Dorsal root ganglion-derived exosomes deteriorate neuropathic pain by activating microglia via the microRNA-16-5p/HECTD1/HSP90 axis.

BACKGROUND: The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism. METHODS: A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments. RESULTS: DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP. CONCLUSIONS: miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.

Dorsal root ganglion-derived exosomes deteriorate neuropathic pain by activating microglia via the microRNA-16-5p/HECTD1/ HSP90 axis

Background The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism. Methods A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments. Results DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP. Conclusions miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.

Exosomal microRNA-16-5p from macrophage exacerbates atherosclerosis via modulating mothers against decapentaplegic homolog 7.

OBJECTIVE: Studies have probed the function of microRNA (miR)-16-5p in the progression of atherosclerosis (AS), while the regulatory function of exosomal miR-16-5p from macrophage on AS remains largely unknown. This study commits to exploring the efficiency of exosomal miR-16-5p from macrophage on AS via modulating mothers against decapentaplegic homolog 7 (SMAD7). METHODS: Macrophages were cultured and transfected with miR-16-5p antagomir, then, the exosomes from macrophages were extracted. The AS mouse model was established, and miR-16-5p or SMAD7 expression in AS mice was detected. Thereafter, the effects of macrophage-derived exosomes, miR-16-5p or SMAD7 on serum inflammatory response, oxidative stress response, pathological changes and apoptosis in AS mice were observed by immunohistochemical and biochemical analysis. Finally, the binding relation between miR-16-5p and SMAD7 was examined. RESULTS: MiR-16-5p was elevated while SMAD7 was depleted in AS mice. Macrophage-derived exosomes aggravated AS progression via facilitating inflammatory response and oxidative stress, exacerbating pathological changes and increasing cell apoptosis in AS mice; while downregulation of miR-16-5p reversed the exacerbation of AS progression by macrophage-derived exosomes in AS mice. MiR-16-5p targeted SMAD7, and the down-regulated SMAD7 reversed the impacts of depleted miR-16-5p on AS progression. CONCLUSION: Exosomal miR-16-5p from macrophages aggravates AS progression via downregulating SMAD7 expression. This study provides novel therapeutic targets for AS treatment from the animal level.

LINC00152 knockdown suppresses tumorigenesis in non-small cell lung cancer via sponging miR-16-5p.

Background: Non-small cell lung cancer (NSCLC) is one of the most aggressive types of cancer worldwide. It has been reported that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of NSCLC. In addition, LINC00152 is known to be upregulated in NSCLC. However, the mechanism underlying the effect of LINC00152 on NSCLC tumorigenesis remains to be elucidated. Methods: In the present study, cell viability, apoptosis and invasion were investigated by CCK-8, flow cytometry and Transwell assays, respectively. Reverse transcription­quantitative polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels. In addition, the association between LINC00152, microRNA (miR)-16-5p and BCL2-like 2 (BCL2L2) was evaluated using a dual-luciferase assay. Results: The results demonstrated that LINC00152-knockdown significantly attenuated the viability of NSCLC cells via promoting cell apoptosis. In addition, the migration and invasion ability of NSCLC cells was also decreased following transfection of cells with LINC00152 siRNA. Furthermore, miR-16-5p inhibitor or BCLCL2-overexpression reversed LINC00152 siRNA-induced NSCLC cell growth inhibition. Conclusions: The findings of the present study demonstrated that LINC00152-silencing suppressed NSCLC tumorigenesis via regulating the miR-16-5p/BCL2L2 axis. Therefore, linc00152 has the potential as a molecular marker and may be a potential target for the treatment of non-small cell lung cancer.

Melatonin Inhibits the Malignant Progression of Glioblastoma via Regulating miR-16-5p/PIM1.

OBJECTIVES: Melatonin (MT) is a pineal hormone with antineoplastic potential. This study aims to explore the therapeutic potential and mechanism of MT on glioblastoma (GBM). METHODS: A human GBM cell line, LN229, was used to evaluate the function of MT. Cell viability, apoptosis, and migration were detected by CCK-8, flow cytometry, and transwell assays, respectively. The mRNA and protein expressions of specific genes were measured by qRT-PCR and western blot, respectively. The regulatory relationship between miR-16-5p and PIM1 was validated by dual luciferase reporter gene assay. A mouse xenograft model was established to prove the anti-tumor effect and related mechanisms of MT in vivo. RESULTS: MT inhibited the viability and migration and promoted the apoptosis of LN229 cells in a dose-dependent manner. MiR-16-5p was dose-dependently up-regulated by MT in LN229 cells, negatively regulating its target PIM1. MiR-16-5p inhibitor eliminated the anti-tumor effect of MT in LN229 cells, while si-PIM1 reversed the effect of miR-16-5p inhibitor in MT-treated cells. MT inhibited the tumor growth in vivo and MT-induced PIM1 down-regulation was reversed by miR- 16-5p inhibition in tumor tissues. CONCLUSIONS: MT inhibits the malignant progression of GBM via regulating miR-16-5p-mediated PIM1.

Overexpressing circ_0000831 is sufficient to inhibit neuroinflammation and vertigo in cerebral ischemia through a miR-16-5p-dependent mechanism.

Circular RNAs (circRNAs) hold potential as stroke-related biomarkers due to involvement in various pathophysiological processes associated with cerebral ischemia and stability in peripheral blood. Differentially expressed circulating circRNAs were identified by preliminary sequencing analysis, through which we identified underexpressed circ_0000831 in ischemic stroke (IS). Validation was performed in peripheral blood of IS patients by quantitative polymerase chain reaction. Microglia was exposed to oxygen-glucose deprivation (OGD), where polarization phenotypes and inflammation were assessed. Middle cerebral artery occlusion was performed in mice to mimic ischemic stroke-induced vertigo, where cerebral blood flow, neurological deficits, vertigo degree, infarct area, inflammation and cell apoptosis were assayed in response to ectopic expression and knockdown of circ_0000831, miR-16-5p, and AdipoR2. Mechanically, circ_0000831 bound to miR-16-5p and downregulated miR-16-5p, and AdipoR2 was targeted by miR-16-5p and increased PPARγ expression in microglia. Furthermore, circ_0000831, AdipoR2, or PPARγ overexpression or miR-16-5p inhibition alleviated neuroinflammation, vertigo, neurological deficit, and cell apoptosis in MCAO mice. Consistently, circ_0000831, AdipoR2, or PPARγ upregulation or miR-16-5p downregulation diminished apoptosis and inflammation of OGD-induced microglia. Consequently, these findings pinpoint the circ_0000831/miR-16-5p/AdipoR2 axis as an essential signaling pathway during ischemia stroke. Thus, the circRNA circ_0000831 may work as a possible target for novel treatment in patients with ischemic stroke.

Bone marrow mesenchymal stem cells-derived exosomal microRNA-16-5p restrains epithelial-mesenchymal transition in breast cancer cells via EPHA1/NF-κB signaling axis.

OBJECTIVE: This study intends to conquer the mystery of microRNA-16-5p/erythropoietin-producing hepatocellular A1/nuclear factor-κB signaling (miR-16-5p/EPHA1/NF-κB signaling) in breast cancer. METHODS: Expression of miR-16-5p, EPHA1 and NF-κB signaling-related proteins were detected. Gene overexpression or silencing was used to examine the biological roles of bone marrow mesenchymal stem cells (BMSCs)-derived exo-miR-16-5p in breast cancer. The effect of exo-miR-16-5p on tumorigenesis of breast cancer was confirmed by the xenograft nude mouse model. RESULTS: Low miR-16-5p and high EPHA1 expression were examined in breast cancer. BMSCs-derived exosomes, up-regulated miR-16-5p or down-regulated EPHA1 restrained epithelial-mesenchymal transition (EMT) of breast cancer cells and tumor growth in nude mice. Down-regulated miR-16-5p or up-regulated EPHA1 activated NF-κB signaling. Knockdown of EPHA1 or inhibition of NF-κB signaling reversed the effects of down-regulated miR-16-5p on breast cancer cells. CONCLUSION: BMSCs-derived exosomal miR-16-5p hinders breast cancer cells progression via EPHA1/NF-κB signaling axis.

Positive feedback loop of lncRNA SNHG1/miR­16­5p/GATA4 in the regulation of hypoxia/reoxygenation­induced cardiomyocyte injury.

Numerous studies have demonstrated that long non­coding RNAs (lncRNAs) serve an important regulatory role in ischemic injury of cardiomyocytes. lncRNA small nucleolar RNA host gene 1 (SNHG1) could effectively protect cardiomyocytes against various injuries. However, the role of SNHG1 in ischemic cardiomyocyte injury is unclear. It was hypothesized that SNHG1 may have a protective effect on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) by sponging microRNA (miRNA/miR). The purpose of the present study was to explore the role and molecular mechanism of SNHG1 in ischemic cardiomyocyte injury. A H9c2 cardiomyocyte H/R model was established. The expression levels of SNHG1 in cardiomyocytes treated with H/R were detected using reverse transcription­quantitative PCR. A luciferase reporter assay was used to analyze the associations among SNHG1, miR­16­5p and GATA binding protein 4 (GATA4). Chromatin immunoprecipitation experiments were performed to analyze the interaction between SNHG1 and GATA4. Cell Counting Kit­8, enzyme­linked immunosorbent assay, terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling and western blotting experiments were used to detect cell activity, lactate dehydrogenase release, apoptosis and apoptosis­related proteins (Bcl­2, Bax, Cleaved caspase­3 and Cleaved caspase­9), respectively. The expression levels of SNHG1 were downregulated in cardiomyocytes treated with H/R. Overexpression of SNHG1 had a protective effect on cardiomyocyte injury induced by H/R. In addition, SNHG1 could regulate the expression levels of GATA4 via sponging of miR­16­5p. Further experiments revealed that GATA4 could bind to the promoter region of SNHG1 and subsequently regulated the expression levels of SNHG1, indicating the important role of the positive feedback loop of SNHG1/miR­16­5p/GATA4 in cardiomyocyte ischemic injury. To conclude, the present study revealed the protective effect of the SNHG1/miR­16­5p/GATA4 positive feedback loop on cardiomyocyte injury induced by H/R and provided a potential therapeutic target for ischemic cardiomyocyte injury.

Anti-inflammatory effects of artesunate on atherosclerosis via miR-16-5p and TXNIP regulation of the NLRP3 inflammasome.

BACKGROUND: Atherosclerosis (AS) is chronic inflammatory arterial disorder. Artesunate could exhibit anti-inflammatory activity in AS, but its role in AS is still in its incipient stage. In this study, we explored the anti-inflammatory effect of artesunate in AS and its underlying mechanism. METHODS: We isolated CD14+ monocytes from peripheral blood (PB) of 115 coronary heart disease (CHD) patients and 33 non-CHD patients confirmed by coronary angiography. Phorbol myristate acetate (PMA) was used to induce the differentiation of THP-1 monocytes to macrophages. Cells were treated with artesunate at a final concentration of 2.5, 5 or 10 µmol/L. The activation of NLRP3 inflammasome was assessed by immunoblotting of apoptosis-associated speck-like protein containing caspase recruitment domain (ASC). The expression of pro-caspase-1/pro-interleukin (IL)-1ß/pro-IL-18 and their mature forms was measured using immunoblotting. A rat model of AS was induced by vitamin D3 (VD3) and a 21-day high-fat diet. RESULTS: Downregulated miR-16-5p and upregulated thioredoxin-interacting protein (TXNIP) was determined in CD14+ monocytes from CHD patients and associated with disease severity. Artesunate abrogated the activation of NLRP3 inflammasome in the presence of inflammasome activators in cultured macrophages. Artesunate reduced TXNIP expression and impaired the interaction between TXNIP and NLRP3, thereby inhibiting release of inflammatory cytokines and ASC production in cultured macrophages. In addition, miR-16-5p negatively regulated the messenger RNA (mRNA) of TXNIP. Artesunate increased the expression of miR-16-5p in a dose-dependent manner, and inhibition of miR-16-5p enhanced the secretion of inflammatory cytokines. Our in vivo experiments also demonstrated that artesunate reduced lipid accumulation, atherosclerotic plaque formation, and antagonized inflammation in a dose-dependent manner by upregulating miR-16-5p. CONCLUSIONS: In summary, the present study unveiled a mechanism underlying the anti-inflammatory role of artesunate in AS.

RETRACTED: MicroRNA-16-5p/BTG2 axis affects neurological function, autophagy and apoptosis of hippocampal neurons in Alzheimer's disease.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief as there are concerns about the reliability of the results. Concerns have been raised about the western blot bands in Figures 6 B + D having the same eyebrow shaped phenotype as found in many other publications as detailed here (https://pubpeer.com/publications/B32F93859FBAA13471ED0FFCA5BCB6). The journal requested the corresponding author to comment on these concerns and send the raw data, however the author was not able to provide uncropped images of the original gels. The Editor-in-Chief therefore no longer has confidence in the data and conclusions of this study.

RETRACTED: Exosomal microRNA-16-5p from adipose mesenchymal stem cells promotes TLR4-mediated M2 macrophage polarization in septic lung injury.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). The authors have requested that this paper be retracted as they were unable to replicate the experimental data reported in Figure 1A. The authors posit that changes in reagents or experimental conditions might be the source of their inability to do so. Additional concerns were raised about the reliability of the Western blot results in Figure 1E, Figure 4B and F, Figure 5B, and Figure 6B, as regarding 'morphology space' similarities contained within a series of papers with distinctive eyebrow blots, tabulated here (https://docs.google.com/spreadsheets/d/149EjFXVxpwkBXYJOnOHb6RhAqT4a2llhj9LM60MBffM/edit#gid=0 [nam11.safelinks.protection.outlook.com] [nam11.safelinks.protection.outlook.com]). The journal requested the authors comment on these concerns and provide raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

MicroRNA­16­5p/BIMP1/NF­κB axis regulates autophagy to exert a tumor­suppressive effect on bladder cancer.

Bladder cancer (BC) is the second most common urological disease worldwide. Previous studies have reported that microRNA (miR)­16­5p is associated with the development of BC, but whether miR­16­5p regulates BC cell autophagy remains unknown. Thus, the aim of the present study was to investigate this issue. miR­16­5p expression in BC cells was assessed by reverse transcription­quantitative PCR. Cell viability and apoptosis were detected via Cell Counting Kit­8 and flow cytometry assays, respectively. For cell autophagy detection, autophagic flux was detected using a mCherry­green fluorescent protein­microtubule­associated proteins 1A/1B light chain 3B (LC3) puncta formation assay, followed by determination of autophagy­related protein markers. The targeting relationship between miR­16­5p and caspase recruitment domain family member 10 (BIMP1) was confirmed using a dual­luciferase reporter assay, followed by detection of the BIMP1/NF­κB signaling pathway. The results showed that miR­16­5p overexpression inhibited cell viability, whereas miR­16­5p knockdown promoted cell viability in BC. Furthermore, miR­16­5p overexpression induced autophagy, which was accompanied by increased autophagic flux and expression of the autophagy­related proteins LC3­II and beclin 1, as well as decreased p62 expression, whereas miR­16­5p silencing led to an inhibition of autophagy in BC cells. Moreover, autophagy inhibitor 3­methyladenine treatment inhibited cell autophagy and apoptosis in miR­16­5p­overexpressing cells. Mechanistic studies demonstrated that miR­16­5p could inhibit the BIMP1/NF­κB signaling pathway and this inhibition was achieved by directly targeting BIMP1. Furthermore, it was found that blockade of the BIMP1/NF­κB signaling pathway inversed the inhibitory effects of miR­16­5p knockdown on autophagy in BC cells. In vivo experiments further verified the tumor­suppressive effect on BC of the miR­16­5p/BIMP1/NF­κB axis. Therefore, the results of the present study indicated that miR­16­5p promotes autophagy of BC cells via the BIMP1/NF­κB signaling pathway, and an improved understanding of miR­16­5p function may provide therapeutic targets for clinical intervention in this disease.

LncRNA NEAT1 inhibition upregulates miR-16-5p to restrain the progression of sepsis-induced lung injury via suppressing BRD4 in a mouse model.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are known to sponge microRNAs (miRNAs) to regulate biological processes. However, the role of nuclear paraspeckle assembly transcript 1 (NEAT1) binding miR-16-5p in sepsis-induced lung injury remains largely unknown. We aim to explore the effect of NEAT1 sponging miR-16-5p on sepsis-induced lung injury via regulating bromodomain containing 4 (BRD4). METHODS: A mouse model of sepsis-induced lung injury was established. Expression of NEAT1, miR-16-5p and BRD4 was determined. The pulmonary edema, myeloperoxidase (MPO) activity, pathological changes, levels of inflammatory factors, cell viability and apoptosis in mouse lung tissues were evaluated. The binding relationships between NEAT1 and miR-16-5p, and between miR-16-5p and BRD4 were confirmed. RESULTS: NEAT1 and BRD4 were upregulated while miR-16-5p was downregulated in sepsis-induced lung injury. NEAT1 inhibition or miR-16-5p elevation suppressed pulmonary edema, MPO activity, pathological changes, inflammation and apoptosis, and promoted cell viability in mouse lung tissues. NEAT1 bound with miR-16-5p and miR-16-5p targeted BRD4. CONCLUSION: NEAT1 inhibition upregulates miR-16-5p to repress the progression of sepsis-induced lung injury via downregulating BRD4.

Exosomal microRNA-16-5p from human urine-derived stem cells ameliorates diabetic nephropathy through protection of podocyte.

Diabetic nephropathy (DN) remains one of the severe complications associated with diabetes mellitus. It is worthwhile to uncover the underlying mechanisms of clinical benefits of human urine-derived stem cells (hUSCs) in the treatment of DN. At present, the clinical benefits associated with hUSCs in the treatment of DN remains unclear. Hence, our study aims to investigate protective effect of hUSC exosome along with microRNA-16-5p (miR-16-5p) on podocytes in DN via vascular endothelial growth factor A (VEGFA). Initially, miR-16-5p was predicated to target VEGFA based on data retrieved from several bioinformatics databases. Notably, dual-luciferase report gene assay provided further verification confirming the prediction. Moreover, our results demonstrated that high glucose (HG) stimulation could inhibit miR-16-5p and promote VEGFA in human podocytes (HPDCs). miR-16-5p in hUSCs was transferred through the exosome pathway to HG-treated HPDCs. The viability and apoptosis rate of podocytes after HG treatment together with expression of the related factors were subsequently determined. The results indicated that miR-16-5p secreted by hUSCs could improve podocyte injury induced by HG. In addition, VEGA silencing could also ameliorate HG-induced podocyte injury. Finally, hUSC exosomes containing overexpressed miR-16-5p were injected into diabetic rats via tail vein, followed by qualification of miR-16-5p and observation on the changes of podocytes, which revealed that overexpressed miR-16-5p in hUSCs conferred protective effects on HPDCs in diabetic rats. Taken together, the present study revealed that overexpressed miR-16-5p in hUSC exosomes could protect HPDCs induced by HG and suppress VEGFA expression and podocytic apoptosis, providing fresh insights for novel treatment of DN.

Long non-coding RNA DLX6-AS1 knockdown suppresses the tumorigenesis and progression of non-small cell lung cancer through microRNA-16-5p/BMI1 axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a huge threat to sufferers' life and overall health. Long non-coding RNA (lncRNA) distal-less homeobox 6 antisense RNA 1 (DLX6-AS1) has been revealed to function as a carcinogenesis factor in some cancers. This research aimed to scrutinize the role and mechanism underlying DLX6-AS1 in NSCLC tumorigenesis and progression. METHODS: The levels of DLX6-AS1, microRNA-16-5p (miR-16-5p), and BMI1 mRNA were estimated via reverse transcription-quantitative PCR (RT-qPCR) assay. The protein levels were disclosed by western blot assay. Cell proliferative potential was estimated by colony formation and Cell Counting Kit-8 (CCK-8) assays. Cell migration was estimated by Transwell and wound healing assay. A Transwell assay was executed to estimate cell invasion. The relationships of DLX6-AS1, miR-16-5p, and BMI1 were forecasted by bioinformatics analysis, and confirmed by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A xenograft mice model was employed to to inspect the function of DLX6-AS1 knockdown on NSCLC tumorigenesis in vivo. RESULTS: DLX6-AS1 was overexpressed in NSCLC tissues and cells, and was inextricably linked with the poor prognosis of NSCLC patients. Depletion of DLX6-AS1 oppressed cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) but promoted apoptosis in NSCLC. MiR-16-5p is a target of DLX6-AS1 and directly targets BMI1. Moreover, the anti-tumor impacts of miR-16-5p were overturned by overexpression of DLX6-AS1 or BMI1 in NSCLC cells. Additionally, DLX6-AS1 silencing inhibited tumor growth of NSCLC in vivo. CONCLUSIONS: In conclusion, lncRNA DLX6-AS1 downregulation suppressed the tumorigenesis and progression of NSCLC via miR-16-5p/BMI1 axis in vitro and in vivo, elucidating the vital roles and downstream targets of DLX6-AS1 in NSCLC.

Exosomal miRNA-16-5p Derived From M1 Macrophages Enhances T Cell-Dependent Immune Response by Regulating PD-L1 in Gastric Cancer.

Macrophages have an affinity to developing tumors and have been shown to play a role in tumor combat and immune surveillance. However, the exact mechanism by which macrophages participate in the anti-tumor immune response remains unclear. Hence, the current study aimed to identify the effect of macrophages on gastric cancer (GC) cells via exosomes. Paired cancerous, tumor-adjacent, and non-cancerous stomach tissues were initially from 68 GC patients. T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from both the GC patients as well as the healthy donors. Next, the exosomes were isolated from LPS and IFN-γ-induced PBMCs (M1 macrophages) and co-cultured with human GC cells. Another co-culture system comprised of CD3+ T cells and exosomes-treated GC cells was then performed. BALB/c mice and NOD/SCID nude mice were prepared for effects of exosomal miR-16-5p on tumor growth and anti-tumor immune response in GC in vivo. A relationship between M1 macrophages and the poor survival of GC patients was identified, while they secreted exosomes to inhibit GC development and activate a T cell-dependent immune response. Our results revealed that miR-16-5p was transferred intercellularly from M1 macrophages to GC cells via exosomes and targeted PD-L1. M1 macrophage-derived exosomes containing miR-16-5p were found to trigger a T cell immune response which inhibited tumor formation both in vitro and in vivo by decreasing the expression of PD-L1. Taken together, the key findings of the current study suggest that M1 macrophage-derived exosomes carrying miR-16-5p exert an inhibitory effect on GC progression through activation of T cell immune response via PD-L1. Our study highlights the promise of M1 macrophages as a potential cell-based therapy for GC treatment by increasing miR-16-5p in exosomes.

MicroRNA­16­5p regulates cell survival, cell cycle and apoptosis by targeting AKT3 in prostate cancer cells.

Prostate cancer (PCa) is a malignancy with the highest morbidity rate in 105 countries worldwide and was a major cause of cancer­associated death in men in 2018. Accumulating evidence suggests that microRNAs (miRNAs/miRs) have important functions in the carcinogenesis of PCa, and may provide novel treatment targets. Previous studies have indicated that miR­16­5p is associated with PCa. However, the relevance and importance of miR­16­5p in PCa carcinogenesis are still not completely understood. In the current study, we aimed to investigate the role and mechanism of miR­16­5p in PCa carcinogenesis. The results showed that miR­16­5p was markedly downregulated in PCa cells, and MTS assay, colony formation, flow cytometric analyses demonstrated that miR­16­5p inhibited PCa cell survival, regulated cell cycle distribution and induced apoptosis. Moreover, luciferase reporter assay and western blot analysis showed that miR­16­5p directly targets AKT3 (AKT serine/threonine kinase 3), which is associated with PCa carcinogenesis, and the effects of the downregulation of AKT3 were similar to the effects of upregulation of miR­16­5p in PC­3 cells. In conclusion, our data clarify that miR­16­5p has anticancer functions in PCa cells, and our findings provide experimental evidence to highlight the potential value of miR­targeting treatment strategies for PCa.

Circulating Plasma microRNA to Differentiate Cushing's Disease From Ectopic ACTH Syndrome.

Corticotropinomas and adrenocorticotropic hormone (ACTH)-secreting neuroendocrine tumors exhibit differential levels of some microRNAs (miRs) compared to normal tissue. Because miRs can be released from tissues into circulation, they offer promise as novel disease biomarkers. Objective: To evaluate whether miRs are differentially detected in plasma samples of patients with ACTH-dependent Cushing's syndrome (CS). Design: Case-control study. Methods: Morning fasting plasma samples were collected from 41 consecutive patients with confirmed ACTH-dependent CS and 11 healthy subjects and stored at -80°C. Twenty-one miRs previously reported to be differentially expressed in ACTH-secreting tumors vs. healthy tissue samples were quantified in plasma by qPCR. Results: Among enrolled subjects, 28 were confirmed to have Cushing's disease (CD), 13 had ectopic ACTH secretion (EAS) and 11 were healthy controls. We found statistically significant differences in the circulating levels of miR-16-5p [45.04 (95% CI 28.77-61.31) in CD vs. 5.26 (2.65-7.87) in EAS, P < 0.001; q = 0.001], miR-145-5p [0.097 (0.027-0.167) in CD vs. undetectable levels in EAS, P = 0.008; q = 0.087] and differences in miR-7g-5p [1.842 (1.283-2.400) in CD vs. 0.847 (0.187-1.507) in EAS, P = 0.02; q = 0.14]. The area under the receiver-operator (ROC) curve was 0.879 (95% CI 0.770-0.987), p < 0.001, when using miR-16-5p to distinguish between CD and EAS. Circulating levels of miR-16-5p in the healthy control group differed from that of both the CD and EAS groups. Conclusions: Plasma miR levels differ in patients with CD and EAS. In particular, miR-16-5p, miR-145-5p and miR-7g-5p are promising biomarkers for further research to differentiate ACTH-dependent CS.

[microRNA-16-5p targeted tetraspanin 15 gene to inhibit the proliferation, migration and invasion of osteosarcoma cell through phospoinositide 3-kinase/protein kinase B signaling pathway].

Objective: To study the effects of miR-16-5p on proliferation, migration and invasion of osteosarcoma cells and its mechanism. Methods: Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression of miR-16-5p and TSPAN15 in human normal osteoblasts hFOB 1.19 and osteosarcoma cells MG63, Saos2 and HOS. The miR-16-5p or si-TSPAN15 was transfected into MG63 cells to observe its role in cell proliferation, migration and invasion. Cell proliferation was measured with MTT assay, cell migration and invasion were examined by Transwell, and the protein expression of CyclinD1, matrix metalloproteinase 2 (MMP-2), MMP-9, tetraspanin 15 (TSPAN15), phospha-tidylinositol3-kinase(p-PI3K) and phospha-protein kinase B(p-AKT) were determined by using Western blotting. The starbase website prediction combined with dual luciferase gene reporter assay was performed to analyze the targeting relationship between miR-16-5p and TSPAN15. miR-16-5p and pcDNA-TSPAN1 were co-transfected to assess the effect of high expression of TSPAN15 on overexpression of miR-16-5p-induced proliferation, migration and invasion of MG63 cells. Data comparison between the two groups was performed by using t test. Results: Compared with hFOB 1.19 cells (1.00±0.12), the expression of miR-16-5p was significantly decreased in MG63, Saos2 and HOS cells (0.32±0.05, 0.40±0.04, 0.45±0.06, respectively)(F=156.204, P<0.05), and TSPAN15 mRNA and protein levels were greatly increased (F=71.718, 110.350, both P<0.05). Overexpression of miR-16-5p obviously reduced the expression of CyclinD1, MMP-2, MMP-9 protein, cell viability, cell migration and invasion (F=150.136,117.228, 154.971, 89.479, 98.373, 130.880, all P<0.05) in MG63 cells. Knockdown of TSPAN15 greatly reduced CyclinD1, MMP-2, MMP-9 protein levels, cell survival rate, cell migration, and invasion number (F=93.206, 107.030, 109.326, 115.625, 146.113, 139.300, all P<0.05). Overexpression of miR-16-5p markedly decreased the expression of p-PI3K and p-AKT protein in MG63 cells (F=156.755, 181.419, both P<0.05). miR-16-5p targeted to regulate the expression of TSPAN15. High expression of TSPAN15 partially reversed the inhibitory effect of miR-16-5p on TSPAN15, CyclinD1, MMP-2, MMP-9, p-PI3K, p-AKT protein expression, cell viability, cell migration number and invasion number in MG63 cells. Conclusion: miR-16-5p inhibits the proliferation, migration and invasion of osteosarcoma cells by targeting the TSPAN15 gene and regulating the PI3K/AKT signaling pathway.

microRNA-16-5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1.

Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. miR-16-5p has been reported to be involved in cell proliferation, apoptosis, differentiation and angiogenesis. However little is known about miR-16-5p functional role in 3T3-L1 adipocyte differentiation. In this study, we found that miRNA-16-5p was significantly upregulated during 3T3-L1 preadipocytes differentiation towards mature adipocytes. Over-expression of miRNA-16-5p promoted mature adipocytes specific genes expression and fat droplet accumulation in vitro and in vivo. Meanwhile we have identified EPT1 as the target gene of miRNA-16-5p. Taken together, our data provided evidence to support that miRNA-16-5p promotes adipocyte differentiation by suppressing EPT1.