Construction Of Gastric Cancer MGC803 Cell Line Which Is Transfected By Cbl-b ShRNA

ObjectConstruction of targeting Cbl-b gene short hairpin RNA (shRNA) eukaryotic plasmid expression vector, and transfected into gastric cancer cells MGC803 using G418 screening, the establishment of Cbl/Cbl-b shRNA stable transfection of MGC803 cell lines and for testing. With this stable transfection of gastric cancer cell lines MGC803 conduct Cbl-b regulate EGFR signal transduction pathway related researchMethods1,Design PrimerAccording to Cbl-b gene sequences, through the software design, three target sequences, using BLAST homology analysis, design of Group 2 Cbl-b shRNA sequence and a group and the control target sequences. shRNA sequences are:BamH I restriction site+anti-sense sequence+loop+justice sequence+TC+Hind III and BamH I restriction site+justice, anti-sense sequence+loop+serial+AG+Hind III.2,Construct Cbl-b ShRNA plasmidSelected pRNA-U6.1/Neo (Genscript, Piscatcway) as a carrier. Synthesis of shRNA sequence of chemical synthesis will be paired single-stranded synthetic double-stranded, using Bam-HI/Hindlll restriction enzyme digestion, with its T4 ligase to the same through the use of Bam-HI/Hindlll restriction endonuclease enzyme digestion within the post-pRNA-U6.1/Neo carrier. Will build into the to the feelings of state,αexpression plasmid was transformed into strain DH5 you will bacilli coating on the LB plate containing ampicillin, screening positive colonies3,SequencingConstruction completed on the plasmid were sequenced by the test results can be seen that three sections of primers were successfully inserted into plasmid inside. Confirmed by sequencing after a large number of replication, amplification, extraction of recombinant plasmid.4,TransfectionContaining 10%newborn calf serum in DMEM culture medium MGC803 cultured gastric cancer cells. Will be exponential growth in the transfected cells 24 hours before, per-hole density of 10X104 cells then seed 48-well plate, when the cells reached 85-95%of Lipofectamine 2000 in accordance with the instructions for the recombinant plasmid transfection.5,Western Blot Detection of transfected cells into the gene expressionCollected after transfection cells, the control plasmid transfected cell line and non-transfected cell line as control with Western Blot, select the expression rate of the control group of 10%or less of the cell lines were positive cell line.6,Detect four kinds of gastric cancer cell proliferatbn with MTTTo take non-transfected gastric cancer MGC803 cells after transfection of the blank control, Cbl-b gene silencing MGC803 gastric cancer cells to MTT test.7,Detecte the expression of the p-ERK and p-AKT in silent Cbl-b cell linesCollected three kinds of transfected cells and non-transfected gastric cancer cells with Western Blot test,applicate AKT antibody and ERK antibody. Compare changes in the expression of AKT and ERK.Results1,Design pRNA-U6.1/Neo-cbl target sequence and blank controlsequenceThe results showed that Cbl-b 414,852, and blank control template is built on the success of pRNA-U6.1/Neo vector, sequence entirely correct, and purpose of the same sequence.2,SequencingThe results show that shRNA414,852, and blank control template is built on the success of pRNA-U6.1/Neo vector, sequence entirely correct, and purpose of the same sequence.3,Screening blank control group and Cbl-b gene silencing cell lines of r MGC803 gastric canceThe use of Western Blot testing, selection and non-transfected MGC-803 cells, Cbl-b expression levels of the same, blank control group transfected cells are cells in the control group, select the level of Cbl-b expression in non-transfected group below 10% of the cell line for the Cbl-b gene silencing cell lines.4,The proiferation of control group and Cbl-b gene silencing of MGC803 gastric cancer cellBy conducting MTT test was found after 24h in cell proliferation, Cbl-b gene silencing gastric MGC803 cells (shRNA414, shRNA852) proliferation has quickened, with the blank control and the non-transfected cells, a significant difference.5,The impaction of slience Cbl-b in the p-ERK and p-AKTThrough the Western Blot test, in the Cbl-b gene silencing cells, p-AKT expression compared with non-transfected cells and control cells were significantly increased in the blank. In the p-ERK expression, the four kinds of cells, no significant difference.Conclusion1,Construction of ubiquitin ligase Cbl-b ShRNA plasmid2,Establishment of Cbl-b expression was significantly reduced after the intervention MGC803 of gastric cancer cell lines. To further study the Cbl-b and the EGFR signaling pathway lay the foundation for the relationship.3,In Cbl-b gene silencing in MGC803 gastric cancer cells, EGFR pathway downstream AKT protein's expression increased.