PIK3R3在生长激素型垂体神经内分泌肿瘤中的表达及其生物学功能研究  

Expression and biological functions of PIK3R3 in growth hormone pituitary neuroendocrine tumors

作　　者：张港 汪攀 廖彬 龚胜 吴南 Zhang Cang;Wang Pan;Liao Bin;Gong Sheng;Wu Nan(Chongqing Medical University,Chongqing 400016,China;Department of Neurosurgery,Chongqing General Hospital,Chongqing 401147,China)

机构地区：[1]重庆医科大学,重庆400016 [2]重庆市人民医院神经外科,重庆401147

出　　处：《中华神经外科杂志》2024年第6期618-625,共8页Chinese Journal of Neurosurgery

基　　金：重庆市科学技术局基金(CSTB2022NSCQ-MSX0548)。

摘　　要：目的探讨磷酸肌醇3激酶调节亚基3(PIK3R3)在生长激素型垂体神经内分泌肿瘤(GH-PitNETs)中的表达及其与细胞增殖、迁移和侵袭之间的关系.方法利用基因表达数据库(GEO)中相关的PitNETs数据集,分析PIK3R3在GH-PitNETs中的表达情况.利用来源于不同PitNETs亚型的细胞株,通过蛋白质免疫印迹实验(WB)检测PIK3R3在GH3、MMQ和AtT-20细胞中的表达情况.体外培养GH3细胞,应用慢病毒感染的方法对PIK3R3进行敲低,并将细胞分为PIK3R3敲低2组(Sh-PIK3R3-2)、PIK3R3敲低3组(Sh-PIK3R3-3)和未转染对照组(Sh-NC),通过CCK-8、EdU及集落形成实验检测各组细胞增殖速度,采用流式细胞术检测细胞凋亡,采用Transwell实验检测细胞迁移和侵袭,采用WB检测上皮型钙粘蛋白(E-cadherin)、神经型钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)、AKT和磷酸化AKT(P-AKT)的表达.结果GEO数据库的数据分析结果显示,相较于正常垂体或瘤旁组织,PIK3R3在GH-PitNETs中表达上调(均P<0.05),而在催乳素型PitNETs、促肾上腺皮质激素型PitNETs和促性腺激素型PitNETs中表达差异均无统计学意义(均P>0.05).WB检测结果显示,相较于MMQ和AtT-20细胞,PIK3R3在GH3细胞中表达更高(均P<0.05).CCK-8检测结果显示,在24 h、48 h、72 h时间点,Sh-PIK3R3-2组和Sh-PIK3R3-3组细胞的吸光度值分别为0.19±0.02、0.46±0.03、1.25±0.06和0.17±0.02、0.37±0.02、1.03±0.05,均低于对照组细胞的0.24±0.02、0.64±0.04、1.67±0.09(均P<0.05).EdU检测结果显示,Sh-PIK3R3-2组和Sh-PIK3R3-3组细胞的EdU阳性细胞率分别为(16.87±1.63)%和(13.45±1.33)%,均低于对照组的(34.51±2.76)%(均P<0.05).集落形成实验结果显示,Sh-PIK3R3-2组和Sh-PIK3R3-3组细胞的单细胞集落数分别为(84±10)个和(75±8)个,均低于对照组的(173±19)个(均P<0.05).流式细胞术检测结果显示,Sh-PIK3R3-2组和Sh-PIK3R3-3组细胞的细胞凋亡率分别为(25.62±2Objective To investigate the expression of phosphoinositide 3 kinase regulatory subunit 3(PIK3R3)in growth hormone pituitary neuroendocrine tumors(GH-PitNETs)and its relationship with PitNET cell proliferation,migration and invasion.Methods The expression of PIK3R3 in GH-PitNETs was analyzed using the PitNETs dataset in Gene Expression Omnibus(GEO).Using cell lines derived from different PitNETs,the expression of PIK3R3 in GH3,MMQ and AtT-20 cells was detected by Western blot.Lentiviral infection was used to knockdown the PIK3R3 gene in the GH3 cells,and the cells were divided into PIK3R3 knocked down 2 group(Sh-PIK3R3-2),PIK3R3 knocked down 3 group(Sh-PIK3R3-2)and untransfected control group(Sh-NC).Cell proliferation was detected by CCK-8,EdU and colony formation assay.Apoptosis was detected by flow cytometry.Cell migration and invasion were detected by Transwell assay.The expression of epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),Vimentin,matrix metalloproteinase-2(MMP2),matrix metalloproteinase-9(MMP9),AKT and phosphory-lated AKT(P-AKT)were detected by Western blot.Results GEO database analysis showed that PIK3R3 expression was upregulated in GH-PitNETs compared to normal pituitary or paraneoplastic tissues(all P<0.05),whereas there was no statistically significant difference in prolactin-type PitNETs,adrenocorticotropic hormone-type PitNETs,and gonadotropin PitNETs(all P>0.05).Western blot showed that PIK3R3 was highly expressed in GH3 cells compared to MMQ and AtT-20 cells(both P<0.05).CCK-8 assay showed that the absorbance of the cells in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were 0.19±0.02,0.46±0.03,1.25±0.06 and 0.17±0.02,0.37±0.02,1.03±0.05 at 24 h,48 h and 72 h,respectively,which were all lower than the control group's 0.24±0.02,0.64±0.04,1.67±0.09(all P<0.05).EdU assay showed that the rates of EdU-positive cells in the Sh-PIK3R3-2 and Sh-PIK3R3-3 groups were(16.87±1.63)%and(13.45±1.33)%,respectively,which were all lower than the control group's(34.51±2.76)%(all P<0.05).Colon

关 键 词：垂体肿瘤 磷酸肌醇3激酶调节亚基3 细胞增殖 细胞迁移 细胞侵袭 体外研究 

分 类 号：R736.4[医药卫生—肿瘤]