Rap1b在子宫内膜癌组织中的表达及介导自噬调控人子宫内膜癌细胞化疗敏感性的研究

The expression of Rap1b in endometrial cancer tissues and the autophagy-mediated regulation of chemotherapy sensitivity of human endometrial cancer cells

目的检测子宫内膜癌(EC)组织中Ras相关蛋白1b(Rap1b)表达,观察其对EC细胞Ishikawa化疗敏感性的影响及机制。方法选取2016年9月至2019年1月江门市中心医院诊治的60例EC患者作为研究对象。采用实时荧光定量PCR(qRT-PCR)检测EC组织与癌旁组织中Rap1b mRNA表达,免疫组化染色检测组织中Rap1b与自噬相关蛋白Beclin1、LC3表达。建立Ishikawa/DDP耐药细胞株,脂质体介导细胞转染法构建Rap1b低表达的Ishikawa/DDP细胞,以转染NC-siRNA的Ishikawa/DDP细胞作为阴性转染组,未转染的Ishikawa/DDP细胞作为对照组;qRT-PCR和Western blot检测细胞中Rap1b的表达;CCK-8实验检测DDP处理后细胞存活率,并计算IC50值;流式细胞术检测细胞凋亡;免疫荧光染色检测LC3表达;Western blot检测自噬相关蛋白LC3Ⅰ、LC3Ⅱ、Beclin1及P62蛋白表达。结果与癌旁组织比较,EC组织中Rap1b mRNA与蛋白表达均升高,Beclin1、LC3表达也升高(P<0.05);与对照组和NC-siRNA组相比,Rap1bsiRNA组细胞中Rap1b mRNA和蛋白相对表达量均下降,25、50、100μmol/L DDP处理后细胞存活率下降,IC50值降低,细胞凋亡率升高,LC3荧光染色强度减弱,LC3Ⅱ/LC3Ⅰ比率下降,Beclin1蛋白表达水平下降而P62蛋白表达水平升高(P<0.05)。结论Rap1b在EC组织中表达水平较高,降低Rap1b表达可提高Ishikawa细胞的化疗敏感性,其机制可能与抑制自噬相关。

Objective To detect the expression of Ras-related protein 1b(Rap1b)in endometrial cancer tissues,and observe the effect of Rap1b on the chemotherapy sensitivity of endometrial cancer cells Ishikawa and its mechanism.Methods Sixty endometrial cancer patients admitted to Jiangmen Central Hospital from September 2016 to January 2019 were selected as the research objects.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expression of Rap1b mRNA in endometrial cancer tissues and adjacent tissues.Immunohistochemical staining was used to detect the expression of Rap1b and autophagy-related proteins Beclin1 and LC3.Establishment of Ishikawa/DDP resistant cell line,liposome-mediated cell transfection was used to construct Ishikawa/DDP cells with low Rap1b expression,Ishikawa/DDP cells transfected with NC-siRNA were used as negative transfection group,and untransfected Ishikawa/DDP cells were used as control group;qRT-PCR and Western blot to detect the expression of Rap1b;CCK-8 experiment detects the cell survival rate of each group after DDP treatment,and calculates the IC50 value;Flow cytometry to detect cell apoptosis;Immunofluorescence staining to detect the expression of LC3;Western blot was used to detect the expression of autophagy-related proteins LC3Ⅰ,LC3Ⅱ,Beclin1 and P62.Results Compared with adjacent tissues,the expressions of Rap1b mRNA and protein in endometrial cancer tissues were increased,the expressions of Beclin1 and LC3 were also increased(P<0.05);Compared with the control group and the NC-siRNA group,the relative expression of Rap1b mRNA and protein in Rap1b-siR·NA group decreased,the cell survival rate decreased under 25,50,100 μmol /L DDP treatment,the IC50 valuedecreased,the rate of cell apoptosis increased,the intensity of LC3 fluorescence staining decreased,the ratio ofLC3Ⅱ/LC3Ⅰ decreased,the expression level of Beclin1 protein decreased while P62 protein increased ( P <0. 05) . Conclusions Rap1b is highly expressed in endometrial carcinoma tissues,and reducing Rap1b expressioncan improve the chemotherapy sensitivity of Ishikawa cells,which may be related to the inhibition of autophagy.