Dataset Information

Identification of Circulating miRNAs in a Mouse Model of Nerve Allograft Transplantation under FK506 Immunosuppression by Illumina Small RNA Deep Sequencing.

ABSTRACT:

Background

This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation in the presence and absence of FK506 immunosuppression.

Methods

A 1?cm BALB/c donor sciatic nerve graft was transplanted into the sciatic nerve gaps created in recipient C57BL/6 mice with or without daily FK506 immunosuppression [1?mg/(kg·d)]. At 3, 7, and 14?d after nerve allotransplantation, serum samples were collected for miRNA expression analysis by Illumina small RNA deep sequencing.

Results

Sequence analysis showed that the dominant size of circulating small RNAs after nerve allotransplantation was 22 nucleotides, followed by 23-nucleotide sequences. Nine upregulated circulating miRNAs (let-7e-5p, miR-101a-3p, miR-151-5p, miR-181a-5p, miR-204-5p, miR-340-5p, miR-381-3p, miR-411-5p, miR-9-5p, and miR-219-2-3p) were identified at 3?d, but none was identified at 7 or 14?d. Among them, miR-9-5p had the highest fold-change of >50-fold, followed by miR-340-5p with 38.8-fold. The presence of these nine miRNAs was not significant at 7 and 14?d after nerve allotransplantation with or without immunosuppression, showing that these miRNAs are not ideal biomarkers for monitoring rejection of deep-buried nerve allografts, a response usually observed later.

Conclusions

We identified nine upregulated circulating miRNAs, which may have a biological function, particularly during the early stages after nerve allotransplantation under FK506 immunosuppression.

SUBMITTER: Wu SC

PROVIDER: S-EPMC4578739 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

Identification of Circulating miRNAs in a Mouse Model of Nerve Allograft Transplantation under FK506 Immunosuppression by Illumina Small RNA Deep Sequencing.

Wu Shao-Chun SC Rau Cheng-Shyuan CS Yang Johnson Chia-Shen JC Lu Tsu-Hsiang TH Wu Yi-Chan YC Chen Yi-Chun YC Tzeng Siou-Ling SL Wu Chia-Jung CJ Lin Chia-Wei CW Hsieh Ching-Hua CH

Disease markers 20150908

<h4>Background</h4>This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation in the presence and absence of FK506 immunosuppression.<h4>Methods</h4>A 1 cm BALB/c donor sciatic nerve graft was transplanted into the sciatic nerve gaps created in recipient C57BL/6 mice with or without daily FK506 immunosuppression [1 mg/(kg·d)]. At 3, 7, and 14 d after nerve allotransplantation, serum samples were collected for miRNA expression analysis ...[more]

PMID: 26435568

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Project description:This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation. Experimental design Male Balb/c and C57BL/6 mice (age, 10M-bM-^@M-^S12 weeks; weight, 30M-bM-^@M-^S35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The Balb/c mice served as sciatic nerve allograft donors. The C57BL/6 mice were randomized into the group of nerve allograft transplantation with FK506 treatment and the group without FK506 treatment. These species of mice were selected on the basis of disparity at the MHC locus and prior experience with reciprocal rejection of grafts between these murine strains [18]. The mice were anesthetized by intraperitoneal injection of an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine. The anesthetized mice were restrained in a supine position on a heated pad to maintain the body temperature at 37M-BM-0C. Under aseptic conditions, with sterile povidone/iodine preparation and 70% ethanol and sterile instruments and drapes, the skin over the proximal right hindlimb was incised, and the underlying biceps femoris muscle was bluntly dissected to expose the sciatic nerve. An established mouse sciatic nerve allotransplantation model was used. In brief, 1-cm Balb/c donor sciatic nerve grafts were transplanted in reverse orientation into 0.5-cm sciatic nerve gaps created in the recipient Balb/c mice. Tension-free repair was then performed under an operating microscope with three 11M-bM-^@M-^S0 nylon (Ethicon Inc., Somerville, NJ) interrupted epineurial sutures under 40X magnification. The muscle was closed with 5M-bM-^@M-^S0 vicryl sutures, and the skin with interrupted 5M-bM-^@M-^S0 nylon sutures. The animals were monitored to ensure appropriate feeding and diet after surgery. FK506 was administered subcutaneously at 1 mg/(kg-d) throughout the experimental course. At 3, 7, and 14 d after initial surgery (n = 3 animals per group at each time point), the whole blood samples were collected at the indicated times in tubes containing anticoagulant. (F)3d, (F)7d and (F)14d were used to indicate the serum samples of allotransplantation mice with FK506 immunosuppression for 3, 7, and 14 d, respectively. (N)3d, (N)7d and (N)14d were used to indicate the serum samples of allotransplantation mice without FK506 immunosuppression and sacrificed at postoperative 3, 7, and 14 d, respectively. After the whole blood samples were incubated at room temperature for 15 min, they were centrifuged at 3,000 M-CM-^Wg for 10 min, white blood cells were slowly removed from the corresponding layers, and the serum was extracted and stored at M-bM-^@M-^S80M-BM-0C before processing for RNA analyses. Small RNA libraries generated from the serum of mice of above groups were analyzed using an Illumina HiSeq2000 Sequencer.

Project description:We profiled the expression of circulating microRNAs (miRNAs) in mice exposed to gram-positive and gram-negative bacteria using Illumina small RNA deep sequencing. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. Male C57BL/6 mice (age, 10–12 weeks; weight, 30–35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The mice were anesthetized by intraperitoneal injection of an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine. The anesthetized mice were restrained in a supine position on a heated pad to maintain body temperature at 37°C. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) purchased from Caliper (Caliper, USA) were used to induce bacterial infection in the mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). To create mixed gram-negative and gram-positive bacterial infection, 1 × 108 Xen14 bacteria and 1 × 108 Xen29 bacteria/100 μL of PBS were used for wound contamination. Three animal models were used to create bacterial infection routes: subcutaneous injection (hereafter referred to as (I)), cut wound (hereafter referred to as (C)), and skin grafting (hereafter referred to as (S)). In the (I) model, E. coli and/or S. aureus suspensions were injected subcutaneously into the backs of the mice using an Fr. 25 needle. In the (C) model, a 1 cm incision wound was created in the midline of the back, smeared with E. coli and/or S. aureus suspension, and the wound was closed directly with a 4-0 nylon suture. In the (S) model, a 1×1 cm rectangular full- thickness skin graft was lifted from the backs of the mice, E. coli and/or S. aureus suspensions were spread over the wound bed, and the skin graft was reattached and closed with a 4-0 nylon suture. An additional group of animals in each of these three models was inoculated with PBS to serve as a negative control. Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer.

Dataset Information

Identification of Circulating miRNAs in a Mouse Model of Nerve Allograft Transplantation under FK506 Immunosuppression by Illumina Small RNA Deep Sequencing.

Background

Methods

Results

Conclusions

Publications

Identification of Circulating miRNAs in a Mouse Model of Nerve Allograft Transplantation under FK506 Immunosuppression by Illumina Small RNA Deep Sequencing.

Similar Datasets

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