Identification of effective siRNA against K-ras in human pancreatic cancer cell line MiaPaCa-2 by siRNA expression cassette.
OBJECTIVE
We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities.


METHODS
Three different sites of SECs were constructed by PCR. K1/siRNA, K2/siRNA and K3/siRNA are located at sites 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively.


RESULTS
Introduction of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells.


CONCLUSIONS
K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that K1/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.
Wang
Wei
W
Department of Pancreas Surgery Center, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China. gianty2001@yahoo.com.cn
Wang
Chun-You
CY
Dong
Ju-Hua
JH
Chen
Xiong
X
Zhang
Min
M
Zhao
Gang
G
D017209:Apoptosis; D045744:Cell Line, Tumor; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000628:therapy*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology*
D012333:RNA, Messenger; D034741:RNA, Small Interfering