Establishing MDA-MB-231pcDNA3.1/syk(s) Cell Lines Of Breast Cancer And Study Of The Proliferation On The Cell

Malignant tumors are a kind disease which seriously imperil the health aswell as lives of human beings.Tumorigenesis is a multifactor, multistep,polygene involved process that consists of complex pathological changes, itsnature is reflected in the genetic changes,such as activation of oncogenes andinactivation of tumor suppressor gene and changement of their correspondingprotein products,which led to appear Continuing differentiation,autonomousproliferation, apoptosis inhibition,eventually caused tumorigenesis,progression, invasion and metastasis. More than50%of proto-oncogene andoncogene proteins have been characterized with the protein-tyrosine kinase(PTKs) activity, and Spleen tyrosine kinase (Syk) is a non-receptor typeprotein tyrosine kinase, its has two isomers,full-length of spleen tyrosinekinase (Syk(L))and the short form of spleen tyrosine kinase(Syk(S)). Syk(S) isidentical to Syk(L), except that it lacks23amino acid residues (deletion)within the interdomain B (IDB) of Syk, but their function could not be moredistant. Studies suggest that Syk(L) act as inhibiting effect on the growth andmetastasis of breast cancer, gastric cancer, lung cancer, liver cancer, and itshas also become increasingly clear about mechanism and therapeutic target invitro studies. However, the studies on Syk(S) are still inconclusive,somescholars think it may has a contribution to mammary tumor progression.Objective: This experiment aided to establish a breast cancer cell linesstably expressing Syk(S) protein, and explore the effects of Syk(S) gene onproliferation of breast cancer.Methods:1Syk(S) gene restructuring plasmid amplification, then transfected intoMDA-MB-231cell and formed a breast cancer cell lines stably expressing Syk(S) gene. 1.1Plasmid amplification and transfection: A plasmid containing Syk(S) genewas transformed into E.coli.DH5α and amplified. Then transfected the Syk(S)plasmid into breast cancer cell MDA-MB-231by lipofectin medium, afterscreened with G418,we establish a new breast cancer cell lines with stableexpressing Syk(S)(MDA-MB-231pcDNA3.1/Syk(S)), Using MCF7cells aspositive control，MDA-MB-231pcDNA3.1cells which stably transfectednegative plasmid and MDA-MB-231cell lines as negative controls.1.2Verifiy the stable cell line: The controlled groups:MDA-MB-231cellsgroup，MDA-MB-231pcDNA3.1cells group which stably transfectednegativeplasmid; The experimental group:MDA-MB-231pcDNA3.1/Syk(S) cellsgroup which stably transfected Syk(S) plasmid.Using Immunohistochemistry,Western blot methods to detect the expression of Syk(S) protein.2Study of cell cycle and apoptosis of effects of the stably transfection cell.2.1The groups of the experiment: The controlled groups:MDA-MB-231cellsgroup,MDA-MB-231pcDNA3.1cells group; The experimental groupgroup:MDA-MB-231pcDNA3.1/Syk(S) cells group.2.2Using flow cytometry analyses the different group cell cycle andapoptosis,explores the Syk(S) influence on proliferation and apoptosis ofhuman breast cancer cells.Results:1The Syk(S) plasmid was amplificated successfully and transfected intobreast cancer cell, formed MDA-MB-231pcDNA3.1/Syk(S)cell lines, afterscreened with G418(400μg/mL)3week, still had survived cell and continuedto selecting,amplification,picking clone, got20strains stable expression Syk(S)gene of clone. Cells had not changed significantly.2Results of Immunohistochemical show that:Anti-syk polyclonalantibody results: the experimental group MDA-MB-231pcDNA3.1/Syk(S)show cytoplasmic brown granules, transfected negative plasmid groupMDA-MB-231pcDNA3.1and MDA-MB-231cell cytoplasm, nucleus havenot stained; positive control MCF7cells show visible brown granules in thecytoplasm and the nucleus. Syk(L) monoclonal antibody results： the experimental group and the controlled group do not show brown granules inthe cytoplasm and the nucleus.3Results of Western blot show that: Compared to the controlled group,the experimental group MDA-MB-231pcDNA3.1/Syk(S) cell lines haveexpression of Syk(S) protein fromⅠto Ⅳgeneration4Results of Flow cytometry detection about apoptosis show that: the rateof apoptosis in the experimental group MDA-MB-231pcDNA3.1/Syk(S) cells(A)0.35士0.32%is less than the MDA-MB-231pcDNA3.1cells (B)2.54士1.10%and MDA-MB-231cells (C)3.32士1.63%, there were statisticalsignificances in the comparison of the rate of apoptosis (P<0.05).5Results of Flow cytometry detection about cell cycle show that: G0/G1phase29.77%,S phase43.72%in the experimental groupMDA-MB-231pcDNA3.1/Syk (S) cells, while G0/G1phase39.73%, S phase25.53%in MDA-MB-231pcDNA3.1cells group; G0/G1phase38.93%, Sphase21.23%in MDA-MB-231cells, After transfected Syk(S),thepercentage of G0/G1phase was significantly decreased, S phase increased,there were statistical significances(P<0.05), but there were no significantdifferences about G2/M phase between three groups (P＞0.05).Conclusion:1Successfully established of a breast Cell Line named asMDA-MB-231pcDNA3.1/Syk (S) whose Syk (S) gene is stably expressed.2Syk (S) will promote the cell from G0/G1phase into S phase,whichresult in increasing cell proliferation;at the same time, Syk (S) will inhibitapoptosis to promote the growth of breast cancer cells.