Mmu-let-7a-5p inhibits macrophage apoptosis by targeting CASP3 to increase bacterial load and facilities mycobacterium survival.

We have been trying to find a miRNA that can specifically regulate the function of mycobacterial host cells to achieve the purpose of eliminating Mycobacterium tuberculosis. The purpose of this study is to investigate the regulation of mmu-let-7a-5p on macrophages apoptosis and its effect on intracellular BCG clearance. After a series of in vitro experiments, we found that mmu-let-7a-5p could negatively regulate the apoptosis of macrophages by targeting Caspase-3. The extrinsic apoptosis signal axis TNFR1/FADD/Caspase-8/Caspase-3 was inhibited after BCG infection. Up-regulated the expression level of mmu-let-7a-5p increase the cell proliferation viability and inhibit apoptosis rate of macrophages, but down-regulated its level could apparently reduce the bacterial load of intracellular Mycobacteria and accelerate the clearance of residual Mycobacteria effectively. Mmu-let-7a-5p has great potential to be utilized as an optimal candidate exosomal loaded miRNA for anti-tuberculosis immunotherapy in our subsequent research.

Inhibition of miR-96-5p alleviates intervertebral disc degeneration by regulating the peroxisome proliferator-activated receptor γ/nuclear factor-kappaB pathway.

BACKGROUND: Intervertebral disc degeneration (IDD) is the main pathogenesis of low back pain. MicroRNAs (miRNAs) have been found to exert regulatory function in IDD. This study aimed to investigate the effect and potential mechanism of miR-96-5p in IDD. METHODS: In vitro cell model of IDD was established by treating human nucleus pulposus cells (HNPCs) with interleukin-1ß (IL-1ß). The level of peroxisome proliferator-activated receptor γ (PPARγ) was examined in the IDD cell model by Western blot and quantification real-time reverse transcription-polymerase chain reaction (qRT-PCR). The expression level of miR-96-5p was detected by RT-qPCR. Effects of PPARγ or/and PPARγ agonist on inflammatory factors, extracellular matrix (ECM), apoptosis, and nuclear factor-kappaB (NF-κB) nuclear translocation were examined through enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry assay, and immunofluorescence staining. The Starbase database and dual luciferase reporter assay were used to predict and validate the targeting relationship between miR-96-5p and PPARγ, and rescue assay was performed to gain insight into the role of miR-96-5p on IDD through PPARγ/NF-κB signaling. RESULTS: PPARγ expression reduced with concentration and time under IL-1ß stimulation, while miR-96-5p expression showed the reverse trend (P < 0.05). Upregulation or/and activation of PPARγ inhibited IL-1ß-induced the increase in inflammatory factor levels, apoptosis, degradation of the ECM, and the nuclear translocation of NF-κB (P < 0.05). MiR-96-5p was highly expressed but PPARγ was lowly expressed in IDD, while knockdown of PPARγ partially reversed remission of IDD induced by miR-96-5p downregulation (P < 0.05). MiR-96-5p promoted NF-κB entry into the nucleus but PPARγ inhibited this process. CONCLUSION: Inhibition of miR-96-5p suppressed IDD progression by regulating the PPARγ/NF-κB pathway. MiR-96-5p may be a promising target for IDD treatment clinically.

Inhibition of miR-221-3p promotes axonal regeneration and repair of primary sensory neurons via regulating p27 expression.

This study aimed to explore the key microRNA (miRNA) playing a vital role in axonal regeneration with a hostile microenvironment after spinal cord injury. Based on the theory that sciatic nerve conditioning injury (SNCI) could promote the repair of the injured dorsal column. Differentially expressed miRNAs were screened according to the microarray, revealing that 47 known miRNAs were differentially expressed after injury and perhaps involved in nerve regeneration. Among the 47 miRNAs, the expression of miR-221-3p decreased sharply in the SNCI group compared with the simple dorsal column lesion (SDCL) group. Subsequently, it was confirmed that p27 was the target gene of miR-221-3p from luciferase reporter assay. Further, we found that inhibition of miR-221-3p expression could specifically target p27 to upregulate the expression of growth-associated protein 43 (GAP-43), α-tubulin acetyltransferase (α-TAT1) together with α-tubulin, and advance the regeneration of dorsal root ganglion (DRG) neuronal axons. Chondroitin sulfate proteoglycans (CSPGs) are the main components of glial scar, which can hinder the extension and growth of damaged neuronal axons. After CSPGs were used in this study, the results demonstrated that restrained miR-221-3p expression also via p27 promoted the upregulation of GAP-43, α-TAT1, and α-tubulin and enhanced the axonal growth of DRG neurons. Hence, miR-221-3p could contribute significantly to the regeneration of DRG neurons by specifically regulating p27 in the p27/CDK2/GAP-43 and p27/α-TAT1/α-tubulin pathways even in the inhibitory environment with CSPGs.

Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.

Background: The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Methods: Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Results: Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3'-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. Conclusion: mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.

Small RNA sequencing and bioinformatics analysis of RAW264.7-derived exosomes after Mycobacterium Bovis Bacillus Calmette-Guérin infection.

BACKGROUND: The mechanisms through which Mycobacterium tuberculosis evades immune surveillance during tuberculosis (TB) infection remain complex. Previous studies have found that Mycobacteria can manipulate the miRNAs of host cells to promote their survival during host-pathogen interactions, and most of these effects occur at the cellular miRNA level. We attempted to investigate the possible related mechanisms at the exosomal miRNA level. RESULTS: High-throughput sequencing revealed that Bacillus Calmette-Guérin (BCG) infection could alter the composition of the macrophage exosome content, and the expression levels of miRNAs in exosomes derived from the cell culture media of macrophages showed significant differences between the BCG-infected and non-infected groups. Compared with the non-infected group, 20 exosomal miRNAs were up-regulated and 7 exosomal miRNAs were down-regulated in the infection group (p < 0.05), of which mmu-miR-27b-3p, mmu-miR-93-5p, mmu-miR-25-3p, mmu-miR-1198-5p, mmu-let-7c-5p and let-7a-5p were significantly up-regulated. A bioinformatic analysis indicated that these differentially expressed exosomal miRNAs were involved in multiple biological processes and pathways. The target genes of top six miRNAs in up-regulated groups were positively correlated with the regulation of apoptosis. CONCLUSIONS: The expression profile of miRNA in exosomes derived from macrophage were altered after Mycobacterium Bovis Bacillus Calmette-Guérin infection, and the differentially expressed miRNAs were involved in multiple biological processes and signalling pathways. The top six up-regulated miRNAs and their targeted genes were predominantly correlated with the regulation of apoptosis.

The accuracy of MRI in diagnosing and classifying acute traumatic multiple ligament knee injuries.

BACKGROUND: Magnetic resonance imaging (MRI) is widely used for the evaluation of knee injuries, however, the accuracy of MRI in classifying multiple ligament knee injuries (MLKIs) remains unknown. This study aimed to investigate the accuracy of MRI in diagnosing and classifying acute traumatic MLKIs, we hypothesize that MRI had high accuracy in detecting and classifying MLKIs. METHODS: The clinical data of 97 patients who were diagnosed with acute traumatic MLKIs and managed by multi-ligament reconstruction between 2012 and 2020 were retrospectively reviewed. The MR images were read by two experienced radiologists and results were compared with intraoperative findings, which were considered as the reference for the identification of injured structures. The value of MRI in detecting injuries of anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), and meniscus was evaluated by calculating the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and kappa coefficients analysis. The value of MRI in classifying MLKIs was evaluated by calculating the agreement between MRI and intraoperative findings. RESULTS: For detecting the specific injured structures in MLKIs, MRI had high sensitivity (90.7% for ACL, 90.4% for PCL, and moderate specificity (63.6% for ACL, 50% for PCL) in detecting cruciate ligament injuries, moderate sensitivity (79.1% for MCL, 55.6% for LCL) and specificity (46.7% for MCL, 68.4% for LCL) in detecting collateral ligament injuries, fair sensitivity (61.5%) and low specificity (39.4%) in the diagnosis of injuries to the meniscus. For classifying the MIKIs, MRI had a moderate agreement with intraoperative findings in classifying KD-V (kappa value = 0.57), poor agreement in the KD-I (kappa value = 0.39) and KD-IIIM (kappa value = 0.31), meaningless in the KD-II and KD-IIIL (kappa value < 0). The overall agreement between MRI and intraoperative findings in classifying MLKIs was poor (kappa value = 0.23). CONCLUSIONS: MRI is valuable in early detection and diagnosis of acute MLKIs, however, the accuracy of MRI in classifying MLKIs is limited. The management of MLKIs should be based on intraoperative findings, physical examinations, and comprehensive imaging results.