AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.
AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.
Authors: Douglas A Rubinson; Christopher P Dillon; Adam V Kwiatkowski; Claudia Sievers; Lili Yang; Johnny Kopinja; Dina L Rooney; Mingdi Zhang; Melanie M Ihrig; Michael T McManus; Frank B Gertler; Martin L Scott; Luk Van Parijs Journal: Nat Genet Date: 2003-02-18 Impact factor: 38.330