Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression...Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.展开更多
Objective: To analyze the genomic structure of SNC6, a progesterone-receptor associated protein gene and its regulatory elements in its 5'-flanking region. Methods: Genomic sequence from C, enllank database ( acce...Objective: To analyze the genomic structure of SNC6, a progesterone-receptor associated protein gene and its regulatory elements in its 5'-flanking region. Methods: Genomic sequence from C, enllank database ( accession number: Z98048) covering the whole SNC6 gene was used to analyze the genonfic stnmture of SNC6 and design primers for PCR amplification of its 5'-flanking region. A 1894 bp fragrnem of the 5’-flanking region ( - 1814 to + 75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments ( 1423 bp, 632 bp and 416bp, which correspond to - 1344 to + 75, - 552 to + 75 and - 337 to + 75 respectively), were subeloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luefferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All tmnsfected SW620 cells with the above 5-flanking region-containing constructs showed lueiferase activities. The highest lueiferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luefferase activities were measured in tmnsfected cells with vectors containing 416 bp fragments, lmeiferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in tmnsfected cells with vectors containing 1423 bp fragments. Conclusion: The basle tran-scription-promoting element (promoter) for SNC6 expression resides between 0 to - 337, and two transcription-enhancing dements (enhancer) resides between - 337 to - 552 and - 1344 to - 1814, whereas one transcription-inhibiting element (silencer) exists between -552 to - 1344.展开更多
基金Project (No. 30471943) supported partly by the National Natural Science Foundation of China
文摘Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.
文摘Objective: To analyze the genomic structure of SNC6, a progesterone-receptor associated protein gene and its regulatory elements in its 5'-flanking region. Methods: Genomic sequence from C, enllank database ( accession number: Z98048) covering the whole SNC6 gene was used to analyze the genonfic stnmture of SNC6 and design primers for PCR amplification of its 5'-flanking region. A 1894 bp fragrnem of the 5’-flanking region ( - 1814 to + 75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments ( 1423 bp, 632 bp and 416bp, which correspond to - 1344 to + 75, - 552 to + 75 and - 337 to + 75 respectively), were subeloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luefferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All tmnsfected SW620 cells with the above 5-flanking region-containing constructs showed lueiferase activities. The highest lueiferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luefferase activities were measured in tmnsfected cells with vectors containing 416 bp fragments, lmeiferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in tmnsfected cells with vectors containing 1423 bp fragments. Conclusion: The basle tran-scription-promoting element (promoter) for SNC6 expression resides between 0 to - 337, and two transcription-enhancing dements (enhancer) resides between - 337 to - 552 and - 1344 to - 1814, whereas one transcription-inhibiting element (silencer) exists between -552 to - 1344.
