目的 探究塞来昔布通过上调Cyclin D1基因甲基化水平对食管癌细胞增殖和凋亡的作用及机制。方法 将细胞分为对照、转染和塞来昔布组，分别转染阴性对照载体、Cyclin D1过表达载体和Cyclin D1转染后给予60 μmol/L塞来昔布。采用MTT法、流式细胞术分别检测细胞增殖、细胞周期和细胞凋亡率的变化，Western blotting法检测细胞周期和细胞凋亡相关蛋白表达水平；甲基化特异性PCR（MS-PCR）、qRT-PCR法用于检测Cyclin D1甲基化特异性扩增片段和Cyclin D1 mRNA表达水平。结果 塞来昔布能够抑制Cyclin D1诱导的细胞体外增殖，阻滞细胞周期向S期转化，并促进食管癌细胞凋亡；MS-PCR结果显示塞来昔布能够上调Cyclin D1基因甲基化水平，在转录水平抑制细胞内Cyclin D1 mRNA的表达。结论 塞来昔布能够通过上调Cyclin D1基因甲基化水平发挥抑制食管癌细胞增殖、促进其体外凋亡的作用。

Objective To investigate the effects and mechanism of celecoxib on the cell proliferation and apoptosis by regulating methylation status of Cyclin D1 in esophageal carcinoma cells. Methods Cells were divided into control group, transfected group and celecoxib group. Cells in the control group were transfected with negative control vector while cells in the transfected group and celecoxib group were transfected with Cyclin D1-overexpression vector. The celecoxib group was treated with 60 μmol/L celecoxib solution after transfection. MTT and flow cytometry were used to detect the cell proliferation capacity, cell cycle distribution, and cell apoptotic rates in vitro. Western blotting was used to measure the expression levels of related proteins. MS-PCR and qRT-PCR were used to detect the Cyclin D1 promoter region methylation level and Cyclin D1 mRNA expression level. Results Celecoxib can inhibit cell proliferation in vitro, which was promoted by Cyclin D1 overexpression, block cell cycle transition into S phase and promote cell apoptosis. MS-PCR showed that celecoxib could up-regulate the methylation level of Cyclin D1 gene and suppressed Cyclin D1 mRNA expression at transcriptional level. Conclusion Celecoxib can inhibit cell proliferation and promote apoptotic rate of esophageal cancer cells through up-regulating the methylation level of Cyclin D1.

R966

河南省科技计划发展项目（202102310408）