Cd151-integrin Complex Regulates Tumor Cell Progression,Migration And Invasion In Vitro And In Vivo With Its Mechanism Involved

Objective Cancer has always been a class of serious diseases harm to human health. It insidious onset, rapid progress, high degree of malignancy, treatment difficulties, high mortality and poor prognosis characteristics makes some cancer patients already became advanced stages when diagnosed. China has a high incidence of many common tumors such as lung carcinoma and hepatic carcinoma. How to block tumor growth, invasion, and metastasis is the focus of current cancer treatment research. The gene therapy of cancer has always been a hot issue of concern, both in basic research and clinical studies. CD151is a member of tetraspanin protein super family. It can combine with a variety of intracellular protein molecular, participating in promoting tumor cell movement, tumor growth, survival, metastasis and invasion. For example, CD151combined with laminin-banding integrins, and other kinds of intracellular protein such as growth factors, molecular signaling and other tetraspanin protein family members forming complex, together to play a variety of different biological functions. Regulating cell movement and metastasis is the most prominent feature of CD151. Many clinical trials have indicated that, CD151promoted tumor metastasis and invasion, and always has a close relationship with a series of poor prognosis tumors. In order to investigate the definitive role of CD151and its complexes in the progression of human carcinoma, we constructed the RAAV-CD151-QRD194-196CD151-AAA194-196mutant plasmid and synthesized CD151-SIRNA to explore the CD151functions. Through this experimental study, we eventually evaluated the definitive role of CD151in tumor onset, development, and progression. And the effects of CD151-integrin complex on tumor cell growth, survival, migration and invasion in vitro and in vivo, including intracellular signaling transmission and mechanism.Methods1. Constructed RAAV-CD151recombinant plasmid, RAAV-CD151-QRD194-196-CD151-AAA194-196mutant plasmid, and RAAV-GFP negative control plasmid. And extracted a large number of these three kinds of plasmids via plasmid highpure kit.2. Designed and synthesized small molecule interfering RNA specific for CD151known as CD151-SIRNA, and also synthesized out of order SIRNA as negative control.3. Transfected the prepared plasmid DNA, and small interference RNA by Using liposome-mediated gene transfection into HepG2cells, A549cells, and Hela cells regulating CD151expression to observe CD151effects on the biological behavior of the three tumor cells.4. Co-immunoprecipitation assay to explore the combination of CD151and integrinα3, a6, β1subunits in the three kinds of tumor cell lines after transfection.5. MTT assay and transwell assay was used to detected the difference of cells growth ability, migration and invasion ability among each group of tumor cells after transfection.6. Western blot analysis was used to determine the effect of CD151-integrin complex on tumor cell progression associated molecular signaling pathways.7. Examine the association of CD151and its partner integrin in human lung squamous cancers tissues and its adjacent tissues in20patients.8. Constructed BALB/C-NULL xenografts models to learn the definitive role of CD151-integrin complex in tumor cell progression, survival, metastasis and invasion in vivo.Results1. Successfully constructed recombinant plasmid RAAV-CD151, mutant plasmid RAAV-CD151-QRD194-196-CD151-AAA194-196, and negative control plasmid RAAV-GFP. And extracted a large number of these three kinds of plasmids via plasmid highpure kit.2. Successfully designed and synthesized small molecule interfering RNA specific for CD151, CD151-SIRNA, and also synthesized out of order SIRNA as negative control.3. Successfully transfected the prepared plasmid DNA, and small interference RNA into HepG2cells, A549cells, and Hela cells regulating CD151expression.4. In the mutant plasmid RAAV-CD151-QRD194-196-CD151-AAA194-196transfection group the combination between CD151and integrina3, a6, β1subunits was disrupted.5. CD151-integrin complex effectively promoted tumor cells progression, migration and invasion, losing the contact with integrin, the function of CD151was diminished. Together, specific inhibited CD151expression, all the biological behaviors was inhibited.6. CD151-integrin complex had markedly induced the phosphorylation activation of focal adhesion kinase (FAK), and finally upregulated phosphorylation activation of pathway. In the mutant transfection group and the CD151-SIRNA transfection group these functions of CD151were diminished.7. Real time PCR and western blot analysis showed higher expression of CD151protein in20paired human lung squamous cancer tissues compared with the adjacent nontumor tissues. It was also the same in4paired human colon cancer tissues. The majority of CD151located at the cell-cell contacts as a stable complex with α3β1in these tissues.8. Successfully constructed BALB/C-NULL xenografts models, CD151-integrin located at the cell-cell contacts as a stable complex with α3βl in tumor tissues, and effectively promoted tumor cells progression, survival, metastasis and invasion, losing the contact with integrin, the function of CD151was diminished.ConclusionCD151promotes tumor cells proliferation, survival, metastasis and invasion in vitro and in vivo. It also induced the phosphorylation activation of focal adhesion kinase (FAK), and finally upregulated phosphorylation activation of Src-p130CAS pathway. Not only Losing the contact with integrins but also specific inhibiting endogenesis CD151expression, the function of CD151was remarkablely diminished.