【摘要】 目的体外实验研究PLCε基因调节人膀胱癌细胞迁移和侵袭的分子机制。方法设计并合成针对PLCε基因mRNA的寡核苷酸序列,构建重组腺病毒Ad-sh PLCε。T24细胞感染Ad-sh PLCε腺病毒后,用Western blot检测PKCα/β及TBX3、E-cadherin的表达;用划痕实验、Transwell迁移和侵袭实验检测膀胱癌细胞T24的迁移、侵袭能力。结果 Ad-sh PLCε重组腺病毒感染膀胱癌细胞T24,对其PLCεmRNA表达抑制率为75.6%,对其PLCε蛋白表达抑制率为67.4%。Ad-sh PLCε感染组T24细胞迁移能力较空载组和空白对照组明显减弱(P<0.05);重组腺病毒Ad-sh PLCε感染组T24细胞穿膜细胞数较空载组和空白对照组明显减少(P<0.05)。Ad-sh PLCε重组腺病毒感染膀胱癌T24细胞后下游PKCα/β的活化受到抑制(P<0.05),同时,TBX3的表达减少,而E-cadherin的表达增高(P<0.05)。结论通过以重组腺病毒干扰抑制PLCε基因,能有效抑制膀胱癌T24细胞系中PLCε下游PKCα/β的活化情况及TBX3,E-cadherin的表达变化,并且对T24细胞的迁移、侵袭能力具有一定的抑制作用。更多还原

【Abstract】 Objective To investigate the molecular mechanisms of PLCε in regulating the invasion and migration of human bladder cancer cells in vitro. Methods After cells treated with recombinant adenovirus,the migratory / invasive abilities of T24 cells were explored by wound healing and Transwell chamber cell migration and invasion assay; RT-PCR was used to detect the mRNA levels of PLCε; The protein levels of PLCε,PKCα,PKCβ,TBX3 and E-cadherin were determined by Western blot; QRT-PCR was used to detect the mRNA levels of TBX3 and E-cadherin. Results It was confirmed by digesting and sequencing that the recombinant adenovirus had been constructed successfully. The expression of PLCε mRNA and PLCε protein were both decreased after the infection of Adsh PLCε. Wound healing and Transwell chamber cell migration / invasion assay showed that Ad-sh PLCε treatment could inhibit the migratory and invasive activity of bladder cancer cells( P < 0. 05). The results of Western blotindicated that the expression of PKCα / β in membrane decreased( P < 0. 05),and phosphorylation level of PKCαand PKCβ was reduced. QRT-PCR and Western blot analysis demonstrated that the expression level of TBX3 decreased,but the expression level of E-cadherin increased. Conclusions PLCε shRNA can inhibit migratory and invasive ability of bladder cancer cells through PKCα / β / TBX3 / E-cadherin pathway.更多还原