The Invasion And Migration Of Bladder Cancer Cells Through PKCα / β / TBX3 / E-cadherin Signaling Pathway

Objective: To investigate the molecular mechanisms of PLC inregulating the invasion and migration of human bladder cancer cells invitro.Methods: After cells treatment with different factors, themigratory/invasive abilities of T24and BIU-87cells were explored bywound healing and transwell chamber cell migration and invasion assay;QRT-PCR was used to detect the mRNA levels of TBX3and E-cadherin;The protein levels of PLC，PKC，PKCβ，p-PKC，p-PKCβ, TBX3andE-cadherin were determined by using western blot; IHC was used tomeasure the protein levels of PLC，PKC and PKCβ in bladder carcinomatissues.Results: Wound healing and transwell chamber cell migration/invasionassay showed that Ad-shPLC treatment can inhibit the migratory/invasiveactivity of bladder cancer cells. The results of western blot indicated that the expression of PKC/β in membrane was decreased, andphosphorylation levels of PKC and PKCβ were reduced. The results ofwestern blot and IHC showed that PLC-high-expressing tumors showedsignificantly higher positive rates of PKC/β cell membrane staining andphosphorylation levels of PKC/β than did PLC-low-expressing tumors inbladder tumors tissues. The results of western blot and QRT-PCR showedthat Ad-shPLC treatment can inhibit the expression of TBX3at mRNAand protein levels; however, the expression of E-cadherin was up-regulated.Combination treatment of pGenesil-shTBX3with Ad-shPLC, leading tothe partial enhancement of E-cadherin expression and cell migration; aftercells combination treatment of PKC/β inhibitor Go6976with Ad-shPLC,results exhibited that the expression of TBX3was decreased, theexpression of E-cadherin was increased and the migratory/invasive abilityof cells was obviously inhibited compared with Ad-shPLC treatmentgroup; In addition, Ad-shPLC cells were incubated with PMA,suppression of TBX3expression by Ad-shPLC treatment alone was partlyrestore, elevated E-cadherin by Ad-shPLC treatment alone was partlyreversed and prevented the migration/invasion inhibitory effect ofAd-shPLC.Conclusion: PLC shRNA can inhibit migratory/invasive ability ofbladder carcinoma cells through PKC/β/TBX3/E-cadherin pathway. ABSTRACTObjective: To study the effect of Ad-shPLC on human bladder cancerxenografts growth and identification of the mechanisms of PLC-shRNA onmigration and invasion of bladder cancer cells.Method: Ad-shPLC-infected T24cells, Ad-HK-infected T24cells andT24cells (3×106) were subcutaneously inoculated into three groups ofnude mice (three mice per group) for tumorigenesis experiments. Theweight and volume of nude mice was evaluated. After4weeks, all micewere sacrificed, the xenograft tumor weight was measured at the terminaltime and resected tumor tissues for HE straining andimmunohistochemistry examining PLC, ki67, p-PKC, p-PKCβ, TBX3and E-cadherin protein expression.Results: Bladder cancer xenograft tumors were established with T24 cells (3×106). Cells that were infected with Ad-shPLC showed areduction in the final tumor weight compared with control groups (P<0.05).In addition, immunohistochemistry indicated that the levels of PLC, ki67,p-PKC, p-PKCβ, TBX3expression in tumors formed fromAd-shPLC-infected T24cells were significantly down-regulated andE-cadherin was up-regulated with respect to the Ad-HK groups and controlgroups.Conclusion: In vivo, PLC shRNA could inhibit bladder cancergrowth, up-regulate E-cadherin and down-regulate ki67, p-PKC，p-PKCβand TBX3protein expression.