shRNA ile HBV replikasyonunun in vitro olarak baskılanması
Özet
RNA interference (RNAi) can be defined as sequence- spesific mRNA degradation. RNAi mechanism is initiated by small interfering RNA (siRNA) and results in silencing of an active gene transcript. siRNAs are spesific for only target mRNAs. So no mRNA other than target one can be degraded with RNAi mechanism. Our purpose in this study is to investigate effects of shRNAs targeting three different sites of hepatitis B virus mRNAs on the viral replication in HepG2.2.15cells. MATERIAL- METHOD: Three HBV-specific siRNAs were designed targeting X (1686- 1705), Core (2228- 2247) and S (765-784 nt.) transcripts. These siRNAs were cloned to PENTRY/U6 vector for expression of shRNAs (small hairpin RNAs). HepG2.2.15, a stable HBV producing cell line, was used to see the effects of shRNAs on HBV. The cells were transfected with shRNA expressing plasmids (P765, P2228 and P1686 targeting X, core and S region respectively) or a mock plasmid targeting lacZ gene. Transfection efficiency was determined by using green flourescent protein expressing plasmid. After transfection viral DNA levels were detected. RESULTS: The culture media was collected in the days 2, 3, 4, 5, 6, 7 posttransfection and analyzed by Real-time PCR. Viral DNA production suppressed for 7 days. The HBV DNA levels were decreased by 81 %, 76 %, 75 % with P765, P2228 and P1686 vectors respectively. Whereas viral DNA level was increased with mock plasmid (PlacZ). DISCUSSION: In conclusion, the siRNAs designed for X, core and S regions, spesifically and significantly suppressed HBV DNA. Our results support the potential use of this treatment strategy in hepatitis B. Keywords: RNAi, siRNA, shRNA, HBV, HepG2.2.15 cell line. 78