| Objectives:It has been demonstrated that circulating miRNAs hold promise to serve as practicable molecular markers for diverse physiological and pathological conditions. In this investigation, we chose partial hepatectomy (PH) on rat as traumatic injury model. Then we measured rat serum CRP, ALT and AST, and stained liver tissue section with hematoxylin and eosin (HE) post PH. The miRNA microarray was used to assess the level and composition of rat serum miRNAs. Real-time RT-PCR was used to confirm the expression levels of miR-9 in rat serum.METHODS:1. Male Sprague-Dawley rats of 7 to 10 weeks of age (body weight, 250±20 g) were kept in groups of 2 in standard breeding cages and maintained in a temperature-controlled animal facility (21±1?C), with a light/dark cycle of 12 h and ad libitum access to standard laboratory chow and water. 2. 2/3 PH was performed according to the technique described by Higgins and Anderson. After being anesthetized, the liver was exposed through a 2-3cm longitudinal incision in the abdomen and the vascular pedicles were ligated. Then the left and middle lobes, totaling about two-thirds of the liver, were resected. We also used 1/3 PH (only medial lobe was removed), a procedure that causes less injury. Rats were killed at 6, 12, 24 and 48h after PH.3. The blood samples were collected from rats in different time points. The blood was centrifuged at 1500 rpm for 15 min at 4°C and then the supernatant (serum) was carefully transferred into 1.5 ml Eppendorf tubes for further experiment. Serum samples were stored at - 80°C until use.4. Serum CRP, AST and ALT levels were measured to evaluate the severity of trauma. Tissue samples from remaining liver were sectioned from each experimental group and immediately fixed in 10% neutral-buffered formalin, embedded in paraffin for histological examination. Tissue sections (4 mm-thick) were stained with hematoxylin and eosin (HE) and examined under a light microscope.5. Rat blood samples were collected into vacutainer tubes containing EDTA. All the samples were stored at room temperature and were immediately used as a source of leukocytes and polymorphonuclear leukocytes (PMN). Rat leukocytes and PMN were separated by animal white blood cell isolation kit and PMN isolation kit according to the protocol provided by the manufacturer (GenMed, USA). Purity of the populations of leukocytes and PMN were routinely assessed by flow cytometry (BD Biosciences, Mountain View, CA, USA).6. The miRNA microarray was used to assess the level and composition of miRNA. After having passed RNA measurement on the Nanodrop instrument, the samples were labeled using the miRCURY? Hy3? Power labeling kit (Exiqon) and hybridized on the miRCURY? LNA Array (v.11.0). The miRCURY LNA? miRNA Array contained more than 1700 probes for all organisms and viruses listed in miRBase, and have very high miRBase coverage.7. Real-time RT-PCR was used to confirm the expression levels of miR-9 in rat serum. Reverse transcription PCR was performed according to the protocol of PrimeScript? RT reagent Kit (TaKaRa, Otsu, Japan),Real Time PCR was performed with SYBR premix Ex Taq II (TaKaRa) on Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, USA) in accordance with the manufacturer's instructions. TheΔΔCt method determined miRNA expression level. Quantitative values were determined using the 2-△△Ct equation.Results:1. The serum ALT, AST levels and histological changes showed differences among 1/3 and 2/3 PH group; however, the variation of CRP measurements were not sensitive enough.2. Comparing the 2/3 PH groups with controls, the serum miRNAs expression pattern was found to be significantly different. 27 miRNAs were found to be expressed up-regulated more than two fold in 2/3 PH rats serum compared to controls. Furthermore, five of them, miR-9, miR-133a, miR-122, miR-133b and miR-183, were found up-regulated more than ten fold. Especially, the expression of miR-9 demonstrated the highest up-regulated (70-fold overexpressed).3. The data showed that PH-induced miR-9 change was significantly different between 2/3 PH and 1/3 PH (2/3 PH induced higher up- regulation of miR-9 than 1/3 PH). Pearson's correlation analysis was performed to estimate the potential relationship between miR-9 expression level and severity of 2/3PH-induced traumatic injury. Scatter plots illustrated that serum miR-9 expression level was significantly positively correlated with serum AST, ALT, and CRP levels.4. Our data showed that miR-9 was produced at low level in rat serum under normal condition. 2/3 PH resulted-in significantly up-regulated of miR-9 expression in leukocytes. The expression levels of miR-9 in leukocytes which induced by 2/3 PH, could been found up-regulated obviously after 6 h and steadily increasing over the time period assessed. The change of miR-9 expression in PMN was similar to leukocytes.5. Except for expression in leukocytes, miR-9 was also detected up-regulated in brain compare to other tissues from rat that was injured by PH. RT-PCR analysis showed that miR-9 basal expression levels vary considerably among different areas in rat brain (cerebellum showed the highest expression level). The trauma imposed on rat rapidly induced up-regulation of miR-9 expression in cerebral cortex, hypothalamus and pituitary gland. But the expression levels of miR-9 were not significantly altered in cerebellum and hippocampus. Furthermore, the expression levels of miR-9 in lung, liver and heart were low, and 2/3 PH could not alter their miR-9 expression obviously even the traumatic liverConclusions:We provided evidence that injury caused by PH could make significant change in the spectra and levels of serum miRNAs; and the level of specific serum miRNAs such as miR-9, could be used as a novel serum-based biomarker potentially offering more sensitive tests than those current protein-based biomarkers for early diagnosis of trauma injury. And furthermore, our data showed that miR-9 might play an important role in modulation of stress response post trauma. |
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