Effects And Mechanism Of Integrin Linked Kinase Gene Silencing By RNA Interference In Pancreatic Cancer PANC-1 Cells

ObjectiveSmall interfering RNA (siRNA) targeting ILK was constructed and transfected into PANC-1 cells. Effects of ILK knockdown on cell proliferation and mechanism were investigated. It will provide theoretical basis to study the pathogenesis and treatment of pancreatic cancer.MethodsDesign three pairs of genes for ILK small interfering RNA (small interfering RNA, siRNA) sequences, and synthesis of two small hairpin RNA (small hairpin RNA, shRNA) of single-stranded DNA template with the sequence of, while designing two different restriction sites at the respective ends of the template chain. Annealed to form siRNA vector inserted fragments. Linearize siRNA empty vector with a restriction enzyme, insert the fragments into an empty siRNA plasmid with T4 ligase.Tto identify whether the construction of recombinant plasmid.is successful by PCR and sequencing.The recombinant plasmid and the negative control plasmids were transformed into Panc-1 cellsby cationic liposome Lipofectamine. Observe the plasmid expression efficiency by fluorescence microscopy after G418 selection for 4 weeks. Selection positive monoclonal,continue to expand under the pressure of G418, observe the plasmid expression efficiency by flow cytometry and fluorescent microscopy plasmid.Detect the expression of ILK mRNA level in PANC-1 cells by Semi-quantitative RT-PCR. Detect the expression of ILK gene protein in PANC-1 cells by Western-Blot.In the end filter out the most interference efficient plasmid.MTT test:each of about 2,000 cells were put in 96 cell culture dishes at 37℃,5%CO2 of incubator. Take out cells respectively in Oh,24h,48h,72h and 96h, each well was added l0μL MTT dye, cubated in 37℃,5%CO2 for 4 h and centrifuged, then added DMSO 150μL/hole. Measure optical density (OD) values at the wavelength of 570nm,with enzyme-linked immunosorbent detector (Elx-800 type) to compare three groups of cell growth.ResultsRecombinant plasmid Results:recombinant plasmid was successfully constructed by PCR and DNA sequencing identification.Interfere efficiency results:ILK expression was effectively suppressed(the interference efficiency was 74.61%, ILK protein inhibition efficiency was 54.66%) after the recombinant plasmid was transfected into Panc-1 cells.MTT results:in the 0,24 h, the cells of A570 values of the three groups have no difference; at 48,72 h and 96h, the of A570 value of transfected plasmid Panc-1 cells was significantly reduced than the negative control plasmid, and the nomal Panc-1 cells (P<0.01).ConclusionsILK interference plasmid was successfully constructed. ILK siRNA expression plasmid could effectively inhibit the expression of ILKin Panc-1 cells. Meanwhile, Panc-1 cell proliferation was increased.It reveals that ILK gene and malignant behavior of pancreatic cancer cells is closely related and ILK will take as a target for treatment of pancreatic cancer.RNA interference is an effective means to gene therapy.