Tan Jing, Zhou Xiaohu, Zhu Hong
Division of Hepatopancreatobiliary, Second Affiliated Hospital, Kunming Medical College, Kunming 650101, China.
Exp Clin Transplant. 2012 Aug;10(4):386-93. doi: 10.6002/ect.2011.0157. Epub 2012 Jul 2.
This study sought to observe transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-cytomegalovirus neo-hTERT-siRNA and examine the combined effect of gemcitabine and siRNA inhibition of telomerase on pancreatic cancer cells.
Transfected pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-cytomegalovirus neo-hTERT-siRNA were selected as target and divided into 9 groups: (1) T1 group (pSilencer4.1-CMV neo-hTERT1-siRNA), (2) T2 group (pSilencer4.1-CMV neo-hTERT2-siRNA), (3) L group (Lipofectamine) (4) M group (mismatch group pSilence4.1-CMV, as negative control), (5) C group (cell group without transfection), (6) blank and gemcitabine group, (7) mismatch siRNA and gemcitabine group, (8) hTERT1-siRNA and gemcitabine group, and (9) hTERT2-siRNA and gemcitabine group. Expression of hTERT mRNA was detected by reverse transcriptase polymerase chain reaction. Viability of cells was measured by colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide assay. Cell cycle and cell apoptosis were measured by flow cytometry. Expression of telomerase protein was measured by Western blot.
Compared with the L group, M group, and C group, expression of hTERT-mRNA and the level of telomerase protein in T1 and T2 group was down-regulated significantly (P < .05), viability of BxPC-3 cells decreased significantly (P < .05), the ratio of cells in G(0)/G(1) stage increased, the ratio of cells in the S stage and the G(2)/M stage decreased, and the ratio of apoptotic cells increased significantly in the T1 and T2 groups. Gemcitabine treatment had a comparable effect. Combination hTERT siRNA and gemcitabine killed twice as many cancer cells, showing a cumulative effect of the treatments.
Transfection of pancreatic cancer cells BxPC-3 with recombinant plasmid pSilencer4.1-CMV neo-hTERT-siRNA represents good RNAi silencing and anti-pancreatic cancer effects in vitro and could potentiate the cytotoxic effect of gemcitabine to pancreatic cancer cells.
本研究旨在观察重组质粒pSilencer4.1 - 巨细胞病毒neo - hTERT - siRNA对胰腺癌细胞BxPC - 3的转染情况，并检测吉西他滨和siRNA抑制端粒酶对胰腺癌细胞的联合作用。
将转染重组质粒pSilencer4.1 - 巨细胞病毒neo - hTERT - siRNA的胰腺癌细胞BxPC - 3作为靶细胞，分为9组：（1）T1组（pSilencer4.1 - CMV neo - hTERT1 - siRNA），（2）T2组（pSilencer4.1 - CMV neo - hTERT2 - siRNA），（3）L组（Lipofectamine），（4）M组（错配组pSilence4.1 - CMV，作为阴性对照），（5）C组（未转染细胞组），（6）空白加吉西他滨组，（7）错配siRNA加吉西他滨组，（8）hTERT1 - siRNA加吉西他滨组，（9）hTERT2 - siRNA加吉西他滨组。采用逆转录聚合酶链反应检测hTERT mRNA的表达。采用比色法3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐法检测细胞活力。采用流式细胞术检测细胞周期和细胞凋亡。采用蛋白质免疫印迹法检测端粒酶蛋白的表达。
与L组、M组和C组相比，T1组和T2组中hTERT - mRNA的表达及端粒酶蛋白水平显著下调（P < 0.05），BxPC - 3细胞活力显著降低（P < 0.05），G(0)/G(1)期细胞比例增加，S期和G(2)/M期细胞比例降低，T1组和T2组凋亡细胞比例显著增加。吉西他滨处理也有类似效果。hTERT siRNA与吉西他滨联合使用杀死的癌细胞数量增加了一倍，显示出治疗的累积效应。
用重组质粒pSilencer4.1 - CMV neo - hTERT - siRNA转染胰腺癌细胞BxPC - 3在体外具有良好的RNA干扰沉默和抗胰腺癌作用，并且可以增强吉西他滨对胰腺癌细胞的细胞毒性作用。
