Department of Gastroenterology, the Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Anticancer Drugs. 2013 Jul;24(6):566-76. doi: 10.1097/CAD.0b013e3283608bc5.
We investigated the effects of inhibiting heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) expression on apoptosis, invasion, migration, and the chemotherapy sensitivity of pancreatic cancer cells to gemcitabine, 5-FU, and oxaliplatin chemotherapy using small interfering RNA (siRNA). Chemically synthesized siRNA hnRNP A2/B1 was transfected into the human pancreatic cancer cell lines SW1990 and BxPC-3. The IC(50) of gemcitabine, 5-FU, and oxaliplatin was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and cycle were detected using flow cytometry. The expressions of apoptosis-related genes, p53, Bax, Bcl-2, TRAIL, Survivin, multidrug resistance 1 (MDR1), E-cadherin, and matrix metalloproteinases-2 (MMP-2) were detected using real-time PCR and western blot. Plate colony formation assay, wound scratch assay, invasion, and migration were also examined. Gemcitabine, 5-FU, and oxaliplatin inhibit the proliferation of SW1990 and BxPC-3 cells in a concentration-dependent manner. Inhibition of hnRNP A2/B1 expression significantly reduced the IC(50) of gemcitabine, 5-FU, and oxaliplatin (P<0.01). hnRNP A2/B1 siRNA combined with gemcitabine, 5-FU and oxaliplatin significantly increased (P<0.01) apoptosis of pancreatic cancer cell lines SW1990 and BxPC-3, increased the expression level of Bax mRNA, decreased Bcl-2 mRNA and MDR1 mRNA expression (P<0.01), and induced no change in p53, TRAIL, and Survivin mRNA expression in SW1990. In the western blot analysis, the expression level of Bax protein increased (P<0.01); the expression of both P-glycoprotein (Pg-p) protein and Bcl-2 protein decreased (P<0.01). Silencing hnRNP A2/B1 decreased invasion and migration in the cell line SW1990. Silencing hnRNP A2/B1 in SW1990 also correlated with an increase in E-cadherin expression and a decrease in MMP-2 expression at the same time. Inhibition of hnRNP A2/B1 expression can induce apoptosis in pancreatic cancer cells and improve chemosensitivity to gemcitabine, 5-FU, and oxaliplatin. hnRNP A2/B1 may play a role in invasion and migration in the pancreatic cancer cell line SW1990 through the regulation of E-cadherin and expression of MMP-2.
我们通过小干扰 RNA（siRNA）研究了抑制异质核核糖核蛋白 A2/B1（hnRNP A2/B1）表达对胰腺癌细胞凋亡、侵袭、迁移和对吉西他滨、5-FU 和奥沙利铂化疗敏感性的影响。化学合成的 hnRNP A2/B1 siRNA 转染至人胰腺癌细胞系 SW1990 和 BxPC-3。使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定吉西他滨、5-FU 和奥沙利铂的 IC50。采用流式细胞术检测细胞凋亡和细胞周期。采用实时 PCR 和 Western blot 检测凋亡相关基因 p53、Bax、Bcl-2、TRAIL、Survivin、多药耐药 1（MDR1）、E-钙黏蛋白和基质金属蛋白酶-2（MMP-2）的表达。还进行了平板集落形成试验、划痕试验、侵袭和迁移检测。吉西他滨、5-FU 和奥沙利铂呈浓度依赖性抑制 SW1990 和 BxPC-3 细胞的增殖。抑制 hnRNP A2/B1 表达显著降低了吉西他滨、5-FU 和奥沙利铂的 IC50（P<0.01）。hnRNP A2/B1 siRNA 联合吉西他滨、5-FU 和奥沙利铂显著增加（P<0.01）胰腺癌细胞系 SW1990 和 BxPC-3 的凋亡，增加 Bax mRNA 的表达水平，降低 Bcl-2 mRNA 和 MDR1 mRNA 的表达（P<0.01），但 SW1990 中 p53、TRAIL 和 Survivin mRNA 的表达无变化。Western blot 分析显示 Bax 蛋白表达水平增加（P<0.01）；P-糖蛋白（Pg-p）蛋白和 Bcl-2 蛋白表达均降低（P<0.01）。沉默 hnRNP A2/B1 降低了细胞系 SW1990 的侵袭和迁移。SW1990 中 hnRNP A2/B1 的沉默同时伴有 E-钙黏蛋白表达增加和 MMP-2 表达降低。抑制 hnRNP A2/B1 表达可诱导胰腺癌细胞凋亡，提高吉西他滨、5-FU 和奥沙利铂的化疗敏感性。hnRNP A2/B1 可能通过调节 E-钙黏蛋白和表达 MMP-2 来发挥作用，从而影响胰腺癌细胞系 SW1990 的侵袭和迁移。
