Objective To construct small interfering RNA(siRNA) eukaryotic expression vector specific for human hnRNP K gene,and to observe its silencing effects on hnRNP K gene in A549 cells.Methods The expression vectors of pSUPER/hnRNP K siRNAa,pSUPER/hnRNP K siRNAc and pSUPER/siRNAn were constructed by gene recombination and then transfected into the A549 lung carcinoma cell line by using Lipofectamine2000(a and c respectively represented A and C fragments in hnRNP K coding sequence contained 19 nts,n represented nonsense fragment as control).The mRNA and protein were harvested after 24 h and analyzed for the expression of hnRNP K by RT-PCR and Western blotting respectively.Results The siRNA vector targeted to hnRNP K successfully decreased hnRNP K mRNA and protein levels 24 h after transfection in A549 cells.Relative expressed doses of hnRNP K mRNA in lung cancer cells transfected by hnRNP K siRNAa and hnRNP K siRNAc respectively were 0.24±0.53 and 0.28±0.57 after 24 h,which were significantly lower than that in the control group(both P lt;0.01).The gray scale values of hnRNP K protein were 0.23±0.11 and 0.28±0.09 respectively,which were also significantly lower than those in the control group(both P lt;0.05).And pSUPER/hnRNP K siRNAa was the most effective one.Conclusion Eukaryotic expression vector of siRNA specific for hnRNP K is successfully constructed,which lays the basis for the function study of hnRNP K gene and its application in the treatment of lung carcinoma.
Citation:
TANG Fengming,LI Weimin,WANG Dongmei,HAN Juan. Construction of eukaryotic expression vector of siRNA specific for hnRNP K and characterization of its efficiency in A549 lung carcinoma cell line. Chinese Journal of Respiratory and Critical Care Medicine, 2008, 08(5): 350-353. doi:
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- 1. Matunis MJ,Michael WM,Dreyfuss G.Characterization and primary structure of the poly(C)-binding heterogenous nuclear ribonicleoprotein complex K protein.Mol Cell Biol,1992,12:164-171.
- 2. Brendel C,Rehbein M,Kreienkamp HJ,et al.Characterization of Staufen 1 ribonucleoprotein complexes.Biochem J,2004,384:239-246.
- 3. Pino I,Pío R,Toledo G,et al.Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein(hnRNP) family in lung cancer.Lung Cancer, 2003,41:131-143.
- 4. Mandal M,Vadlamudi R,Nguyen D,et al.Growth factors regulate heterogeneous nuclear ribonucleoprotein K expression and function.J Biol Chem,2001,276:9699-9704.
- 5. Schullery DS,Ostrowski J,Denisenko ON,et al.Regulated interaction of protein kinase Cdelta with the heterogeneous nuclear ribonucleoprotein K protein.J Biol Chem,1999,274:15101-15109.
- 6. de Hoog CL,Foster LJ,Mann M.RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers.Cell,2004,117:649-662.
- 7. Hannon GJ.RNA interference.Nature,2002,418:244-251.
- 8. Rye PD,Stigbrand T.Interfering with cancer:a brief outline of advances in RNA interference in oncology.Tumour Biol,2004,25:329-336.
- 9. Tan FL,Yin JQ.RNAi,a new therapeutic strategy against viral infection.Cell Res,2004,14:460-466.
- 10. Pomerantz RJ.RNA interference:a potential novel therapeutic combating HIV-1 in the central nervous system.Arch Immunol Ther Exp(Warsz),2004,52:401-409.
- 11. Sui G,Soohoo C,Affar el B,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.Proc Natl Acad Sci U S A,2002,99:5515-5520.