目的：探究胱硫醚β合成酶（CBS）通过AKT/cyclin D1 通路影响肺腺癌A549 细胞的增殖及其分子机制。方法: 通过 cBioPortal 数据库分析肺腺癌组织中CBS 和AKT/cyclinD1 相关蛋白的表达，收集的国人肺腺癌组织通过免疫荧光法验证CBS 的 表达。WB 法检测A549 细胞及其移植瘤中半胱氨酸代谢和AKT/cyclin D1 通路相关蛋白的表达。将pCW57.1-myrAKT 质粒分 别与RNA 干扰对照质粒pGIPZ-CBS-nc-shRNA、干扰质粒pGIPZ-CBS-shNRA1 和pGIPZ-CBS-shNRA2 共转染A549 细胞，共转染 组又分多西环素（DOX）诱导组（DOX+）和不诱导组（DOX-），通过细胞集落形成实验检测转染后各组A549 细胞的集落形成能力。通过CBSwt A549 细胞、CBSI278TA549 细胞移植瘤实验检测过表达CBS 对移植瘤生长的影响，AzMC 荧光染色法和免疫组织化学法 分别检测移植瘤组织中H2S 水平和Ki-67 阳性率。结果：数据库分析和国人组织标本检测均显示，与癌旁组织比较，肺腺癌中 CBS 呈低表达（均P<0.01）；与SV-40 细胞比较，A549、SKMES1 和NCIH460 细胞中CBS 蛋白呈低表达（均P<0.01），而p-AKT 和 cyclin D1 蛋白均呈高表达（均P<0.01）。A549 细胞中过表达AKT 显著促进细胞集落的形成（P<0.01），在此基础上敲减CBS 可进 一步促进细胞集落的形成(均P<0.01)。移植瘤实验显示，与DOX- CBSWT 组比较，DOX+ CBSWT 组中CBS、RB 和P21 蛋白表达升 高、H2S 含量增加、肿瘤体积变小、Ki-67 阳性率降低（均P<0.01）；与DOX- CBS I278T 组比较，DOX+ CBSI278T 组中CBS 表达升高（P< 0.01），但H2S 水平和移植瘤体积变化均不明显。结论: 肺腺癌组织和A549 细胞中CBS 呈低表达，而AKT/cyclin D1 通路呈激活 状态，过表达或敲减CBS 可抑制或促进A549 细胞及其移植瘤的生长，CBS 和AKT/cyclin D1 通路相关蛋白可能是肺腺癌的潜在靶点和标志物。

Objective: To investigate the effect of cystathionine β-synthase (CBS) on the proliferation of lung adenocarcinoma A549 cells through the AKT/cyclin D1 pathway and its molecular mechanism. Methods: Expression of CBS and AKT/cyclinD1-related proteins in lung adenocarcinoma tissues was analyzed by cBioPortal database. The CBS expression in collected lung adenocarcinoma tissues from Chinese patients was verified by immunofluorescence method. WB assay was performed to detect the expression of cysteine metabolism and AKT/cyclin D1 pathway-related proteins in A549 cells and their transplanted tumors, and CBS DNA methylation sequencing was performed to detect the effect of its methylation on CBS mRNA expression in lung cancer cells. The pCW57.1-myrAKT plasmid was co-transfected with RNA interference control plasmid pGIPZ-CBS-nc-shRNA, interference plasmid pGIPZ-CBS-shNRA1 and pGIPZ-CBS-shNRA2, respectively, into A549 cells, which were further sub-divided into DOX+ and DOX-groups. The colony formation ability of A549 cells in each group after transfection was examined by cell colony formation assay. The effect of overexpression of CBS on the growth of transplanted tumors was examined by CBSwt A549 cell- and CBSI278TA549 cell-transplanted tumor assay, and AzMC fluorescence staining and immunohistochemistry were used to detect H2S levels and Ki-67 positivity in transplanted tumor tissues, respectively. Results: Both database analysis and tissue specimens showed that CBS was lowly expressed in lung adenocarcinoma tissues compared with para-cancerous tissues (all P<0.01); CBS protein was lowly expressed in A549, SKMES1 and NCIH460 cells compared with SV-40 cells (all P<0.01), while p-AKT and cyclin D1 protein levels were highly expressed (all P<0.01). Overexpression of AKT in A549 cells significantly promoted the formation of cell colonies (P<0.01), and knockdown of CBS on this basis further promoted the formation of cell colonies (all P<0.01). Transplantation tumor experiments showed that CBS, RB and P21 protein expression was elevated, H2S level was increased, tumor volume was decreased and Ki-67 positivity was reduced in the DOX+ CBSWT group compared with those in the DOX- CBSWT group (all P<0.01); CBS expression was elevated in the DOX+ CBSI278T group compared with the DOX- CBS I278T group (P<0.01), but the changes in H2S level and transplantation tumor volume were not significant. Conclusions: CBS is lowly expressed in lung adenocarcinoma tissues and A549 cells, while the AKT/cyclinD1 pathway is activated. Overexpression or knockdown of CBS can inhibit or promote the growth of A549 cells and their transplanted tumors, and CBS and AKT/cyclin D1 pathway-related proteins may be the potential targets and markers for lung adenocarcinoma.

中南大学湘雅医学院附属第三医院科研资助项目（No. 2018C112）