SH2结构域2A在胰腺癌中的表达及对其吉西他滨耐药的影响

Expression of SH2 domain containing 2A in pancreatic ductal adenocarcinoma and its effect on gemcitabine resistance

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、HPAFⅡ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、HPAFⅡ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。

Objective To explore the effect of SH2 domain containing 2A(SH2D2A)on gemcitabine 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 disease-free survival of PDAC patients;Forty cases of PDAC tissues and paracancerous tissues surgically resected 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 analyzed 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 stemness of the GEM-treated cells;t-test was used for comparison between groups.Results Bio-confidence analysis 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,HPAFⅡand 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 upregulated in human PDAC and is involved in the regulation of PDAC resistance to gemcitabine.