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.
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 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 investigate the expression level and methylation level of micro RNA-34b（miR-34b） gene in papillary thyroid carcinoma （PTC）， and to analyze the relationship between methylation and clinicopathological characters of PTC. Methods PTC tissues and tumor adjacent tissues were collected from 25 patients with PTC who underwent operation in Huai’an First People’s Hospital of Nanjing Medical University from Sep. 2008 to Oct. 2010. Expression of miR-34b gene and level of methylation in gene promoter were detected by real time PCR and methylation-specific PCR in the 2 kinds of tissues， respectively. Results The expression value of miR-34b mRNA in PTC tissues was 0.85±0.05， which was significantly lower than those of tumor adjacent tissues （1.62±0.09）， P=0.030. There were methylation in 18 （72%，18/25） PTC tissues， and 10 （40%，10/25） in tumor adjacent tissues， and the ratio of methylation was higher in PTC tissues （P=0.021）. In PTC tissues， methylation was not related to age， gender， tumor size， TNM stage， and invasion of the capsule （P＞0.05）， but was related to lymph node metastasis （P＜0.05）. Ratio of methylation in patients with lymph node metastasis was significantly higher than those of patients with no lymph node metastasis. Conclusion Methylation of miR-34b gene promoter is one of the reasons for inactivation of PTC， and it may be related to the development and metastasis of PTC， which needs to be further investigated.
The early detection, diagnosis and treatment of lung cancer are the key points to reduce mortality and improve the prognosis. Detecting tumor markers in blood is a minimally-invasive, cost-effective, easy to administer and approachable test. A microsatellite consists of a tract of tenderly repeated DNA motifs that range in length from two to five nucleotides. Microsatellite alterations (MA) have been described as two types: loss of heterozygosity (LOH) and microsatellite instability (MSI). MicroRNA (miRNA) is a noncoding RNA and protein involved in regulating gene expression in the transcription level. In recent years, some miRNA markers and microsatellite alterations show significant tumor association, tissue specific and stability. Therefore, we write this review to discuss the microsatellite alterations and microRNA in blood of lung cancer.
Circular RNAs (circRNAs) are a novel class of non-coding RNAs, which are more stable than linear RNAs for their closed circular structure by covalent bond. CircRNAs exist in a large variety of cells and regulate the expressions of target genes. Moreover, circRNAs are closely related to various diseases and have a potential value as biomarkers and prognostic markers clinically. In this article, the classification and biological functions of circRNA molecules (including being as microRNA sponges, regulating gene transcription, regulating RNA binding protein and the potential translation function) are summarized, and the latest research progress of circRNAs in rheumatoid arthritis is reviewed.
ObjectiveTo investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot.ResultsCompared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased (P<0.05); the above indexes in group D were significantly decreased (P<0.05); the above indexes between groups C, E and group A were not significantly different (P>0.05).ConclusionmiR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.