Polymerase synthesis and potential interference of a small-interfering RNA targeting hPim-2.
OBJECTIVE
To synthesize three small-interference RNAs (siRNAs) by T7 RNA polymerase-catalyzed reaction, and to investigate their efficacy on modulating the expression of serine/threonine kinase Pim-2 in human colon cancer cell line.


METHODS
siRNA I, II and III were synthesized by T7 RNA polymerase-directed in vitro transcription, then transfected into human colon cancer cells SW-480. After incubation for 6 h at 37 degrees, 100 mL/L FBS in RPMI 1640 was substituted in each well. After the transfection was repeated twice to three times in each kind of siRNA, hPim-2 mRNA and protein expression were measured by RT-PCR and Western blotting, respectively.


RESULTS
Compared to the control group, after transfected for 48 h with hPim-2 siRNA I, II and III, the relative inhibition rates of hPim-2 mRNA expression in colon cancer cells were 65.4% (P<0.05), 46.2% (P<0.05) and 56.1% (P<0.05), respectively. The protein level of hPim-2 was decreased at 72 h compared to the untransfected cells. The relative inhibition percentages of hPim-2 protein by siRNA I, II, III were 61.6% (P<0.05), 45.8% (P<0.05) and 55.6% (P<0.05), respectively.


CONCLUSIONS
The in vitro transcribed siRNAs can be useful for silencing oncogene hPim-2 expression specifically and efficiently. This may open a new path toward the use of siRNAs as a gene-specific therapeutic tool.
Zhang
Shu-Qun
SQ
Second Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China. zhangshuqun1971@yahoo.com.cn
Du
Qing-You
QY
Ying
Yang
Y
Ji
Zong-Zheng
ZZ
Wang
Sheng-Qi
SQ
D045744:Cell Line, Tumor; D002470:Cell Survival; D003110:Colonic Neoplasms; D012321:DNA-Directed RNA Polymerases; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
C490587:PIM2 protein, human; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D014764:Viral Proteins; D017346:Protein Serine-Threonine Kinases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases