RNA Interference Of Grb2 On K562 Cell's Ability Of Invasion And Metastasis

Objective: Chronic myelognous leukemia (CML) is a malignant disease of proliferation which originated in the bone marrow multipotent,hematopoietic stem cell,the emergence of fusion protein bcr-abl is the main characteristics in genetic line of CML . Myeloid leukaemias in blast crisis arecharacterised by an excessive egress of cells from the marrow into peripheral blood and subsequently by infiltration into various tissues, multiple organ failure caused by the transfer of malignant tumors patients is an important factor in patient's death [1], prevention of tumor metastasis is one of the keys to the treatment of tumors in clinic. The growth factor receptor-bound protein-2(Grb2)close ties with many cancer . In chronic myelognous leukemia , the Grb2 binding with bcr-abl fusion protein directly or through SHC , activating of RAS signaling pathway , leading to malignant transformation of hematopoietic cells. It is great significance that Grb2-related signal transduction pathway is blocked in treatment of CML . In this study, we approach whether inhibiting of Grb2 could impact K562 cells's invasion and metastasis and analyze the likely factors. It provides new ideas for the gene therapy of invasion and metastasis in oncosis.Methods: 1.Construct siRNA prokaryotic expression vector targeting of Grb2 gene.Four pairs of complementary siRNA oligonucleotide chains were synthesized according to the sequence of Grb2 in GenBank and cloned into the vector pSilencer4.1-CMV,then transformed into E.coli TOP10 , picked and amplified Ampicillin-resistant colonies, rapidly prepared a small amount of plasmid and confirmed DNA sequencing, its prokaryotic expression vector pSilence-4.1CMV- Grb2 was constructed successfully.2.Transfecting into K562 cells.Prokaryotic expression vector of pSilence -4.1CMV-Grb2 was stable transfected into chronic myelognous leukemia cells K562 by using lipofectamin protocols. After hygromycin selecting, K562/pSilence- 4.1CMV-Grb2 cells stable express siRNA. Using Western blot and RT-PCR methods to detect the expression of Grb2 on three group cells:transfect Grb2 siRNA recombinant plasmid group(K562/pSilence-4.1CMV-Grb2 cell),transfect pSilence-4.1CMV-Grb2 group (K562/pSilence-4.1CMV cell)and none transfected group (K562 cell).3.Cell's migration and invasion detection. Using migration and invasion experiment to detect the cells'migratory and invasive abilities.Results: Prokaryotic expression vector of pSilence-4.1CMV-Grb2 was constructed successfully. Prokaryotic expression vector of pSilence-4.1CMV-Grb2 can be stable transfected into chronic myelognous leukemia cells K562 by using lipofectamin protocols and be expressed.Compared with K562/pSilence-4.1CMV-Grb2 /541,K562/ pSilence-4.1CMV-Grb2/215,K562/pSilence-4.1CMV and K562 cells, K562/pSilence -4.1CMV-Grb2/405 cells lower express Grb2 by Western blot and RT-PCR methods. Compared with K562/pSilence-4.1CMV and K562 cells, the migratory and invasive abilities of K562/pSilence-4.1CMV-Grb2/405 cells have decreased by cell migration and invasion detection.Conclusions:Prokaryotic expression vector of RNAi could transfect into human chronic myelognous leukemia cells K562 by liposome stably and inhibit expression of the Grb2 protein and nucleic acid in K562 cells efficiently. The migratory and invasive abilities of K562 cells transfected recombinant plasmid reduced obviously.It has laid a solid foundation for further study of antineoplaston targeted to Grb2 in chronic myelognous leukemia.