转染tbx2 siRNA对人前列腺癌细胞增殖、迁移、侵袭及上皮-间质转化的影响

摘要

目的 观察转染T-BOX转录因子2(tbx2)小干扰RNA(siRNA)对人非雄激素依赖性前列腺癌PC3细胞和雄激素依懒性前列腺癌LNCaP细胞增殖、迁移、侵袭及上皮-间质转化(EMT)的影响.方法 将体外培养的人前列腺癌PC3细胞随机分为观察组和对照组,通过SiLentFectTM Lipid Reagent技术分别转染tbx2 siRNA和阴性对照siRNA,继续培养48 h.采用Western blotting法检测两组细胞中tbx2蛋白相对表达量.采用细胞计数试剂盒(CCK-8)法检测两组细胞增殖活性(以OD450表示).采用Transwell迁移实验检测两组细胞迁移细胞数.采用Transwell侵袭实验检测两组细胞侵袭细胞数.采用Western blotting法检测两组细胞EMT相关标志物E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、纤维黏连蛋白(Fibronectin)及EMT相关上游转录因子Snail、Twist的表达.另取LNCaP细胞进行相同实验.结果 转染48 h后观察组、对照组PC3细胞中tbx2相对表达量分别为0.345±0.016、0.723±0.031,LNCaP细胞中tbx2相对表达量分别为0.315±0.006、0.692±0.010.观察组PC3、LNCaP细胞中tbx2相对表达量均低于对照组(P均<0.05).将转染后的两种前列腺癌细胞继续培养24、48、72 h后,PC3细胞对照组OD450值分别为0.487±0.026、0.868±0.031、1.387±0.107,观察组分别为0.315±0.022、0.550±0.027、0.765±0.046.LNCaP细胞对照组OD450值分别为0.388±0.032、0.722±0.032、1.187±0.135,观察组分别为0.251±0.021、0.450±0.037、0.625±0.066.相同时间点观察组PC3、LNCaP细胞OD450低于对照组(P均<0.05).观察组与对照组PC3细胞迁移细胞数分别为(118.67±6.11)、(292.33±5.03)个;LNCaP细胞迁移细胞数分别为(85.67±4.31)、(192.33±8.07)个.观察组PC3、LNCaP细胞迁移数目均低于对照组(P均<0.05).观察组,对照组PC3细胞侵袭细胞数分别为(89.00±3.02)、(186.33±5.84)个;LNCaP细胞侵袭细胞数分别为(81.67±3.42)、(232.33±7.42)个.观察组PC3、LNCaP细胞迁移数目均低于对照组(P均<0.05).观察组PC3、LNCaP细胞E-adherin表达高于对照组(P均<0.05);N-cadherin、Vimentin、Fibronectin及Snail、Twist相对表达量低于对照组(P均<0.05).结论 转染tbx2 siRNA可以抑制细胞增殖,降低细胞迁移及侵袭能力,抑制细胞EMT过程.%Objective To observe the effects of transfection of tbx2 siRNA on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of human non-androgen dependent prostate cancer cells PC3 and androgen-dependent prostate cancer cells LNCaP.Methods Human prostate cancer PC3 and LNCaP cells cultured in vitro were randomly divided into the observation group and the control group, which were transfected with tbx2 siRNA and negative control siRNA by SiLentFectTM Lipid Reagent, respectively, and then were cultured for 48 h.The protein expression of tbx2 in cells of the two groups was detected by Western blotting.The proliferative activity (OD450) was detected by using cell counting Kit (CCK-8).Transwell migration and invasion assay were performed to detect the migration and invasion cells of both groups.The expression of EMT-related markers including E-cadherin, N-cadherin, Vimentin, and Fibronectin, and EMT-related upstream transcription factors Snail and Twist, was detected by Western blotting.We did the same experiments for LNCaP cells.Results Tbx2 siRNA was successfully transfected into two kinds of prostate cancer cells.The expression levels of tbx2 cultured for 48 h in PC3 cells of the observation group and control group were 0.345±0.016 and 0.723±0.31, respectively, and they were 0.315±0.006 and 0.92±0.10, respectively, in LNCaP cells.The relative expression of tbx2 in PC3 and LNCaP cells of the observation group was lower than that in the control group (both P<0.05).After being cultured for 24, 48, and 72 h, the OD450 of PC3 was 0.487 0.026, 0.868 0.031, and 1.387±0.107, respectively, in the control group, versus 0.315±0.022, 0.550±0.027, and 0.765±0.046 in the observation group.The OD450 of LNCaP was 0.388±0.032, 0.722±0.032, and 1.187 0.135, respectively, in the control group, versus 0.251±0.021, 0.450±0.037, and 0.625±0.066 in the observation group.The OD450 of PC3 and LNCaP cells in the observation group was lower than that in the control group, respectively (both P<0.05).The number of migration cells in PC3 cells was 118.67±6.3 and 292.33±5.03, versus 85.67 4.31 and 192.33 8.07 in LNCaP cells, respectively, in the observation group and the control group.The number of migration cells of PC3 and LNCaP in the observation group was lower than that in the control group, respectively (both P<0.05).The number of invasion cells in PC3 cells was 89.00±3.02 and 186.33±5.84, versus 81.67±3.42 and 232.33±742 in LNCaP cells, respectively.The number of invasion cells of PC3 and LNCaP in the observation group was lower than that in the control group, respectively (both P<0.05).The expression of N-cadherin, Vimentin, Fibronectin, Snail, and Twist in PC3 and LNCaP cells of the observation group was significantly lower, while the expression of E-cadherin protein was higher than that in the control group (all P<0.05).Conclusion Silencing tbx2 gene can inhibit the proliferation, decrease the abilities of migration and invasion, and inhibit EMT process in prostate cancer cells.