多聚嘧啶区结合蛋白1对胃癌细胞增殖和转移的作用机制

Mechanism of polypyrimidine tract-binding protein 1 on the proliferation and metastasis of gastric cancer cells

目的探讨多聚嘧啶区结合蛋白1(PTBP1)在胃癌组织和人胃癌细胞株中的表达,以及PTBP1对胃癌细胞增殖与转移的作用机制。方法收集2019年1至6月于西安交通大学第一附属医院行外科手术切除的胃癌患者的癌组织与对应的癌旁组织。运用Kaplan-Meier Plotter数据库分析胃癌患者的生存情况。选择PTBP1表达水平相对较高的人胃癌细胞株SGC7901和AGS进行小干扰RNA(siRNA)转染下调PTBP1表达,实验分为空白对照组、阴性对照组和PTBP1敲低组。分别运用实时荧光定量PCR和蛋白质印迹法检测胃癌细胞中PTBP1 mRNA和PTBP1的表达。通过MTT法转染24、48、72、96 h后,观察PTBP1对胃癌细胞增殖的作用;Transwell实验检测下调PTBP1后胃癌细胞侵袭和迁移的变化,蛋白质印迹法检测下调PTBP1后胃癌细胞上皮-间质转化(EMT)标志物E-钙黏蛋白、N-钙黏蛋白和波形蛋白的改变。采用独立样本t检验、方差分析和秩和检验进行统计学分析。结果Kaplan-Meier Plotter预后分析显示,高表达PTBP1胃癌患者总生存期短于低表达PTBP1胃癌患者[9.2个月(6.2个月,17.2个月)比19.0个月(14.5个月,28.4个月)],差异有统计学意义(Z=5.31,P<0.05)。实时荧光定量PCR检测结果显示,人胃癌细胞株SGC7901和AGS中,PTBP1敲低组PTBP1 mRNA表达水平均低于空白对照组和阴性对照组(SGC7901:0.78±0.11比3.10±0.19、2.99±0.23。AGS:0.80±0.09比3.55±0.24、3.50±0.18),差异均有统计学意义(tSGC7901=10.57、8.08,tAGS=10.91、13.42;P均<0.01)。蛋白质印迹结果显示,人胃癌细胞株SGC7901和AGS中,PTBP1敲低组PTBP1表达水平均低于空白对照组和阴性对照组(SGC7901:0.38±0.04比1.42±0.05、1.35±0.09。AGS:0.17±0.02比1.52±0.08、1.38±0.45),差异均有统计学意义(tSGC7901=15.94、10.57,tAGS=16.60、20.80;P均<0.01)。MTT结果显示,转染48、72、96 h后,PTBP1敲低组的吸光度值较阴性对照组分别下降了0.25±0.01、0.38±0.02、0.84±0.04,其中转染96 h后的差值最显著,差异有统计学意义(t=10.21、14.32,P均<0.01)。Transwell实验结果显示,人胃癌细胞株SGC7901和AGS中,PTBP1敲低组发生侵袭和迁移的细胞数均少于空白对照组和阴性对照组(SGC7901:42.00±5.91比116.40±10.23、114.40±10.43;39.60±6.77比125.80±11.51、122.40±5.90。AGS:40.20±7.25比115.60±14.63、117.40±9.12;36.00±5.20比122.40±12.10、125.40±12.74),差异均有统计学意义(tSGC7901=14.07、13.50、14.43、20.62,tAGS=10.27、14.75、14.68、16.76;P均<0.01)。蛋白质印迹结果显示,PTBP1敲低组E-钙黏蛋白表达水平均高于空白对照组和阴性对照组(SGC7901:1.42±0.05比0.53±0.05、0.57±0.03。AGS:1.34±0.04比0.54±0.03、0.61±0.01),而N-钙黏蛋白和波形蛋白的表达水平均低于空白对照组和阴性对照组(SGC7901:0.50±0.03比1.64±0.05、1.46±0.07,0.32±0.07比1.42±0.07、1.33±0.07。AGS:0.37±0.06比1.47±0.04、1.36±0.04,0.41±0.04比1.53±0.06、1.37±0.04),差异均有统计学意义(tSGC7901=11.63、13.19、18.83、11.68、11.43、10.43,tAGS=15.02、16.23、14.67、12.97、14.45、17.18;P均<0.01)。结论胃癌组织和细胞中PTBP1表达水平均升高,并且参与调控胃癌细胞的增殖、转移和EMT。

Objective To explore the expression of polypyrimidine tract-binding protein 1(PTBP1)in gastric cancer(GC)tissues and GC cell lines,and the role of PTBP1 in the proliferation and metastasis of GC cells.Methods From January to June in 2019 at The First Affiliated Hospital of Xi′an Jiaotong University,the cancer tissues and corresponding para-cancer tissues of GC patients underwent surgical resection were collected.The Kaplan-Meier Plotter database was used to analyze the survival of GC patients.The expression of PTBP1 was down-regulated by transfecting small interfering RNA(siRNA)in human GC cell lines SGC7901 and AGS with relatively high expression of PTBP1.The cells were divided into blank control group,negative control group,and PTBP1 knockdown group.The expression of PTBP1 at mRNA and protein level were detected by real-time fluorescence quantification polymerase chain reaction(RT-qPCR)and Western blotting.At 24,48,72 and 96-hour after transfection,the effect of PTBP1 on the proliferation of GC cells was observed by 3-(4,5 dimethylthiazol)-2,5 diphenyltetrazolium bromide(MTT)experiment.The changes of invasion and migration of GC cells after down-regulation of PTBP1 were detected by transwell assay.The expression changes of epithelial-mesenchymal transition(EMT)markers E-cadherin,N-cadherin and vimentin after down-regulation of PTBP1 in GC cells were determined by Western blotting.Indenpendent samples t test,analysis of variance and rank sum test were used for statistical analysis.Results The Kaplan-Meier Plotter prognostic analysis showed that the overall survival of GC patients with high PTBP1 expression was shorter than that of GC patients with low PTBP1 expression(9.2 months,6.2 months to 17.2 months vs.19.0 months,14.5 months to 28.4 months),and the difference was statistically significant(Z=5.31,P<0.05).The results of RT-qPCR showed that in GC cell lines SGC7901 and AGS,the expression of PTBP1 at mRNA level of PTBP1 knockdown group was lower than that of blank control group and negative control group(SGC7901:0.78±0.11 vs.3.10±0.19 and 2.99±0.23;AGS:0.80±0.09 vs.3.55±0.24 and 3.50±0.18),and the differences were statistically significant(tSGC7901=10.57 and 8.08,tAGS=10.91 and 13.42;all P<0.01).The results of Western blotting indicated that in GC cell lines SGC7901 and AGS,the expression of PTBP1 at protein level of PTBP1 knockdown group was lower than those of blank control group and negative control group(SGC7901:0.38±0.04 vs.1.42±0.05 and 1.35±0.09;AGS:0.17±0.02 vs.1.52±0.08 and 1.38±0.45),and the differences were statistically significant(tSGC7901=15.94 and 10.57,tAGS=16.60 and 20.80;all P<0.01).The results of MTT showed that at 48,72 and 96-hour after transfection the absorbance values of PTBP1 knockdown group decreased by 0.25±0.01,0.38±0.02,and 0.84±0.04 as compared with those of negative control group,and the decrease was the most significant at 96-hour after transfection,and the differences were statistically significant(t=10.21、14.32,both P<0.01).The results of transwell experiment demonstrated that the number of invasion and migration cells of PTBP1 knockdown group were both less than that of the blank control group and the negative control group(SGC7901:42.00±5.91 vs.116.40±10.23 and 114.40±10.43;39.60±6.77 vs.125.80±11.51 and 122.40±5.90;AGS:40.20±7.25 vs.115.60±14.63 and 117.40±9.12;36.00±5.20 vs.122.40±12.10 and 125.40±12.74),and the differences were statistically significant(tSGC7901=14.07,13.50,14.43 and 20.62;tAGS=10.27,14.75,14.68 and 16.76;all P<0.01).The results of Western blotting showed that the expression of E-cadherin of PTBP1 knockdown group was higher than that of the blank control group and the negative control group(SGC7901:1.42±0.05 vs.0.53±0.05 and 0.57±0.03;AGS:1.34±0.04 vs.0.54±0.03 and 0.61±0.01),however the expression levels of N-cadherin and vimentin were both lower than those of the blank control group and the negative control group(SGC7901:0.50±0.03 vs.1.64±0.05 and 1.46±0.07;0.32±0.07 vs.1.42±0.07 and 1.33±0.07;AGS:0.37±0.06 vs.1.47±0.04 and 1.36±0.04;0.41±0.04 vs.1.53±0.06 and 1.37±0.04),and the differences were statistically significant(tSGC7901=11.63,13.19,18.83,11.68,11.43 and 10.43;tAGS=15.02,16.23,14.67,12.97,14.45 and 17.18;all P<0.01).Conclusions The expression levels of PTBP1 increase in GC tissues and cells,which may be involved in regulating the proliferation,metastasis and EMT of GC cells.