小干扰RNA靶向突变K-Ras基因对人胰腺癌细胞TGFβ1表达的影响

背景与目的研究显示ras基因活化可促使肿瘤细胞分泌多种细胞因子,而ras基因与TGFβ1基因表达之间的关系还未可知。本研究利用小干扰RNA敲除突变k-ras基因,探讨突变k-ras基因对TGFβ1表达及分泌的影响。方法小干扰RNA瞬时转染靶向突变k-ras后,应用real-time PCR和Western blot方法检测胰腺癌细胞株CFPAC-1、Aspc-1中ras mRNA和蛋白的表达;应用real-time PCR和流式细胞仪检测对胰腺癌细胞TGFβ1表达的影响;应用ELISA方法检测转染前后胰腺癌细胞上清液中TGFβ1浓度。结果①以人已知基因无影响的序列阴性对照组为基准计算k-ras mRNA的相对表达量,实验组胰腺癌细胞CFPAC-1和ASPC-1转染后k-ras mRNA表达明显减低,k-ras mRNA表达在转染后48h为最低,抑制率达(75.33±5.50)%。Western blot检测各组质粒转染的细胞ras蛋白表达,结果显示实验组ras蛋白量较转染试剂对照组、已知基因无影响的序列阴性对照组明显降低。②Real-timePCR结果显示实验组CFPAC-1和ASPC-1细胞转染后,TGFβ1mRNA表达量(CFPAC-10.68±0.08;Aspc-10.59±0.09)明显降低(P0.05)。而流式细胞仪结果显示TGFβ1蛋白表达降低(CFPAC-1组0.30±0.08,ASPC-1组0.33±0.10,P0.05)。③ELISA法检测实验组CFPAC-1上清液TGFβ1浓度为(1906.87±50.75)ng/ml,较阴性对照组(2072.79±96.98)ng/ml有所降低(P=0.036);实验组Aspc-1细胞上清液TGFβ1浓度(851.47±41.08)ng/ml,较阴性对照组(950.93±31.34)ng/ml有所降低(P0.05)。结论人胰腺癌细胞CFPAC-1、Aspc-1中TGFβ1mRNA和蛋白的表达与ras基因相关。TGFβ1可能是ras基因活化后驱动胰腺癌发生及演变的重要物质。本研究为k-ras突变的胰腺癌治疗提供有益的信息。

The effect of targeting mutant k-ras by siRNA on the expression of transforming growth factor beta 1 in human pancreatic cancer cells

Backgroud and aim： It has been reported that oncogenic RAS signal in cancer cells could upregulate some cytokines. However the relationship of ras oncogene with TGFβ1 is unknown. This study explored the inhibitory effect of mutant k-ras gene depletion by small interfering RNA on the expression and secretion of TGFβ1 in pancreatic cancer cells. Methods After transient transfection into pancreatic cancer cells, the levels of mutant k-ras mRNA and protein were measured by real-time PCR and Western blot. The expression of TGFβ1 was determined by real-time PCR and flow cytometry. ELISA was used to detect the concentration of TGFβ1 in cultured medium before and after transfection. Results ①The expression of k-ras mRNA was efficiently inhibited by targeting mutant k-ras using siRNA in pancreatic cancer cell line CFPAC-1 and Aspc-1. The maximum inhibitory effect on k-ras mRNA was 75.33% at 48 hours after transient transfection. The similar effect was observed in protein expression at 72 hours after transient transfection. ②TGFβ1 mRNA was significantly decreased in treatment groups as compared with negative control group by real-time PCR （P 0.05 ）. Moreover, the protein expression of TGFβ1 was suppressed after transient transfection in both CFPAC-1（0.70 ± 0.08） and Aspc-1 cells （0.73 ± 0.10 P 0.05 ） by flow cytometry. ③ Secreted TGFβ protein was decreased in pancreatic cancer cell conditioned medium after transient transfection in CFPAC-1 （1906.87 ± 50.75 ng/ml in treatment group vs. 2072.79 ± 96.98 ng/ml in negative control ） and Aspc-1 （851.47 ± 41.08 ng/ml in treatment group vs. 950.93 ± 31.34 ng/ml in negative control, P 0.05）. Conclusions The expression of TGFβ1 in pancreatic cancer cells is associated with mutant k-ras gene. TGFβ1 may play an important role in oncogenic RAS-driven pancreatic cancer. The results of this study provide useful information for pancreatic cancer therapy.