Chen Yan, Bieerkehazhi Shayahati, Li Xiumei, Ma Lili, Yibulayin Waresijiang, Ran Jihua
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Xinjiang Medical University, Xinjiang, China.
Morphology Center, School of Basic Medicine, Xinjiang Medical University, Xinjiang, China.
J Oncol. 2021 Jan 5;2021:1384289. doi: 10.1155/2021/1384289. eCollection 2021.
Esophageal cancer (EC) is the eighth most prevalent cancer and the sixth leading cause of cancer-related mortality worldwide. As an antiapoptotic and a proapoptotic protein, respectively, survivin and Bad play an important role in carcinogenesis of the most human cancers including EC. However, the regulatory relationships between them remain unclear. We sought to investigate the effects of survivin knockdown and overexpression on the expression of Bad gene, cell cycle progression, and apoptosis of esophageal carcinoma cell. The mRNA expression levels of survivin and Bad were determined in EC tissue samples. The knockdown and overexpression experiments were performed in ECA109 and KYSE450 cells via transfection with survivin overexpression and shRNA plasmids. A Bad overexpression experiment was conducted to confirm the biological effect on knockdown of survivin via modulating Bad expression. RT-qPCR and Western blot analysis were used to detect mRNA and protein expression, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. The chromatin immunoprecipitation (ChIP) was conducted to determine the binding sites of survivin on the promoter of Bad gene. By analyzing the mRNA expression of survivin and Bad in 40 ESCC patient specimens, we found that the positive expression rate of survivin in tumor tissues (88%, 35/40) was remarkably high, compared with the distal nontumor tissues (48%, 19/40, < 0.01). On the other hand, the positive expression rate of Bad in tumor tissues (70%, 28/40) was remarkably low, compared with the distal nontumor tissues (95%, 38/40, < 0.01). Overexpression of survivin decreases Bad mRNA and protein expression and promotes transformation of cell cycle to S phase. Conversely, knockdown of survivin increases Bad mRNA and protein expression and induces cell cycle arrest and apoptosis. Bad overexpression inducing apoptosis of esophageal carcinoma cell shows the similar apoptotic effect with survivin knockdown. ChIP assays indicate that survivin directly binds to the Bad promoter region, diminishing the transcriptional activity of Bad. In conclusion, the result suggested that survivin regulates Bad gene expression by binding to its promoter and modulates cell cycle and apoptosis in esophageal carcinoma cell.
食管癌（EC）是全球第八大常见癌症，也是癌症相关死亡的第六大主要原因。生存素（survivin）和Bad分别作为一种抗凋亡蛋白和促凋亡蛋白，在包括食管癌在内的大多数人类癌症的致癌过程中发挥重要作用。然而，它们之间的调控关系仍不清楚。我们试图研究生存素基因敲低和过表达对Bad基因表达、细胞周期进程以及食管癌细胞凋亡的影响。检测了食管癌组织样本中生存素和Bad的mRNA表达水平。通过转染生存素过表达质粒和shRNA质粒，在ECA109和KYSE450细胞中进行了基因敲低和过表达实验。进行了Bad过表达实验，以通过调节Bad表达来确认生存素基因敲低的生物学效应。分别采用RT-qPCR和蛋白质印迹分析检测mRNA和蛋白质表达。通过流式细胞术分析细胞周期和凋亡情况。进行染色质免疫沉淀（ChIP）实验以确定生存素在Bad基因启动子上的结合位点。通过分析40例食管鳞状细胞癌（ESCC）患者标本中生存素和Bad的mRNA表达，我们发现肿瘤组织中生存素的阳性表达率（88%，35/40）显著高于远端非肿瘤组织（48%，19/40，P<0.01）。另一方面，肿瘤组织中Bad的阳性表达率（70%，28/40）显著低于远端非肿瘤组织（95%，38/40，P<0.01）。生存素过表达降低了Bad的mRNA和蛋白质表达，并促进细胞周期向S期转化。相反，生存素基因敲低增加了Bad的mRNA和蛋白质表达，并诱导细胞周期停滞和凋亡。Bad过表达诱导食管癌细胞凋亡显示出与生存素基因敲低相似的凋亡效应。ChIP分析表明，生存素直接结合到Bad启动子区域，降低了Bad的转录活性。总之，结果表明生存素通过与其启动子结合来调节Bad基因表达，并调节食管癌细胞的细胞周期和凋亡。
