Regulation of HSVtk gene by endogenous microRNA-122a in liver cell lines as suicide gene therapy

Aim: Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs

Background: Gene therapy with adenovirus [Ad] vectors that express herpes simplex virus thymidine kinase [HSVtk] and ganciclovir [GCV] have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation

Methods: We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'- UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines

Results: Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively

Conclusion: Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression

prevalence of hepatitis B virus surface antigen [HBsAg] variations and correlation with the clinical and serologic pictures in chronic carriers from Khorasan Province, North-East of Iran

This study was designed to determine the correlation of hepatitis B virus surface Ag [HBsAg] variations with the clinical/serological pictures among chronic HBsAg positive patients. The surface gene [S-gene] was amplified and directly sequenced in twenty-five patients. Eight samples [group I] contained at least one mutation at the amino acid level. Five showed alanine aminotransferase [ALT] levels above the normal range of which only one sample was anti-HBe positive. Group II [17 samples] did not contain any mutation, 4 were anti-HBe positive and 9 had increased ALT levels. In both groups, from a total of 18 mutations, 5 [27.5%] and 13 [72.5%] occurred in anti-HBe and HBeAg positive groups respectively. The small number of amino acid mutations might belong to either the initial phase of chronicity in our patients; or that even in anti-HBe positive phase in Iranian genotype D-infected patients, a somehow tolerant pattern due to the host genetic factors may be responsible

Multiplex reverse transcriptase-PCR assay for typing and subtyping of influenza a [H5 and H9] virus in Iran

Avian influenza virus [AIV] infection is a major cause of bird or human mortality and morbidity, therefore the rapid identification of the virus is of important clinical and epidemiological implication. A multiplex Reverse Transcriptase PCR [RT-PCR] was optimized for the detection of influenza A virus and the H5 and H9 subtypes. The influenza type A specific primers were directed to the region of the influenza A matrix gene that is conserved among most type A influenza viruses. The H5 and H9 primers were directed to H5 and H9 hemagglutinin [HA] gene regions that are conserved among H5 and H9 subtypes. The selected primer sets were used in the RT-PCR for simultaneous detection of matrix, H5 and H9 responding specific sequences in a multiplex format. Three reaction conditions were optimized which include: i] RT-PCR typing using matrix gene primers for five subtypes of flu A [H1, H3, H5, H7 and H9], ii] RT-PCR subtyping for H5 and H9 subtypes, and iii] multiplex subtyping of H5 and H9. In this study, the multiplex RT-PCR was applied to 147 cloacal and tracheal swabs of clinical poultry cases with similar influenza symptoms. These results suggest that multiplex RT-PCR assay can be a useful test for rapid detection and subtyping of AIV in clinical samples