Increase in RNASEL gene expression by miR-29-3p inhibitors in HEK293T cells.

BACKGROUND: RNase L is known as a terminal component of antiviral and Interferon (IFN) pathways in mammalian cells. On the other hand, the human miR-29 family of microRNAs (miRs) has three mature members, miR-29a, miR-29b, and miR-29c. miR-29 is encoded by two gene clusters and the family members have multifunctional roles in various biological processes. OBJECTIVES: To determine the potential role of miR-29 in the regulation of RNASEL gene expression by designing inhibitors against its targeting miRNA, miR-29a-3p and evaluate the RNase L expression. MATERIAL AND METHODS: After selecting miR-29a-3p as a main regulating miRNA for RNASEL in silico, two inhibitors were designed against it and synthesized. Synthesized strands were made double-stranded DNA oligos, treated with T4 polynucleotide kinase (PNK), cloned into the pCDH-CMV-MCS-EF1-cGFP-T2A-Puro vector and transformed into DH5α. Colony PCR and sequencing was done for affirmation. Then the miR-29a-3p inhibitors were transfected into HEK-293T cell line and RNASEL gene expression was analyzed. RESULTS: The miR-29a-3p inhibitors decreased miR-29a-3p expression in vitro. In addition, miR-29a-3p expression reduction resulted in an increase of RNASEL gene expression. CONCLUSIONS: miR-29a-3p inhibitors could increase in RNASEL gene expression which potentially affects the antiviral/IFN pathway. The inhibitors could be considered as drug candidates in different diseases especially viral infections.

The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-ß) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-ß cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

[Evaluation of miR-122 level in the plasma of chronically HCV infected patients].

MicroRNAs (miRNAs) are small non-coding RNA molecules, which have an important function in regulating RNA stability and gene expression. They also can circulate in a cell-free form in the blood thatmakes them potential disease markers. The liver contains various classes of miRNAs in which miR-122 accounts for about 70% of all miRNAs and it has been proved that its level increases in case of liver damage. Here, we investigated plasma levels of miR-122 as a useful disease parameter in patients with chronic hepatitis C (CHC) infection. Thirty five hemophilia and thalassemia patients with CHC were studied. The total RNA was extracted from plasma samples, and miR-122 levels were measured by qPCR and then compared with the specific liver markers. The plasma levels of alanine transaminase (ALT) and aspartate transaminase(AST) were correlated with plasma miR-122 level in CHC patients, and the level of circulating miR-122 in healthy individual groups were rarely lower than those of patients with CHC. In our study, miR-122 levels correlated well with markers of liver inflammatory activity. Plasma miR-122 can be assumed to be another marker in liver similar to the currently used specific markers such as ALT and AST for evaluation of liver damage in hepatitis C virus (HCV) infected patients. Moreover, the correlation between miR-122 and ALT was shown to be higher than between miR-122 and AST.

Laboratory effect of HBV infection in kidney transplant recipients and donors.

BACKGROUND: Viral infections are the most common cause of opportunistic infections after kidney transplantation. Among hepatotropic viruses that induce kidney graft failure and rejection, hepatitis B virus (HBV) has an important and critical role. Extrahepatic HBV-related disorders increase morbidity and mortality in kidney transplant recipients. OBJECTIVE: To analyze the molecular prevalence of HBV infection in kidney transplant recipients and donors before and after transplantation. PATIENTS AND METHODS: This cross-sectional study included 273 serum samples collected between 2005 and 2008 in 96 kidney transplant recipients and 59 donors. Detection of HBV DNA was via amplification of the S gene fragment of HBV genome using a qualitative simple polymerase chain reaction assay. Also analyzed were statistical relationships between HBV infection and laboratory and clinical demographic data in all kidney transplant donors and recipients. RESULTS: The HBV genome was detected in 102 of 273 serum samples. Molecular HBV infection was demonstrated in 2 of 13 serum samples (15.4%) from recipients tested before transplantation. HBV DNA was detected in 42 of 96 patients (43.7%) after kidney transplantation. The HBV genome was demonstrated in 21 of 59 donors (35.6%).Significant relationships were observed between HBV infections and hematologic and biochemical indices after kidney transplantation. CONCLUSION: Detection of a high molecular prevalence of HBV infection in kidney recipients enforces the importance of HBV infection in clinical outcome.