title: Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.affiliation: Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Ni
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.forename: Hang-Ping
authors.initials: HP
authors.lastname: Yao
authors.forename: Zheng-Gang
authors.initials: ZG
authors.lastname: Yang
authors.forename: Ke-Zhou
authors.initials: KZ
authors.lastname: Liu
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: Wu
mesh_terms: D001324:Autoantigens; D002460:Cell Line; D015536:Down-Regulation; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D034622:RNA Interference; D012367:RNA, Viral / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000037:antagonists & inhibitors*; D000097585:SS-B Antigen
chemical_list: D001324:Autoantigens; D012367:RNA, Viral; D012261:Ribonucleoproteins; D000097585:SS-B Antigen