Objective To investigate the changes of microRNA-150 ( miR-150) in peripheral blood leukocytes in sepsis patients, and their relationship with expression of immune cytokines and sepsis severity. Methods The level of mature miR-150 was quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to that of control miRNA, U6, in peripheral blood leukocytes of 40 patients with sepsis, 20 patients with systemic inflammatory response syndrome ( SIRS) , and 20 normal individuals. Serum concentrations of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) were measured by enzyme-linked immunoabsorbent assay in all subjects. The sequential organ failure assessment ( SOFA) score systemwas used to evaluate the severity of sepsis. The relationships between miR-150 and the white blood cell count ( WBC) , TNF-α, IL-10 and SOFA score of the sepsis patients were analyzed. Results MiR-150 was stable for at least 5 days when specimen stored at 4 ℃ and the determination of miR-150 had a broad linear detecting range ( 6. 97-6. 97 ×104 pg/ μL RNA, the lowest detecting limit: 6. 97 pg/μL RNA,r=0.999) .MiR-150 expression in the peripheral blood leukocytes in the sepsis group was significantly lower than that in the healthy control group ( Plt;0.01) , while WBC, IL-10 and IL-10/TNF-α ratio were significantly higher ( Plt;0.05) . There was no significant difference in levels of miR-150, IL-10, IL-10/TNF-α ratio, and WBC between the sepsis group and the SIRS group (Pgt;0.05) . There was no significant difference in serum concentrations of TNF-α among three groups ( Pgt;0.05) . MiR-150 expression in non-survivor sepsis patients was significantly lower than that in survivor sepsis patients (Plt;0.05) , while serum IL-10 and IL-10/TNF-αratio were significantly higher (Plt;0.01) , but there was no significant difference in serum TNF-α between the non-survivor group and the survivor group ( Pgt;0.05) . There was significantly negative correlation between miR-150 and SOFA score, TNF-α and IL-10( r=-0. 619, - 0.457, -0. 431, Plt;0.05, respectively) , but no correlation between miR-150 and WBC ( r =-0. 184, Pgt;0.05) . There was no relationship between serum TNF-α, IL-10, IL-10 /TNF-α ratio or SOFA score ( Pgt;0.05) . Conclusions MiR-150 expression in the peripheral blood specimens is significantly decreased in sepsis patients. The expression level of miR-150 not only reflect the situation of inflammatory response, but also may be used as a prognostic marker in sepsis, as it can reflect the severity of sepsis in certain degree.
Abstract： Objective To observe the expression changes of microRNA 1 （miRNA-1） and microRNA 21（miRNA-21） after ischemic preconditioning （IPC）, ischemic postconditioning （IPO） and remote ischemic preconditioning （RIPC）in an ischemia-reperfusion rat heart model in vitro, as well as the expression of their target protein heat shock protein 70 （HSP70） and programmed cell death 4 （PDCD4）, and evaluate whether miRNA are involved in endogenous cardio-protective mechanism. Methods The Langendorff-perfused Sprague-Dawley rat hearts were randomly assigned into one of the four groups, control group （CON group, n=12）, ischemia preconditioning group （IPC group， n=12） , ischemia postconditioning group （IPO group， n=12） and remote ischemia preconditioning group （RIPC group，n=12）. Cardiac function was digitalized and analyzed. The expression of HSP70, PDCD4, B-cell lymphoma/leukemia-2 （Bcl-2） and Bax was detected by Western blotting. The expression of miRNA-1 and miRNA-21 was detected by real-time reverse transcriotion-polymerase chain reaction （RT-PCR）. Assessment of cardiac infarct size and myocardial apoptosis was determined using triphenyltetrazolium chloride （TTC） assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） assay respectively. Results The expressions of miRNA-1 and miRNA-21 were up-regulated in IPC group, but the expression of miRNA-1 was down-regulated in RIPC group and IPO group （P＜0.05）. The expressionsof PDCD4, HSP70 and Bax were down-regulated in ‘conditioning’ groups compared with CON group （P＜0.05）. The expression of Bcl-2 was not statistically different among the four groups. The infarct size and the myocardial apoptosis in ‘conditioning’ hearts were significantly decreased compared with CON group （P＜0.05）. Conclusion The expressions of the miRNA-1 and miRNA-21 are different in IPC, RIPC and IPO groups, and their target proteins are not inversely correlated with the miRNAs in all the ‘conditioning’ groups.
Objective To investigate the role of micro RNA-451 (miRNA-451) in promoting the osteogenesis of mesenchymal stem cells (MSCs) by targeting regulatory calcium binding protein 39 (CAB39). Methods pMIR-report and pRL-TK vectors were selected to identify the relationship between miRNA-451 and CAB39 by using dual-luciferase reporter assay. pre-miRNA-451 (group A), anti-miRNA-451 (group C), pre-miRNA negative control (group B), and anti-miRNA negative control (group D) were transfected into the C3H10T1/2 cells, respectively. Then, the cells were collected after osteogenic induction for 7 and 14 days. At 7 and 14 days, the real-time fluorescent quantitative PCR and Western blot assays were performed to detect the related osteogenetic biomarkers [Runx2 and alkaline phosphatase (ALP) mRNA] and expressions of CAB39 protein. At 14 days, the extracellular calcium deposition during the osteogenesis of MSCs was tested by Alizarin red staining method. Results CAB39 was the target gene of miRNA-451. At 7 and 14 days after osteogenic induction, the mRNA expressions of Runx2 and ALP in group A were significantly higher than those in group B (P lt; 0.05), and the expressions in group C was significantly lower than those in group D (P lt; 0.05). Furthermore, at 14 days after osteogenic induction, the protein expression of CAB39 in group A (0.55 ± 0.05) was significantly lower than that in group B (1.00 ± 0.07), and the protein expression in group C (1.21 ± 0.05) was significantly higher than that in group D (1.00 ± 0.04), all showing significant difference (P lt; 0.05). Finally, at 14 days after osteogenic induction, the extracellular calcium deposition in group A was obviously more than that in group B, and group C was downregulated when compared with group D. Conclusion miRNA-451 can promote the osteogenesis process of MSCs by downregulating the CAB39.
Objective To summary the functional roles and molecular mechanisms of microRNA (miRNA) in osteoblast differentiation so as to supply information for basic and cl inical researches. Methods Recent l iterature concerning miRNA in osteoblast differentiation was reviewed. The information was classified and summarized. Results miRNAs critically regulate bone morphogenetic protein, transforming growth factor β, and Wnt/β-catenin signal ing pathways during osteoblast differentiation. In pathological conditions, especially in some disorders of abnormal osteoblast differentiation, downregulated miRNA expression has been observed. Conclusion miRNA may represent a novel biomarker for diagnosis, and a candidate target therapies for the disorders with abnormal osteoblast differentiation.
Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogeneticprotein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly whencompared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.
Objective To review the regulation and mechanism of the microRNAs (miRNAs) in the bone and cartilage tissue. Methods Recent l iterature concerning the regulation and mechanism of the miRNAs in the bone and cartilage tissue was extensively reviewed, summarized, and analyzed. Results Recently miRNAs is a hot topic in the bone and cartilage tissue. More and more materials show its important regulatory role in osteogenesis and cartilage growth andregeneration, but the definite mechanisms have not been clear yet. Conclusion The study on miRNAs of bone and cartilage tissue can provide a new access to understanding the degenerative osteoarthritic diseases.
Objective To review the research progress of heterotopic ossification (HO) pathogenesis.Methods Recent articles about HO including the risk factors and pathogenesis were reviewed and comprehensively analyzed. Results The pathogenesis of HO is not completely understood, but the extracellular factors, signaling pathways, and transcription factors in the pathogenesis of HO are understood deeply, such as bone morphogenic protein, Smad signaling, and core binding factor α1/runt-related transcription factor 2, which are probably involved in HO. Furthermore, some related microRNAs are also probably involved in HO. Conclusion The pathogenesis of HO should be further investigated so as to lay a foundation for preventing and treating HO.
Objective To review the progress, controversy and trend in the regulation and mechanism of the microRNAs (miRNAs) during the osteogenesis. Methods Recent l iterature concerning regulation and mechanism of the miRNAs during the osteogenesis was extensively reviewed, summarized and analyzed. Results Recently miRNAs was a hot topic for osteogenesis. More and more materials showed its important role in ossification, but its definite mechanism was notclear. Conclusion Osteogenesis can be strengthened by miRNAs technology, which has a bright future and may also provide the molecular mechanism. The study on miRNAs of osteogenesis can provide a model to analyze and compare the osteogenetic effects of novel drugs.