| Backgroud and objectivesColorectal cancer(CRC)is one of the major malignancies in digestive carcinomas,the mobility is only after gastric and esphageal cancer.According to the world cancer epidemoiology survey,the mobility of colorectal cancer is highest in West Europe,North America and Australia. In recent decades,the morbidity of CRCs was increasing at a rate of 2% annual.In Chian,the increasing rate of CRCs mobility is as twice as in the rest of the world with the development of economy,the change of lifestyle and diets,reached a rate of 4% each year.As far, surgery,chemotherapy and radiotherapy are the major treatments and efficacy.While,the side effects and toxicity of conventional chemotherapy regiments to the body are severely.Forthermore,the treatments above are powerless to control tumor migration and metastasis.With the rapid development of molecular geology recently,more and more researches are focus on signaling pathways that involved in tumor progression.Truely,an increasing knowledge about all kinds of signaling pathways which play roles in migration and metastasis of colorectal cancer has the potential to provide an effective approach and breakthrough for management of CRCsPhosphatidylinositol-3-kinase (PI3K)is a lipid kinase and responsible for the phosphorylation of 3 position of the inositol ring of PI(4,5)P2, to generate PI(3,4,5) P3,act as second messenger to trigger diverse signaling cascades that mmediate multiple cellular activities.There are three members in PI3K family,most researches are focus on class-ⅠPI3K,which can be active by membrane receptor.Class-IA PI3K are heterodimers composed of a catalytic subunit (p110α/β/δ/γ)and an adaptor/regulatory subunit(p85α/β/γ). The catalytic p110 subunit of PI3K is homologous to protein kinases and possesses both serine-threonine protein kinase and phosphoinositide kinase activities.The double-enzymatic activity of PI3K as well as the ability of this enzyme to activate a number of signal proteins including some oncoproteins determines its fundamental significance in regulation of cell functions such as growth survival proliferation and malignant transformation.PI3K is activated through two ways.One is occurs through phosphorylation of a tyrosine residue by either receptor (platelet,insulin-like,or epidermal growth factor receptors) or non-receptor (p60-src) tyro-sine kinases.The other is activated through Ras combinate with p110 directly.Phosphatidylinositol-3-kinase (PI3K)is responsible for generating PI(3,4,5) P3,PIP3 act as a second-messenger combinate with signal proteins Akt and PDK1(phosphoinositide dependentkinase-1)then,Akt transfer to membrane and phosphorylate Serl24 and Thr450,PDK1 phosphorylate Thr308 of Akt protein.Class-1 A PI3K/Akt passway regulate proliferation and survival of tumor cells.dysfunction of PI3K/Akt leads to not only neoplastic transformation of normal cells, but also correlation with tumor cell migration, adhesion, tumor angiogenesis, as well as the degradation of extracellular matrix. Metastasis is a complex process whereby tumor cells have acquired the abilities to detach from the primary site through alterations in cell-cell adhesion molecules, degrade the extracellular matrix, invade new sites through altered cell motility, and survive in a new environment AKT1 has been shown the potential of regulating cell motility and that its overexpression in tumors may enhance their metastatic potential.AKT1 overexpression is associated with increased activity of matrix metalloproteinases through its activation of nuclear factor-κB binding to the matrix metalloproteinase promoter and its ability to up-regulate angiogenesis, may also contribute to tumor survival. The other AKT isoforms have been reported to contribute to invasiveness through up-regulation ofβ1-integrins and other pathways. The PI3K/AKT pathway therefore has an critical role in the metastatic phenotype. Targeting PI3 kinase, the most proximal pathway component, has advantages over targeting more distal components such as Akt and mTOR. Inhibitors of PI3K providinga broader inhibition of downstream signaling than distal inhibition. The pharmacologic agents LY294002 and wortmannin, both target the p110 catalytic subunit of PI3K, sensitize cancer cells to various types of conventional chemotherapy and provide powerful preclinical tools to study the cellular consequences of pathway inhibition..While, these commercially available inhibitors limite their clinical application because of their poor solubility and high toxicityIn this study, we investigate the expression and significance of PI3K p110βin the progression of colorectal cancer, including normal colorectal tissue, colorectal adenoma and primary colorectal carcinoma. Then we transient transfect SW480 cells. After transfection, we observed the effect of PI3K p110βexpression on colorectal cancer cell migration and invasion. We also investigated the expression changes of PI3K signaling pathway proteins including Akt,p-Akt. We aim not only to investigate the molecular mechanism of tumor invasion and migration, but also to search new effective target for gene therapy of CRC.Materials and methodsThe expression and significance of PI3K p110βin progression of colorectal cancerImmunohistochemical staining was used to detect the expression and significance of PI3K p110βin the progression of colorectal cancer, including normal colorectal mucosa, colorectal plopy,colorectal adenoma and primary colorectal carcinoma. The relationship between the expression of PI3K p110βprotein and clinicopathological factors was also analyzed.The effect of PI3K p110βdepletion on cell invasion and migration in colorectal cancer cells1. Construction of human PI3K p110βsiRNA vectors The oligonucleotides encoding sequences of PI3K p110βmRNA and a scramble siRNA with the same nucleotide composition as the siRNA but which lacks significant sequence homology to the genome was rerference to relative paper.The oligonucleotides are synthesized, annealed, and ligated into the linearized siRNA Expression Vector by GnenPharma company.2. Transient transfection SW480 cells The siRNA PI3K p110βwas transfected into SW480 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.3. PI3K p110βexpression detecting Extract cell protein after transient transfected SW480 cells and applied to Western blot analysis for evaluating interference effect of PI3K p110βprotein.4. Western bolt was used to analyze the expression of pathway key protein Akt,p-Akt in siRNA p110βgroup,control group and blank group SW480 cells.5. The invasion and migration of changes were observed by Transwell and Wound healing test in SW480 cells afer transfection.Statistical Analysis All experiments results were from at least three separate experiments. For immunohistochemistry results, Kruskal-Wallis test was used. Linear-by-Linear Association was used for analyzing the relationship between PI3K p110βexpression and colorectal tissue type and Dukes stage. For other results, one-way analysis of variance (ANOVA) and Student's t test were used in group comparison. Dates are expressed as the mean±SD. A value of P<0.05 was considered statistically significant.ResultsThe expression and significance of PI3K p110βin progression of colorectal cancerImmunohistochemistry was performed to examine PI3K p110βexpression levels in paraffin-embeded tissue from colorecta mucosa, benign polypi and adenomas to primary colorectal cancers. PI3K p110βexpression was highest in surface epithelium of colorectal mucosa, and expression in the stroma was limited to inflammatory cells, with a predominantly cytoplasmic distribution. Representative shows that PI3K p110βexpression is negative in normal colorectal mucosa and polypi, begins to increase in adenoma and over-expressed in primary colorectal adenocarcinoma (P<0.001). Furthermore, there was a significant difference in the positive rates of the PI3K p110βexpression duiring different Dukes'stage (P<0.050). No obvious correlation was found between expression of PI3K p85a and pathological diagnosis (P>0.050).The effect of PI3K p110βdepletion on invasion and migration in colorectal cancer cells1.After stransient transfection, western blot analysis was used to investigate the expression changes of PI3K p110β. The expression levels were significantly inhibited in SW480 siRNA p110βcells compared to those of control group and blank group cells. Thus, SW480 cells were used for the following experiments.2.Western blot was performed to examined expression levels of Akt,phosphor-Akt.key pathway proteins in SW480 cells.The result showed that PI3K p110βdown-regulation led to substantial reduction in the level of Akt and phosphor-Akt.(P<0.05).3. Transwell and Wound healing test were examined the invasion and migration of SW480 cells. The transwell assay showed that PI3K p110βdepletion resulted in the number of cell migration was obviouslydecreased in siRNA p110βgroup.(P< 0.05).Monolayer scratch after 24 h, compared with the control and blank group, The numbers of cell invasion experiments in siRNA p110βalso decreased obviously (P< 0.05).Conclusions1. Immunohistochemical results show that the expression levels of PI3K p110βgradually increases from normal colorectal mucosa, adenoma to primary colorectal adenocarcinoma, showing that PI3K p110βplays an important role in the progression of colorectal cancer.2. Transwell and Wound healing test indicate that down-regultion of PI3K p110βcould inhibit SW480 cell invasion and migration3. Depletion of PI3K p110βcould inhibit the activity of Akt show that invasion and migration of colorectal cancer have relationship wihth dysfunction of PI3K/Akt signal transduction pathway. |
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