摘要
目的探讨同源域蛋白同源盒基因(HOPX)在胃癌中的表达情况及调控胃癌细胞的增殖能力及其作用机制。方法采用Real time⁃PCR及Western blot检测HOPX在胃癌细胞和组织中的mRNA及蛋白表达水平;在胃癌细胞HGC27及BGC823中稳定过表达HOPX基因,通过CCK⁃8法、克隆形成实验、EdU法检测HOPX对胃癌细胞增殖的影响;采用流式细胞术检测HOPX对胃癌细胞细胞周期的调控作用。另外将用于检测β⁃catenin/TCF/LEF可转录活性的荧光素酶报告质粒TOP flash和FOP flash转染胃癌细胞,使用双荧光素酶报告系统测定过表达HOPX对细胞内荧光素酶转录活性的影响。此外采用Western blot分别测定过表达HOPX对胃癌细胞内β⁃catenin、GSK⁃3β及p⁃GSK⁃3β蛋白表达水平的变化。进一步检测过表达HOPX后,胃癌细胞核内β⁃catenin蛋白表达水平及其下游靶基因CyclinD1的表达变化,以探讨HOPX影响胃癌细胞增殖的具体机制。结果Real time⁃PCR及Western blot结果显示HOPX在胃癌中的mRNA及蛋白表达水平均显著降低,HOPX过表达后可显著抑制胃癌细胞的增殖并抑制其细胞周期。Western blot结果表明过表达HOPX可通过抑制GSK3β的磷酸化水平促进β⁃catenin的降解,从而抑制β⁃catenin入核,进而下调下游靶基因CyclinD1的表达,达到抑制胃癌细胞的增殖能力。结论HOPX通过调控Wnt/β⁃catenin信号通路以抑制胃癌细胞的增殖。
Objective To investigate the expression of HOPX in gastric cancer and the ability to regulate the proliferation of gastric cancer cells and its mechanism.Methods HOPX expression in gastric cancer cells and tissues were detected by Real⁃Time PCR and Western Blot.CCK⁃8,colony formation,EdU and flow cytometry assays were performed to investigate the tumor suppressor gene functions of HOPX in the growth and proliferation capability of gastric cancer cells.In addition,TOP flash and FOP flash plasmids were transfected into gastric cancer cells,and the effect of HOPX on the transcriptional activity ofβ⁃catenin/TCF/LEF was detected by luciferase reporter assay.Furthermore,the effect of HOPX on the expression of GSK⁃3β,p⁃GSK⁃3βandβ⁃catenin in gastric cancer cells was detected by Western blot.In order to explore the mechanism of gastric cancer cell proliferation mediated by HOPX,we further detected the expression level ofβ⁃catenin protein in gastric cancer nucleus and the expres⁃sion of downstream target gene CyclinD1.Results Real time⁃PCR and Western blot showed that the expression levels of mRNA and protein of HOPX in gastric cancer were significantly decreased,and the overexpression of HOPX significantly inhibited the proliferation and G1⁃S phase cell⁃cycle transition of gastric cancer cells.Western blot results show that overexpression of HOPX can promote the degradation ofβ⁃catenin by inhibiting the phosphor⁃ylation level of GSK3β,thus inhibit the entry ofβ⁃catenin into the nucleus,and then down⁃regulate the expression of downstream target gene CyclinD1,so as to inhibit the proliferation of gastric cancer cells.Conclusion This study found that overexpression of HOPX inhibits the proliferation of gastric cancer cells by inhibiting Wnt/β⁃catenin signaling pathway.
作者
凌丽
张菁
张宗敏
令狐熙涛
王钰莹
LING Li;ZHANG Jing;ZHANG Zongmin;LINGHU Xitao;WANG Yuying(Department of Geriatric Medicine,the Affiliated Hospital of Zunyi Medical College,Zunyi 563003,China;不详)
出处
《实用医学杂志》
CAS
北大核心
2022年第20期2538-2544,共7页
The Journal of Practical Medicine
基金
贵州省卫生计生委科学技术基金(编号:gzwjkj 2015⁃1⁃048)
遵义市科技计划(编号:遵义市科合社字(2018)82)。
