胱硫醚β合成酶在前列腺癌中的异常表达及其对前列腺癌细胞增殖的调控作用

Abnormal expression of cystathionineβsynthase in prostate cancer and its regulation on cells proliferation

目的:探讨胱硫醚β合成酶(CBS)在前列腺癌中的差异表达情况,及其对前列腺癌细胞增殖的调控作用。方法:利用GEPIA网站分析来源于TCGA和GTEx数据库的492例前列腺癌组织和152例正常组织中CBS mRNA表达情况。利用ULCAN网站分析来源于TCGA数据库的497例前列腺癌组织和52例正常组织中CBS mRNA的表达情况。用RPIM-1640培养基培养前列腺癌细胞系DU145、PC3、LNCaP和C4-2,免疫印迹实验检测CBS蛋白表达情况。shRNA转染DU145细胞,分为对照shRNA组、CBS shRNA#1组或#2组,克隆形成实验检测细胞克隆形成数量,BrdU标记实验检测细胞增殖能力,免疫印迹实验检测AKT、mTOR、S6K蛋白表达情况,以及AKT S473位点、mTOR S2448位点、S6K T421/S424位点的磷酸化水平。将稳定表达对照shRNA、CBS shRNA#1或#2的DU145细胞注射入小鼠皮下,建立小鼠前列腺癌移植瘤模型,观察肿瘤生长情况。结果:数据库分析结果显示,与正常前列腺组织比较,前列腺癌组织中CBS mRNA的表达水平显著上升。CBS蛋白在上述前列腺癌细胞系中均有表达,其中在DU145细胞中表达水平最高。与对照shRNA组相比,CBS shRNA#1组或#2组的克隆形成数量显著减少[(23±2.309)个vs(5.667±1.453)个,(6.333±1.856)个],BrdU标记信号强度显著下降(211.3±25.31 vs 90±10.6,113±15.39)。小鼠模型显示,与稳定表达对照shRNA的DU145细胞构建的移植瘤对比,表达CBS shRNA#1或#2的DU145移植瘤生长受到抑制,体积显著减小,第27天时体积分别为(840.4±37.48)mm^(3)vs(415.9±34.88)mm^(3),(297.6±25.74)mm^(3)。shRNA转染的DU145细胞中,与对照shRNA对比,CBS shRNA#1组和#2组AKT、mTOR、S6K蛋白的表达水平无明显变化,AKT S473位点、mTOR S2448位点、S6K T421/S424位点的磷酸化水平显著下降。结论:CBS在前列腺癌中高表达,沉默CBS表达可抑制前列腺癌细胞增殖,以及抑制AKT、mTOR、S6K蛋白的活化。

Objective:To investigate the expression pattern of cystathionineβsynthase(CBS)in prostate cancer cells,and determine the impact of CBS on the proliferation of prostate cancer cells as well as the related mechanism.Methods:The CBS mRNA expression of 492 prostate cancer tissues and 152 normal tissues obtained from TCGA and GTEx databases was analyzed by the GEPIA tool.The CBS mRNA expression of 497 prostate cancer tissues and 52 normal tissues obtained from TCGA was analyzed by the ULCAN tool.Prostate cancer cell lines DU145,PC3,LNCaP,and C4-2 were cultured in RPIM-1640 medium,and Western blot was used to detect the expression of CBS protein.DU145 cells were transfected with Control shRNA,CBS shRNA#1 or#2,respectively.The proliferation of DU145 cells was determined by colony formation assay and BrdU labeling assay.Western blot was used to measure the protein expression of AKT,mTOR and S6K,and the phosphorylation levels of AKT S473,mTOR S2448 and S6K T421/S424.DU145 cells stably expressing Control shRNA and CBS shRNA#1 or#2 were subcutaneously injected into mice to establish a prostate cancer xenograft model and the tumor growth was observed.Results:Analysis of the online protein expression database revealed that the expression of CBS mRNA in prostate cancer tissues was significantly increased compared to normal prostate tissues.CBS protein was expressed in all measured prostate cancer cell lines with the highest in DU145 cells.Compared with Control shRNA group,the average number of clones formed in CBS shRNA#1 or#2 groups was significantly decreased by colony formation assay(23±2.309 vs 5.667±1.453,6.333±1.856),and the intensity of BrdU labeling signal in CBS shRNA#1 or#2 groups was significantly decreased by BrdU labeling assay(211.3±25.31 vs 90±10.6,113±15.39).Mouse xenograft model showed that treatment with CBS shRNA#1 or#2 significantly reduced the growth rate of xenografts.On day 27,the average volume of Control shRNA,CBS shRNA#1 or#2 groups was(840.4±37.48)mm^(3) vs(415.9±34.88)mm^(3),(297.6±25.74)mm^(3),respectively.Compared with Control shRNA group,there was no obvious change in the expression level of AKT,mTOR,S6K proteins in CBS shRNA#1 or#2 groups,but the phosphorylation level of AKT S473,mTOR S2448 and S6K T421/S424 were significantly decreased.Conclusion:CBS expression was upregulated in prostate cancer.Knockdown of CBS inhibited the proliferation of prostate cancer cells,and inhibited the phosphorylation of AKT,mTOR and S6K.