GM130通过14-3-3ζ对卵巢癌细胞增殖的影响及机制研究  

Effect of GM130 on the proliferation of ovarian cancer cells via 14-3-3ζand mechanism study

作　　者：郑依果 易永芬[1,2,3] 罗祎 ZHENG Yiguo;YI Yongfen;LUO Yi(Center for Molecular Medicine and Tumor Research,School of Basic Medicine,Chongqing Medical University,Chongqing 400016,China;Center for Molecular Medicine Detection,Chongqing Medical University,Chongqing 400016,China;Department of Pathology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400042,China;Gynecology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400042,China)

机构地区：[1]重庆医科大学基础医学院分子医学与肿瘤研究中心,重庆400016 [2]重庆医科大学分子医学检测中心,重庆400016 [3]重庆医科大学附属第一医院病理科,重庆400042 [4]重庆医科大学附属第一医院妇科,重庆400042

出　　处：《中国肿瘤外科杂志》2024年第2期170-177,共8页Chinese Journal of Surgical Oncology

基　　金：国家自然科学基金资助项目(编号:30672431)。

摘　　要：目的探讨高尔基体基质蛋白130(GM130)通过14-3-3ζ对卵巢癌细胞增殖的影响及可能机制。方法采用免疫组化法检测在20例正常卵巢上皮组织和42例卵巢上皮恶性肿瘤中GM130和14-3-3ζ的表达,分析二者相关性。采用Western blot和RT-PCR法检测卵巢癌SKOV3/A2780细胞株GM130和14-3-3ζ蛋白及其mRNA表达情况,筛选出高表达细胞株。采用免疫荧光染色法检测GM130与14-3-3ζ在卵巢癌细胞中的共表达情况。将设计好的重组表达质粒shRNA-GM130片段稳转进入卵巢癌细胞中,采用Western blot和RT-PCR筛选出降低GM130的最佳片段。细胞分为三组:空白组(WT)、对照组(NC)、低表达组(313),采用CCK法和流式细胞术分别检测各组卵巢癌细胞生殖情况及周期变化情况,采用Western blot法检测各组14-3-3ζ、GRASP65、P115及增殖相关蛋白表达水平。结果GM130和14-3-3ζ在卵巢恶性肿瘤组织中的表达水平高于正常卵巢组织(P<0.001)。在卵巢恶性肿瘤组织中,GM130和14-3-3ζ呈正相关关系(r=0.873,P<0.001)。GM130蛋白及其mRNA在SKOV3细胞中的表达量高于A2780细胞(P<0.05),14-3-3ζ蛋白及其mRNA在SKOV3细胞和A2780细胞中比较差异无统计学意义(P>0.05)。后续实验选用SKOV3细胞。免疫荧光共定位染色显示,GM130与14-3-3ζ主要在胞质表达,前者近核膜部分呈堆叠样表达量较高,后者胞核中表达较少,二者可能存在共定位关系。转染313组片段后,对GM130的抑制效果最好。转染质粒313组细胞增殖能力降低,细胞在G1期阻滞的数量增多,G2期阻滞数量减少,S期阻滞的细胞数量稍减少,GM130、14-3-3ζ、P115、Cyclin A、Cdc25A蛋白相对表达水平降低,P21蛋白相对表达水平升高(P<0.05)。结论抑制GM130可抑制卵巢癌细胞增殖,其机制可能与抑制14-3-3ζ表达,影响卵巢癌细胞周期有关。Objective To investigate the effects and possible mechanisms of Golgi matrix protein 130(GM130)on ovarian cancer cell proliferation via 14-3-3ζ.Methods The expression of GM130 and 14-3-3ζin 20 normal ovarian epithelial tissues and 42 ovarian epithelial malignant tumours was detected by immunohistochemistry,and their correlation was analyzed.Western blot and RT-PCR were used to detect the expression of GM130 and 14-3-3ζproteins and their mRNAs in ovarian cancer SKOV3/A2780 cell lines,and the highly expressed cell lines were screened out.Immunofluorescence staining was used to detect the co-expression of GM130 and 14-3-3ζin ovarian cancer cells.The designed recombinant expression plasmid shRNA-GM130 fragment was stably transfected into ovarian cancer cells,and Western blot and RT-PCR were used to screen out the best fragment to decrease GM130.The cells were divided into three groups:blank group(WT),control group(NC),and low-expression group(313),and the reproductive status and cycle changes of ovarian cancer cells in each group were detected by CCK-8 and flow cytometry respectively,and the expression levels of 14-3-3ζ,GRASP65,P115 and proliferation-related proteins in each group were detected by Western blot.Results The expression levels of GM130 and 14-3-3ζwere significantly higher in ovarian malignant tumour tissues than in normal ovarian tissues(P<0.001).In ovarian malignant tumour tissues,GM130 and 14-3-3ζshowed a positive correlation(r=0.873,P<0.001).The expression of GM130 protein and its mRNA was higher in SKOV3 cells than in A2780 cells(P<0.05),and there was no significant difference between 14-3-3ζprotein and its mRNA when comparing SKOV3 cells and A2780 cells(P>0.05).Therefore,SKOV3 cells were selected for subsequent experiments.Immunofluorescence co-localization staining showed that GM130 and 14-3-3ζwere mainly expressed in the cytoplasm,with higher stacking-like expression in the proximal nuclear membrane portion of the former,and less expression in the cytosol of the latter,which may be co-loc

关 键 词：卵巢癌 高尔基体基质蛋白130 14-3-3ζ 细胞增殖 细胞周期 

分 类 号：R737.31[医药卫生—肿瘤]