were detected by RT-PCR and Western blot methods. The growth of PC-3 cell was more severely inhibited after combined therapy than those transfected with p14ARF gene. The growth inhibition was also observed in Xenografts of nude mice. The Gene expression microarray analysis displayed 119 genes changed in PC-3 and PC-3/p14. The ras homolog gene family C (ARHC), VCAM-1 and discoidin domain receptors (DDR) were down-regulated. ARHC is a negative-regulation gene to the growth of cancer. The down-regulation of VCAM-1 can facilitate tumor shedding from the primary site, migrating and forming metastasis focus. The down-regulation of DDR2 can also down-regulate MMP1 gene to inhibit the invasion and metastasis property. In addition, glutathione S-transferase p1, (GSTpi) , as a new predictor of cancer, is remarkably associated with...