摘要
目的 探讨SH2结构域2A(SH2D2A)对胰腺癌(PDAC)吉西他滨(GEM)耐药的影响.方法 利用Kaplan-Meier Plotter和GEPIA平台探讨SH2D2A在PDAC的表达,对PDAC患者总体存活率(OS)和无病生存期(DFS)的影响;选取郑州大学人民医院手术切除的40例PDAC组织和癌旁组织,蛋白质印迹法(Western blot)分析组织中SH2D2A蛋白表达水平;实时荧光定量聚合酶链反应(qPCR)和Western blot分析购自上海细胞典藏委员会的人正常胰腺导管上皮细胞HPDE、人胰腺癌细胞系 SW1990、MIA PaCa-2、PANC-1、HPAF Ⅱ 和 BxPC-3 中 SH2D2A 蛋白和 mRNA 表达水平;短发卡RNA(shRNA)构建稳定敲低SH2D2A细胞株,分为实验组(Sh组)和对照组(NC组);qPCR和Western blot分析两组细胞SH2D2A的表达水平;细胞计数试剂盒(CCK-8)分析两组细胞的药物半数抑制浓度(IC50);集落形成实验、CCK-8、流式细胞术和肿瘤成球实验分析GEM处理后细胞的增殖、凋亡和肿瘤干性;组间数据比较采用t检验.结果 生信分析显示SH2D2A高表达患者的OS、DFS明显缩短(P均＜0.01).PDAC组织中SH2D2A蛋白表达高于癌旁组织[0.88±0.27比0.36±0.07,t=6.416,P＜0.05].SW1990、MIA PaCa-2、PANC-1、HPAF Ⅱ 和 BxPC-3 中 SH2D2A 表达水平高于 HPDE[mRNA:5.86±0.45、3.47±0.27、4.74±0.37、2.38±0.18、2.54±0.20 比 1.00± 0.08,t=15.780、12.280、14.490、9.040、9.632,P 均＜0.05;蛋白:2.24±0.11、1.67±0.07、1.81± 0.18、1.31±0.08、1.53±0.17 比 1.00±0.01,t=18.110、16.400、7.320、6.406、5.220,P 均＜0.05].Sh 组 SH2D2A 表达水平低于 NC 组[mRNA:0.15±0.03、0.14±0.03 比 1.01±0.07,t=17.970、18.570,P均＜0.05;蛋白:0.35±0.02、0.30±0.02 比 1.00±0.08,t=11.690、14.300,P均＜0.05].Sh 组 IC50低于 NC 组[18.27±0.04、16.54±0.10 比 25.81±0.32,t=45.280、54.850,P 均＜0.05];Sh 组细胞第 4 天的吸光度值低于 NC 组[1.20±0.06、1.02±0.08 比 2.22±0.08,t=17.440、18.490,P 均＜0.05];Sh 组细胞集落数量低于 NC 组[367.70±31.50、331.00±58.66 比 738.00± 101.50,t=7.211、9.676,P 均＜0.05];Sh 组细胞凋亡比例高于 NC 组[(28.59±3.44)％、(22.68± 1.78)％比(17.67±0.38)％,t=5.166、4.375,P 均＜0.05];Sh 组细胞成球大小小于 NC 组[8.79± 1.43、7.63±1.23 比 45.31±11.85,t=4.772、5.191,P 均＜0.05].结论 SH2D2A 在人 PDAC 中表达上调,并参与调控PDAC对吉西他滨的耐药.
Abstract
Objective To explore the effect of SH2 domain containing 2A(SH2D2A)on gemcit-abine resistance in pancreatic ductal adenocarcinoma(PDAC).Methods Exploring SH2D2A expression in PDAC using the Kaplan-Meier Plotter and GEPIA platforms,and the impact on overall survival and dis-ease-free survival of PDAC patients;Forty cases of PDAC tissues and paracancerous tissues surgically resec-ted at Zhengzhou University People's Hospital from June 2022 to June 2023 were selected,and Western blotting was performed to analyze SH2D2A protein expression levels in the tissues;quantitative real-time polymerase chain reaction(qPCR)and Western blotting were performed to analyze the expression levels of SH2D2A protein and mRNA in the humol/Lan normal pancreatic ductal epithelial cells HPDE and humol/Lan PDAC cell line SW1990,MIA PaCa-2,PANC-1,HPAFⅡ,and BxPC-3;short hair RNA construction of stable knockdown SH2D2A cell lines,divided into Sh and NC groups;qPCR and Western blotting ana-lyzed the expression level of SH2D2A in the two groups of cells;cell counting kit-8(CCK-8)analyzed the half maximal inhibitory concentration(IC50)of the two groups of cells;colony formation assay,CCK-8,flow cytometry and tumol/Lor spheroid formation assay analyzed the proliferation,apoptosis and tumol/Lor stem-ness of the GEM-treated cells;t-test was used for comparison between groups.Results Bio-confidence a-nalysis showed that OS and DFS were significantly shorter in patients with high SH2D2A expression(both P＜0.01).SH2D2A protein expression in pancreatic cancer tissues was higher than that in paracancerous tissues,[(0.88±0.27)vs.(0.36±0.07),t=6.416,P＜0.05].The expression levels of SH2D2A were higher than those of HPDE in SW1990,MIAPaCa-2,PANC-1,HPAF Ⅱ and BxPC-3[mRNA:(5.86±0.45,3.47±0.27,4.74±0.37,2.38±0.18,2.54±0.20)vs.(1.00±0.08),t=15.780,12.280,14.490,9.040,9.632,all P＜0.05);Protein:(2.24±0.11,1.67±0.07,1.81±0.18,1.31±0.08,1.53±0.17)vs.(1.00±0.01),t=18.110,16.400,7.320,6.406,5.220,all P＜0.05].The expression level of SH2D2A in Sh group was lower than NC group[mRNA:(0.15±0.03,0.14±0.03)vs.(1.01±0.07),t=17.970,18.570,both P＜0.05);Protein:(0.35±0.02,0.30± 0.02)vs.(1.00±0.08),t=11.690,14.300,both P＜0.05].The IC50 of Sh group was lower than that of NC group,[(18.27±0.04,16.54±0.10)vs.(25.81±0.32),t=45.280,54.850,both P＜0.05];CCK-8 showed that the absorbance of cells in Sh group on day 4 was lower than NC group[(1.20± 0.06,1.02±0.08)vs.(2.22±0.08),t=17.440,18.490,both P＜0.05];the number of cell colonies in Sh group was lower than in NC group[(367.70±31.50,331.00±58.66)vs.(738.00±101.50),t=7.211,9.676,both P＜0.05];the proportion of apoptosis in Sh group was higher than NC group,[(28.59±3.44)％,(22.68±1.78)％vs.(17.67±0.38)％,t=5.166,4.375,both P＜0.05];the size of cell formation in Sh group was significantly smaller than NC group[(8.79±1.43,7.63±1.23)vs.(45.31±11.85),t=4.772,5.191,both P＜0.05].Conclusion SH2D2A expression is upregulat-ed in human PDAC and is involved in the regulation of PDAC resistance to gemcitabine.
基金项目
河南省中青年卫生健康科技创新领军人才培养项目(2023)(LJRC2023015)
NETL
NSTL
万方数据