MicroRNA-9 controls apoptosis of neurons by targeting monocyte chemotactic protein-induced protein 1 expression in rat acute spinal cord injury model.

OBJECTIVE: For the purpose of an early identification of intervention targets for acute spinal cord injury (ASCI), we investigated the changes in expression levels of microRNA-9 (miR-9) and MCPIP1 in rat ASCI model. METHOD: A total of 108 healthy rats were randomly divided into non-ASCI group (n=18) and five ASCI groups, 6h, 12h, 24h, 3 days and 7 days, representing the experimental time points following ASCI (n=18 per group). Hematoxylin and eosin (HE) staining was used to assess the ASCI damage, and quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) were employed for the detection of miR-9 and MCPIP1 mRNA expression. RESULTS: HE staining results showed normal neuronal morphology in the non-ASCI group, but spinal cord tissue at 6h after ASCI showed developing neuronal necrosis. Acute inflammatory response was evident at 12h and 24h, with immune cells infiltrating into the gray matter. Vascular permeability increased and the nerve cells in gray-white matter exhibited extensive damage and necrosis at 24h and 7 days after ASCI. MiR-9 expression in ASCI tissue was significantly lower than that in normal spinal cord tissue. Statistical analysis showed a significant decrease in miR-9 expression in all the ASCI groups, compared to the non-ASCI group. Results from real-time PCR analysis revealed that MCPIP1 expression in all the ASCI groups was significantly higher than the non-ASCI group, and MCPIP1 expressions gradually increased with times at 6h-24h after ASCI. ISH revealed the following results after ASCI (1) miR-9 and MCPIP1 mRNA expression mainly distributed in ventral horn motor neurons, (2) miR-9 expression was high at 7 day after ASCI and (3) in the non-ASCI group, MCPIP1 expression was high at 6h, 12h, 24h and 3 days. CONCLUSION: MCPIP1 is significantly up-regulated after ASCI. The negative relationship between MCPIP1 and miR-9 suggest that MCPIP1 mRNA could be a target of miR-9 during ASCI. Thus, miR-9 is a marker for apoptosis in neurons, and an excellent therapeutic target for ASCI patients.

Patients with chronic hepatitis B infection display deficiency of plasmacytoid dendritic cells with reduced expression of TLR9.

Chronic hepatitis B virus (HBV) infection is a complex interaction between replicating noncytopathic virus and dysregulatory host antiviral immunity. Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immunity via secreting type I interferons. Toll-like receptor (TLR) 9 is involved in major pattern recognition receptors expressed in pDCs. The frequency of pDCs and TLR9 expression in peripheral blood mononuclear cells (PBMC) was determined, using flow cytometry. IFN-alpha production by PBMC was evaluated in vitro in the presence of cytidine phosphate guanosine (CpG) with/without pDCs. The correlation between TLR9, pDCs frequency and viral load was also evaluated. TLR9 expression in pDCs in chronic HBV patients was significantly ( approximately 50%) reduced, supported by approximately 70% reduction of TLR9 mRNA, in comparison to healthy controls, correlating with the impairment of IFN-alpha production in vitro. Furthermore, pDCs frequency in these patients was substantially reduced ( approximately 30%), inversely correlating with serum ALT levels and HBV viral load. HBsAg and HBcAg were detected by immunohistochemistry in pDCs in chronic HBV patients. We conclude that HBV infection results in reduced frequency of circulating pDCs and their functional impairment via inhibiting the expression of TLR9. These data may provide useful information in both basic research and clinical treatment of chronic HBV infection.

[Expression of Toll-like receptor 3 on peripheral blood dendritic cells in HBeAg positive patients with chronic hepatitis B].

OBJECTIVE: To elucidate the roles of Toll-like receptor 3 (TLR3) on dendritic cells (DCs) in HBV infection. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 48 healthy volunteers (HV) and 50 chronically HBV-infected patients (CH). DCs were induced and proliferated in a culture medium with rhGM-CSF and rhIL-4. We stimulated DCs with poly I:C and then TLR3, HLA-DR, and CD86, and CD1a expressions were examined by flow cytometry at 0 h, 12 h, 24 h and 48 h. The mRNA expressions of TLR3 were quantified by real-time PCR. RESULTS: TLR3 expression on DCs before the poly I:C stimulation and afterwards on the 12 h, 24 h, and 48 h were 69.2%+/-20.4%, 76.0%+/-18.6%, 78.2%+/-19.5% and 85.5%+/-6.9% respectively in the CH group, and 70.8%+/-11.2%, 67.5%+/-20.9%, 86.3%+/-14.7%, 68.6%+/-16.9% in the HV group. The expressions of TLR3 were up-regulated significantly at 24 h and 48 h after stimulation with poly I:C in the HV group, and in the CH group they were not significantly increased at 24 h but obviously increased at 48 h. The mRNA expressions of TLR3 increased significantly at 12 h in the HV groups, and at 48 h in CH group. The rate of CD86 expressions increased after poly I:C stimulation, and the increased rates were 12.6%+/-9.8%, 23.8%+/-20.0%, 20.7%+/-14.3% in the CH group, and 31.0%+/-25.0%, 43.4%+/-24.7%, 44.6%+/-25.5% in the HV group at 12 h, 24 h and 48 h after poly I:C stimulation. There was a marked increase of the expression level of CD86 in the HV group. In contrast, the level was only slightly increased in the CH group (31.0% vs 12.6%). The differences between the two groups were significant at 24 h and 48 h. No significant differences were detected in HLA-DR and CD1a between the two groups. CONCLUSIONS: The increase of expression level of TLR3 is slower in the CH group than that in the HV group. A marked increase of the expression level of CD86 is observed in the HV group. Our results suggest that abnormal expression of TLR3 and CD86 may relate to the persistence of HBV infection.

RNA silencing of Waxy gene results in low levels of amylose in the seeds of transgenic wheat (Triticum aestivum L.).

In this study, the level of amylose was reduced in wheat seeds by RNAi strategy. Because the synthesis of amylose is catalyzed by the granule-bound starch synthase I (GBSSI or WAXY protein), the Waxy gene of wheat was isolated from wheat seeds by using RT-PCR. Southern analysis confirmed that there were three Waxy genes in wheat genome. Northern hybridization showed that Waxy mRNA accumulated in seeds following pollination. By RNAi strategy,the 683 bp sense and antisense fragments in reverse orientation separated by a 150 bp intron were cloned into pCAMBIA 3300 just downstream of the maize ub/1 promoter. By Agrobacteriurn-mediated wheat transformation method, four transgenic plants (Cultivar Yangmai 10) were identified by PCR, RT-PCR and leaf painting assay. The level of amylose in the endosperm were significantly reduced in transgenic seeds as checked by iodine staining and analysis of amylose content. The results indicated that RNA silencing of Waxy gene resulted in low level of amylose in the seeds of transgenic wheat.