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

Authors：

TANG Fengming ,  LI Weimin , WANG Dongmei , HAN Juan

Department of Respiratory Medecine，West China Hospital，Sichuan University.Chengdu，Sichuan，610041，China Correponding author ： LI Wei-min ， E-mail ： weimin003@yahoo.com;

Corresponding author：

LI Weimin, Email: weimin003@yahoo.com

Keywords：

Heterogeneous nuclear ribonucleoprotein K; Small interfering RNA; Eukaryotic expression vector

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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: Copy

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.
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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.

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.

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Construction of eukaryotic expression vector of siRNA specific for hnRNP K and characterization of its efficiency in A549 lung carcinoma cell line

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