Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells.
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.
Ni
Qin
Q
Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China.
Chen
Zhi
Z
Yao
Hang-Ping
HP
Yang
Zheng-Gang
ZG
Liu
Ke-Zhou
KZ
Wu
Ling-Ling
LL
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
D001324:Autoantigens; D012367:RNA, Viral; D012261:Ribonucleoproteins; D000097585:SS-B Antigen