Downregulation Of Mir-29c In Human Bladder Cancer And The Inhibition Of Proliferation In T24Cell Via Pi3k/akt Pathway

Objective:To detect the expression of miR-29c in bladder transitionalcell carcinoma and analysis the relationship between miR-29c and clinicalpathological paramerters.Methods: QRT-PCR was used in30pairs of bladder cancer tissuescompared with normal tissues. Ta stage:5,T1:15,T2-4:10.Based on themedian value (0.3496) of the expression of miR-29c in bladder can-certissues, the patients were divided into two groups:15with low miR-29cexpression and15with high miR-29c expression.Results:The expression of miR-29c was downregulated in bladdercancer tissues vs normal tissues (p<0.01).Also, the low level expression of miR-29c was associated with tumor stage (P<0.01).Conclusion: The expression of miR-29c was downregulated in bladdercancer tissues. Objective: To construct miR-29a/c gene recombinant adenovirus andidentify its expression in bladder cancer T24cells.Methods: the PCR product containing miR-29a/c was amplified fromhuman genomic DNA and inserted into the adenoviral shuttle vectorpAdTrace-TO4-CMV. Then, the ecombinant shuttle plasmid linearized bypmeI was co-transformed into competent E. coli. BJ5183with theadenoviral backbone plasmid pAdEasy-1. Then, the recombinantadenoviral DNA was transfected into HEK293cells, packed and amplifiedmiR-29a and miR-29c adenoviruses. T24cells were infected byAd-miR-29a and Ad-miR-29c. The expression of mature miR-29a/c wasdetected by Real-time PCR.Results: miR-29a and miR-29c sucessfully cloned and reconbinant adnoviral plasmid constructed wich conformed by restriction endonucleaseand PCR. Real-time PCR showed that miR-29a and miR-29c significantlyup-regulated in T24cells infected with Ad-miR-29a and Ad-miR-29c(P<0.01).Conclusion: miR-29a and miR-29c adenovirus constructedsuccessfully and the expression of those in bladder cancer T24cells wasconfirmed. Objective: To study the molecular mechanisms of hepaCAM ininhibiting the proliferation of bladder transitional cell carcinoma in vitro.Methods: Cell proliferation was detected by CCK-8assay and fowcytometry. Cell motility was examined by trans-well assay. Cell apoptosiswas analyzed by fow cytometry. Western blot was used to measure theprotein leve of pAKT、t-AKT、p-GSK-3β、 t-GSK-3β、c-myc、cyclinD1. Results: ectopic overexpression of miR-29c can significantly inhibitthe proliferation, decrease motility, suppress the G1/S cell cycle transition,and induce apoptosis of T24cells. Furthermore, it can cause a decrease inAKT and GSK-3β phosphorylation. While LY294002reduced the proteinlevel of pAkt, the overexpression of miR-29c can further decrease its levelin T24cells which were pretreated with LY294002. Our study alsoindicated that the proliferation inhibition of T24may take place viaAKT-GSK3β pathway.Conclusion: miR-29c could be an emerge player in the disease stateof bladder cancer. It can also be a promising tumor suppressor in bladdercancer.