Study On The Effect Of Atf4 On Biological Behavior Of Bladder Cancer Cells By Crispr/cas9

Objective: to study on the association of ATF4 gene with bladder cancer stage that by immunohistochemistry.Further construction of ATF4 Knockout T24 cells line by CRISPR/cas9,to study the of ATF4 gene on the biological behavior of bladder cancer cells.Methods:(1)Using immunohistochemistry,61 cases of different stage bladder cancer tissues were stained to study the expression of ATF4 in bladder cancer tissues with differ stages.(2)To design two pair of gRNA in the exon region of ATF4 by CRISPR/cas9.PX459 vector plasmid was digest by BbsI enzyme and the gRNA was inserted into the PX459 vector plasmid.The constructed PX459-ATF4-gRNA was constructed.The vector plasmid was transfected into T24 cells by liposome,to knocked out the exon regions of ATF4,the knockout effect was verified by PCR,agarose gel electrophoresis,DNA sequencing and Western-blot.(3)Transwell assay was used to detect whether T24 cells changed the migration and invasion ability of ATF4 gene knockout.Clone formation experiments were performed to test the ability of T24 cells to colonize before and after knockout.Results:(1)In immunohistochemistry experiment,we found that the stage of bladder cancer tissue,the expression of ATF4 more higher.(2)The PCR,agarose gel electrophoresis and DNA sequencing showed that the gRNA was successfully inserted into PX459 vector plasmid.The T24 cells transfection with the PX459-ATF4-gRNA vector plasmid were subjected to PCR agarose gel electrophoresis,the target band was found to be shorter that is similar to expected results,DNA sequencing also showed that sequence became shorter,Western-blot clearly showed complete deletion of the ATF4 gene.There results indicate that T24 cells stably knocked out of the ATF4 gene were successfully constructed.(3)ATF4 Knockout cell experiments showed that ATF4 gene silencing inhibited the T24 cell clone formation ability(P<0.05),The T24 cell migration and invasion ability was decreased(P<0.05)Conclusion(1)The higher the stage of bladder cancer that the ATF4 gene was higher the expression.(2)T24 bladder cancer cells stably knocked out ATF4 gene were successfully constructed.(3)The T24 cell with Knockdown of ATF4 gene that the formation of clones,migration and invasion will be decreased.