scholarly journals MiR-106b-5p represses neuropathic pain by regulating P2X4 receptor in the spinal cord in mice

2020 ◽  
Author(s):  
Huiying Du ◽  
Xinran Tan ◽  
Xuhong Wei ◽  
Zhongmin Yuan ◽  
Qingjuan Gong
Keyword(s):  
Spinal Cord ◽  
Neuropathic Pain ◽  
In Situ Hybridization ◽  
Mirna Expression ◽  
Real Time Pcr ◽  
Luciferase Reporter ◽  
Spared Nerve Injury ◽  
Quantitative Real Time Pcr ◽  
Reporter Assays

Abstract Background: P2X4 receptor (P2X4R)-mediated spinal microglial activation makes a critical contribution to pathologically enhanced pain processing in the dorsal horn. It can be upregulated under conditions of neuropathic pain. However, the specific mechanism of pathogenesis and potential molecular targets has not yet been made explicit. MicroRNAs (miRNAs) are commonly recognized as indicators in neuropathic pain pathophysiology.Methods: We established the pain model of spared nerve injury (SNI), and the 50% paw withdrawal thresholds (PWMTs) were used to assess behavior of mouse. MiRNA expression profiling was performed to detect differential expressed miRNA. The western-bolt and quantitative real time PCR to examine P2X4R and miRNA expression in the mouse. Dual-luciferase reporter assays confirmed the correlation between P2X4R and miRNA. Fluorescence in situ hybridization was used to show location between P2X4R and miRNA. Results: In the present study, we found that P2X4R was up-regulated in the spinal dorsal horn of mice following spared nerve injury (SNI), and we identified 69 miRNAs (46 up-regulated and 23 down-regulated miRNAs) were differently expressed (fold change > 2, P < 0.05). P2X4R was a major target of miR-106b-5p (one of down-regulated miRNAs in SNI) with bioinformatics technology and quantitative real time PCR analysis validated the expressed change of miR-106b-5p, and dual-luciferase reporter assays confirmed the correlation between them. Fluorescence in situ hybridization showed that miR-106b-5p was co-localized with P2X4R in the spinal cord. Transfection with miR-106b-5p mimic on BV2 cells reversed the up-regulation of P2X4R induced by LPS. Moreover, miR-106b-5p overexpression significantly attenuated neuropathic pain induced by SNI, with decreased expression of P2X4R mRNA and protein in the spinal cord.Conclusion: Taken together, our results suggest that miR-106b-5p can serve as an important regulator of neuropathic pain development by targeting P2X4R.

scholarly journals CircStrn3 Targeting microRNA-9-5p Is Involved in the Regulation of Cartilage Degeneration and Subchondral Bone Remodeling in Osteoarthritis

2021 ◽  
Author(s):  
Bin Li ◽  
Tao Ding ◽  
Haoyi Chen ◽  
Changwei Li ◽  
Bo Chen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time Pcr ◽  
Tensile Strain ◽  
Protective Factor ◽  
Medial Meniscus ◽  
Joint Disease ◽  
Quantitative Real Time Pcr ◽  
Mechanical Instability ◽  
Human And Mouse

Abstract Background: Osteoarthritis (OA) is the most frequent chronic degenerative joint disease, which is a “whole joint” disease including the pathological changes in the cartilage, subchondral bone and the synovium. Mechanical instability is the initiation of the development of OA. Methods: Minus RNA sequencing, fluorescence in situ hybridization and quantitative real-time PCR were used to detect the expression of circStrn3 in human and mouse OA cartilage tissues and chondrocytes. Stimulate chondrocytes to secrete exosomes miR-9-5p by stretching strain. Intra-articular injection of exosomes miR-9-5p into the OA model induced by the operation of instability of the medial meniscus in mice.Results: In the present study, minus RNA sequencing data showed that tensile strain could decrease the expression of circStrn3 in chondrocytes. The results of fluorescence in situ hybridization and quantitative Real-time PCR showed that circStrn3 expression was significantly decreased in human and mouse OA cartilage tissues and chondrocytes. CircStrn3 could inhibit matrix metabolism of chondrocytes through competitively 'sponging' miRNA-9-5p targeting kruppel-like factor 5 (KLF5), indicating that the decreasing of circStrn3 might be a protective factor in mechanical instability-induced OA. Further studies showed that the tensile strain stimulated chondrocytes to secrete exosomes miR-9-5p. Exosomes with high miR-9-5p expression from chondrocytes could inhibit osteoblasts differentiation by targeting KLF5. In addition, intra-articular injection of exosomal miR-9-5p obviously alleviated the progression of OA induced by destabilized medial meniscus surgery in mice. Conclusions: Taken together, these results demonstrated that the reduction of circStrn3 caused the increasing of miR-9-5p, which acted as a protective factor in mechanical instability-induced OA and provided a novel mechanism of communication among joint components and a potential application for the treatment of OA.

scholarly journals Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization

2006 ◽  
Vol 72 (6) ◽  
pp. 4163-4171 ◽  
Author(s):  
Udo Friedrich ◽  
Jan Lenke
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr ◽  
Starter Cultures ◽  
Calcium Citrate ◽  
Quantitative Real Time Pcr ◽  
Dairy Starter Cultures

ABSTRACT Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.

Low-level copy gain versus amplification of myc oncogenes in medulloblastoma: utility in predicting prognosis and survival

2009 ◽  
Vol 3 (1) ◽  
pp. 61-65 ◽  
Author(s):  
Hidehiro Takei ◽  
Yummy Nguyen ◽  
Vidya Mehta ◽  
Murali Chintagumpala ◽  
Robert C. Dauser ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time ◽  
Real Time Pcr ◽  
Large Cell ◽  
Histological Subtype ◽  
Quantitative Real Time Pcr ◽  
Low Level ◽  
Kaplan Meier ◽  
High Level

Object Medulloblastoma (MB) is a malignant embryonal tumor of the cerebellum. Amplification of c-myc or N-myc is infrequently identified and, when present, is often associated with the large cell/anaplastic (LC/A) phenotype. The frequency of low-level copy gain of myc oncogenes and its relationship to prognosis of MB has not been explored. Methods Archival cases of MB were histologically reviewed and classified into 3 major subtypes: classic, nodular, and LC/A. Using quantitative real-time polymerase chain reaction (PCR), the authors analyzed 58 cases with a pure histological subtype for the copy number (CN) of myc (c-myc and N-myc) oncogenes. Cases with > 5-fold CN were further analyzed using the fluorescent in situ hybridization (FISH) assay. Kaplan-Meier survival analysis was performed. Results A > 5-fold myc CN was noted in 5 (20.8%) of 24 LC/A, 1 (5.3%) of 19 classic, and 2 (13.3%) of 15 nodular subtypes. In a significant number of tumors (14 [56%] of 24 LC/A, 13 [68%] of 19 classic, and 10 [67%] of 15 nodular MBs) the CN was > 2-fold but < 5-fold. High-level amplification, defined as > 10-fold CN, was only seen in the LC/A subtype (5 cases), although moderate amplification (> 5-fold but < 10-fold) could be detected in other histological subtypes. Fluorescence in situ hybridization readily detected most cases corresponding to tumors with > 5-fold amplicon CN by quantitative real-time PCR, and could detect all 5 cases with > 10-fold CN by quantitative real-time PCR. The group of patients with > 5-fold myc amplicon CN showed significantly shorter survival than those with < 5-fold CN (p = 0.045), independent of histological subtype. Conclusions Since FISH could easily detect most cases in the moderate-to-high myc gene amplification (> 5-fold CN) group, the FISH assay has utility in detecting subsets of MB with poorer prognosis.

MicroRNA-155 is upregulated in the placentas of patients with preeclampsia and affects trophoblast apoptosis by targeting SHH/GLi1/BCL2

2020 ◽  
pp. 096032712095425
Author(s):  
Zhenpeng Wang ◽  
Yanhong Shan ◽  
Yi Yang ◽  
Tianshu Wang ◽  
Zhiheng Guo
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Characteristic Curve ◽  
Serum Levels ◽  
Luciferase Reporter ◽  
Quantitative Real Time Pcr ◽  
Multiple Risk Factors ◽  
Reporter Assays

The pathogenesis of preeclampsia (PE) is complicated and multiple risk factors have been associated with its occurrence. Still, the underlying molecular mechanisms involved in PE remain elusive. Aberrant apoptosis and insufficient invasion of trophoblasts have been observed and are considered vital pathological features in PE. Herein, we found that miR-155 can specifically degrade the mRNA of the Hedgehog ligand sonic hedgehog (SHH), using dual luciferase reporter assays. Quantitative real-time PCR found that administering miR-155 mimics or inhibitors could significantly decrease or increase the expression of SHH in the trophoblasts, respectively. The transcription levels of miR-155 in the placenta were higher in patients with PE compared to the levels in healthy pregnant women, as shown by quantitative real-time PCR. Serum levels of miR-155 could predict the diagnosis of PE by receiver operating characteristic curve analysis and diagnosis evaluation tests. A significant increase in apoptosis was observed after administering miR-155 in HTR8/SVneo cells cultured ex vivo, accompanied by reduced proliferation. Mechanistically, transcriptional activity and expression of GLi1 were also inhibited under treatment of miR-155, and could be recovered after supplying additional recombinant human SHH to primary trophoblasts from patients, as determined by luciferase activity assays and western blotting. We further found that inhibiting miR-155 increased the production of SHH and improved the phenotype in primary trophoblasts from patients with PE. Our data show that miR-155 regulates apoptosis of trophoblasts in PE, which has potential value for predicting PE risk and might be deemed as a therapeutic target for treating PE.

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