Effect Of Changes In The Expression Of Cytoskeleton Protein Coronin3 On The Activity Of U87 Glioma Cells

Objective This study intends to explore the effect of changes in coronin 3 expression on the motility of U87 glioma cells by in vitro cell experiments.Methods(1)Construction and identification of p EGFP/coronin 3 eukaryotic overexpression vector:Total RNA of U87 glioma cells was extracted using Trizol reagent under sterile and RNase-free conditions.The coronin 3 gene fragment was amplified by reverse transcription-polymerase chain reaction(RT-PCR).After the agarose gel electrophoresis,the PCR product was recovered and purified,and the E.coli plasmid was extracted.The product and blank plasmid were recovered by agarose gel electrophoresis.In the pEGFP-N2,the target gene and the plasmid were simultaneously digested with Xho I and Bamh I.The two enzyme digested products were ligated with solution I ligase and transformed into competent E.coli DH5?.The LB agar plate was cultured overnight.After screening by colony PCR,the positive clones were extracted and sent to Kunming ShuoQing Biotechnology Services Co.Ltd.for sequencing.The sequencing results were analyzed using BLAST software and Gene Bank database for homology analysis.(2)Transfection of U87 cells and expression and detection of the recombinant gene p EGFP/coronin 3 protein and GFP:The purified recombinant plasmid coronin p EGFP/coronin 3 was transfected into U87 cells by lipofectamine viafect.After 24 hours of transfection,cells were collected and Western blot was used to identify the expression of recombinant coronin 3 in U87 cells.(3)The U87 cell line containing the recombinant plasmid p EGFP/coronin 3 and the U87 cell line containing the empty plasmid GFP and the wild-type U87 cell line were subjected to the cell scratch migration assay and the cell migration experiment,and the coronin 3 was ascertained by comparing the two groups.Effects of changes in expression on the exercise capacity of U87 cells.Results(1)U87 cell cDNA was successfully obtained by reverse transcription polymerase chain reaction.Five recombinant eukaryotic expression vector clones were randomly selected and four positive clones were selected by colony PCR.Sequencing and Blast analysis confirmed that the recombinant plasmid was successfully constructed.(2)After 24 hours of transfection of U87 cells with liposome,immunofluorescence and Western blot were used to identify the expression of recombinant coronin protein in U87 cells.A positive band appeared at 85 kDa.(3)The cell scratch test,recombinant plasmid group,empty plasmid group and non-treated group were three groups.After 24 hours,the recombinant plasmid group cell migration distance(29.85�2.28),the empty plasmid group the cell migration distancewas(16.11�1.80),After 24 hours,the no treatment cell migration distance was(14.76�1.80).Each group was compared with each other,Between empty plasmid group and the recombinant plasmid group,P<0,the difference was statistically significant.Between empty plasmid group and non-treated group,P>0.05,the difference was not statistically significant.The number of cells passing through the cell membrane of the recombinant plasmid group was(93.60�9.02)and the number of cells passing through the cell membrane of the empty plasmid group cells(55.20�5.20)The number of cells passing through the cell membrane in the untreated group was(51.70�7.28).Comparing the three groups,the recombinant plasmid group and the empty plasmid group,and between the recombinant plasmid group and the non-treated group,the difference between P<0.01 was statistically significant,the empty plasmid group was compared with the non-treated group,P>0.05,the difference was not Statistical significance.That is,compared with the empty plasmid group and the non-treated group,the cells of the recombinant plasmid group have significantly improved the ability of cell migration.Conclusion(1)The recombinant eukaryotic expression vector pEGFP/coronin 3 was successfully constructed with U87 recombinant p EGFP/coronin 3 gene.After transient transfection of U87 cells,recombinant coronin 3 protein was expressed.(2)The increased expression of coronin 3 in cells can promote the migration of U87 gliomas.