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.

Research progress in effects of microRNA -15a and microRNA -16 on fibrotic diseases / 中南大学学报(医学版)

MicroRNA (miR) is a class of highly conserved non-coding single-stranded RNA widely existing in mammals, which can negatively regulate the expression of targeting genes after transcription. As a key regulator, miR negatively regulates the expression of the targeting genes and disrupts important molecular signaling pathways, leading to the imbalance of multiple pathways such as tissue repair and inflammation involved in the fibrotic process. Among them, miR-15a/16 can participate in regulating and controlling the fibrotic process of various organs, including liver, lung, heart, kidney and other fibrotic diseases by acting on cell proliferation and transformation, extracellular matrix proteins production and degradation, inflammation and other important cell functions. It has potential diagnostic and therapeutic value. Clarifying the biological function of miR-15a/16 and its mechanism for action and therapeutic application prospects in various fibrotic lesions are of great significance for the molecular targeted treatment of fibrotic diseases.

Predictive value of microRNA-155-5p, microRNA-16 and microRNA-129-5p on the efficacy of continuous renal replacement therapy in children with septic shock / 中国医师进修杂志

Objective:To analyze the predictive value of miR-155-5p, miR-16 and miR-129-5p on the efficacy of continuous renal replacement therapy (CRRT) in children with septic shock.Methods:A total of 179 children with severe sepsis admitted to Shanxi Provincial Children′s Hospital from January 2021 to January 2022 were selected, 89 children with septic shock were selected as group A, 90 children with non-septic shock were selected as group B, and 80 healthy children were selected as group C. The expressions of miR-155-5p, miR-16 and miR-129-5p were measured and compared. The patients in the group A were divided into the effective group and the ineffective group according to the curative effect of CRRT treatment. The expressions of miR-155-5p, miR-16 and miR-129-5p in the two groups were compared, and its predictive value for the efficacy of CRRT treatment was analyzed.Results:Compared with group C and group B, the expressions of miR-155-5p in group A was higher: 2.56 ± 0.98 vs. 1.41 ± 0.35, 0.53 ± 0.11; and the expressions of miR-16 and miR-129-5p were lower: 1.00 ± 0.27 vs. 2.23 ± 0.98, 3.38 ± 1.01; 0.65 ± 0.17 vs.1.39 ± 0.22, 2.25 ± 0.76, there were statistical differences ( P<0.05). The results of Logistic regression analysis showed that the sequential organ failure assessment(SOFA) scores, white blood cell count, C reactive protein, procalcitonin, interleukin-6, tumor necrosis factor-alpha , N-terminal B-type natriuretic peptide precursor and the expressions of miR-155-5p, miR-16, miR-129-5p were independent risk factors for the efficacy of CRRT treatment in the children with septic shock. The results of Pearson correlation analysis showed that miR-155-5p was negative correlation with miR-16 and miR-129-5p ( r = - 0.411, - 0.369, P<0.05); and miR-16 was positive correlation with miR-129-5p ( r = 0.444, P<0.05). The results of receiver operating characteristic curve showed that the combination of the three items had a higher predictive value for the efficacy of CRRT in children with septic shock ( P<0.05). Children with high expression of miR-155-5p and low expression of miR-16 and miR-129-5p had higher mortality ( P<0.05). Conclusions:The expressions of miR-155-5p, miR-16 and miR-129-5p are abnormally in children with septic shock and are related to the efficacy of CRRT, which can be used for early prediction of the efficacy of CRRT.

Astragalus polysaccharides affects multidrug resistance gene 1 and P-glycoprotein 170 in adriamycin nephropathy rats via regulating microRNA-16/NF-κB axis / 中南大学学报(医学版)

OBJECTIVES@#Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms.@*METHODS@#A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB.@*RESULTS@#The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (all P<0.05); the levels of ALB and miR-16 were significantly decreased (both P<0.05). Compared with the model group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of pNF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased (all P<0.05); and the levels of ALB and miR-16 were significantly increased (both P<0.05). Compared with APS+miR-16 antagomir control group, the UP, CHOL, BUN, SCr, IL-6, IL-1β, and TNF-α levels, MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased (all P<0.05). The levels of ALB and miR-16 were significantly decreased in the APS+miR-16 antagomir group compared with the APS+miR-16 antagomir control group (both P<0.05).@*CONCLUSIONS@#APS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.

Serum expression of microRNA-16 in a cohort of Egyptian patients with ulcerative colitis and its correlation with disease extent and severity / Expressão sérica do microRNA-16 em uma coorte de pacientes egípcios com colite ulcerosa e sua correlação com a extensão e gravidade da doença

Abstract Ulcerative colitis is one of the IBDs. Its etiology and pathogenesis remain undefined with an interaction between environmental, genetic and immunological factors is the most accepted explanation. Several recent studies have examined microRNA expression in the peripheral blood and tissues from IBD patients. The study aims at assessing the expression of serum miR-16 in ulcerative colitis patients and its correlation with disease extent, activity and severity. It included 30 treatment naïve ulcerative colitis patients of different presentations. Serum miR-16 expression was assessed using reverse transcriptase quantitative real time PCR (RT-qPCR), and then correlated with that of a group of 20 healthy subjects to assess its role in diagnosis of ulcerative colitis. Also, it was correlated with disease extent (proctitis, left sided colitis, extensive colitis) and disease activity and severity indices (Truelove and Witts criteria, fecal calprotectin and UCEIS). Thirty ulcerative colitis patients were enrolled, 53% had mild, 37% had moderate, while 10% had severe disease. Concerning endoscopic extent, 8 had proctitis, 14 had left sided colitis and 8 had extensive colitis. Serum expression of miR-16 in the 30 patients were compared to that of the healthy control subjects. The patients' group showed median serum miR-16 expression of 1.91, 1.13 for the control group with a significant difference between both groups. Correlation between serum miR-16 expression with disease extent, activity and severity showed no significant relation. From the current study we can conclude that increased serum expression of miR-16 is associated with ulcerative colitis despite no significant relation to disease activity extent or severity.

Resumo A colite ulcerativa é uma das DII. Sua etiologia e patogênese permanecem indefinidas; a interação entre fatores ambientais, genéticos e imunológicos é a explicação mais aceita. Vários estudos recentes avaliaram a expressão de microRNA no sangue e tecidos periféricos em pacientes com DII. O presente estudo teve como objetivo avaliar a expressão do miR-16 sérico em pacientes com colite ulcerativa e sua correlação com a extensão, atividade e gravidade da doença. Foram incluídos 30 pacientes de colite ulcerativa, com diferentes apresentações, que ainda não haviam sido submetidos a nenhum tipo de tratamento. A expressão sérica de miR-16 foi avaliada usando transcrição reversa seguida de reação em cadeia da polimerase quantitativa (RT-qPCR) e, em seguida, correlacionada com a de um grupo de 20 indivíduos saudáveis para avaliar seu papel no diagnóstico de colite ulcerativa. Além disso, foi feita uma correlação com a extensão da doença (proctite, colite do lado esquerdo, colite extensa) e com os índices de atividade e gravidade da doença (critérios de Truelove e Witts, calprotectina fecal e UCEIS). Trinta pacientes com colite ulcerativa foram incluídos no estudo, classificada como leve em 53%, moderada em 37% e grave em 10%. Quanto à extensão endoscópica, oito apresentavam proctite, 14 apresentavam colite do lado esquerdo e oito apresentavam colite extensa. A expressão sérica de miR-16 nos 30 pacientes foi comparada à dos indivíduos controle saudáveis. No, grupo de pacientes, a expressão sérica de miR-16 foi de 1,91 (grupo controle: 1,13), uma diferença estatisticamente significativa entre os dois grupos. Não foi observada relação significativa entre a expressão sérica de miR-16 e a extensão, atividade e gravidade da doença. A partir do presente estudo, pode-se concluir que o aumento da expressão sérica do miR-16 está associado à colite ulcerativa, apesar de não haver relação significativa com a extensão ou gravidade da atividade da doença.

Effects of microRNA-16 on pulmonary surfactant associated protein of human alveolar epithelial cell A549 / 中国新生儿科杂志

Objective To study the regulatory role of microRNA-16 (miR-16) on human pulmonary surfactant associated protein (SP).Method Human alveolar epithelial A549 cells were transfected by miR-16 analogue,analogue negative control,inhibitor and inhibitor negative control.Blank control group was also set up at the same time.The proliferative abilities of the cells in each group were measured using cell counting kit-8 (CCK8) test.The expressions of miR-16,SP-A,SP-B and SP-C mRNA were examined using reverse transcription polymerase chain reaction (RT-PCR).The protein levels of SP-A,SP-B and SP-C were examined using western blotting method.Result RT-PCR showed that the expression of miR-16 after transfected with miR-16 analogue (34.11± 1.79) was higher than the negative control group (1.65 ± 1.07) and the blank control group (1.07 ±0.50).The expression of miR-16 after transfected with miR-16 inhibitor (0.36±0.05) was lower than the negative control group (0.96±0.13) and the blank control group (1.05±0.20).The differences were significant (all P＜0.05),and indicated that miR-16 over-expression and suppression were successfully achieved.Compared with the blank control group,cell proliferation at different time points in the analogue negative control group and the inhibitor negative control group showed no significant differences (all P＞0.05).Compared with the blank control group (1.02±0.19,1.01±0.09,1.01± 0.12) and the analogue negative control group (1.08±0.24,1.00±0.14,1.00±0.05),miR-16 down-regulated the mRNA expressions of SP-A,SP-B and SP-C (0.58±0.16,0.67±0.05,0.61±0.12).On the other hand,compared with the blank control group (1.02±0.19,1.01±0.09,1.01±0.12) and the inhibitor negative control group (1.05±0.22,0.99±0.13,0.98±0.10),miR-16 up-regulated the mRNA expressions of SP-A,SP-B and SP-C (1.66±0.33,1.29±0.11,1.23±0.12)(all P＜0.05).The trends of protein level of SP-A,SP-B and SP-C were related to their mRNA expression.Conclusion This study indicates that miR-16 inhibits pulmonary surfactant associated protein in A549 cells.

Estradiol promotes growth of decidua derived mesenchymal stem cells by inhibiting miR-16 via estrogen receptorα / 中国病理生理杂志

AIM:To study the effect of estradiol (E2) on the viability of mesenchymal stem cells (MSCs) de-rived from the decidua of the placenta by regulating the expression of microRNA-16 (miR-16).METHODS:The concen-tration of E2 in the peripheral blood of normal pregnant women and the patients with severe preeclampsia ( PE) was meas-ured.The effects of E2 at different concentrations on the viability of MSCs were analyzed .The effect of E2 at different con-centrations on the expression of miR-16 in the MSCs was detected , and which estrogen receptor ( ER) mediated the regula-tory effect of E2 on miR-16 expression was determined .RESULTS:The concentration of E2 in peripheral blood of the pa-tients with severe PE was significantly decreased (P<0.01).After treatment with E2 at 5, 10 and 100 nmol/L for 48 h, the viability of MSCs was increased (P<0.05).The expression level of miR-16 was down-regulated in the MSCs treated with E2 at 5, 10 and 100 nmol/L for 12 h.After treatment with E2 at 10 nmol/L for different time (0 h, 3 h, 6 h, 12 h and 24 h) , the expression level of miR-16 in the MSCs showed a clear time-dependent downward trend .E2 significantly promoted the viability of MSCs , and the cell viability was significantly reversed after miR-16 pretreatment.Pretreatment with estrogen receptor antagonists ICI 182780 and tamoxifen for 6 h attenuated the inhibitory effect of E 2 on miR-16 expres-sion.Only ERαagonist propyl pyrazole triol significantly inhibited the expression of miR-16 in MSCs but ERβagonist dia-rylpropionitrile did not .CONCLUSION:E2 promotes the growth of decidua-derived MSCs by inhibiting miR-16 via ERα.

Proliferation and migration of breast cancer cells inhibited by miRNA-16 in vitro Bu Deyong , Jia Hongyan / 肿瘤研究与临床

Objective To investigate the expression of miRNA-16 (miR-16) in breast cancer patients and its effect on the proliferation and migration of breast cancer cells. Methods Polymerase chain reaction (PCR) was used to detect the expression of miR-16 in 30 breast cancer patients. miR-16 mimics was transfected to MDA-MB-231 and MCF-7 cell, and CCK-8 as well as Transwell assay was applied to detect the effect of miR-16 on cell proliferation and migration of MDA-MB-231 and MCF-7 cells. Results Among 30 breast cancer patients, the expression of miR-16 was down-regulated in 23 cases. Cell proliferation and migration of MDA-MB-231 and MCF-7 cells were inhibited significantly after transfection of miR-16 mimics. Conclusion miR-16 is down-regulated in breast cancer patients. miR-16 inhibits significantly the cell proliferation and migration of breast cancer cells in vitro.

Effect of miR-16 on megakaryocytic differentiation of K562 cells / 中国病理生理杂志

AIM: To observe the effect of microRNA-16 (miR-16) on the megakaryocytic differentiation of K562 cells, and to explore the potential mechanism.METHODS:miR-16 was over-expressed or silenced by transfection with miR-16 mimics or inhibitor in K562 cells.The level of miR-16 was detected by real-time PCR.The expression of CD41, CD42b and CD61, as megakaryocytic differentiation markers, was detected by flow cytometry.The effect of miR-16 on the expression of myeloblastosis oncogene ( MYB) was measured by Western blotting, and flow cytometry was performed to confirm whether the effect of miR-16 on expression of CD41, CD42b and CD61 was mediated by MYB.RESULTS:Transfection with miR-16 mimics dramatically elevated the level of miR-16 and the expression of CD41, CD42b and CD61 in the K562 cells.Transfection with miR-16 inhibitor decreased the level of miR-16 and the expression of CD41, CD42b and CD61 in the K562 cells (P<0.05).The expression of MYB was regulated by miR-16, and MYB silencing reversed the regulation of CD41, CD42b and CD61 induced by miR-16.CONCLUSION:miR-16 regulates the megakaryocytic dif-ferentiation of K562 cells by targeting MYB.

miR-16 inhibits glioma cell invasion through regulating NF-κB1/MMP-9 signaling pathway / 中华神经医学杂志

Objective To explore the microRNA-16 (miR-16) and nuclear-transcription factor-κB1 (NF-κB1) expressions in human brain gliomas and their correlations with cell invasion and growth of malignant gliomas SHG44,U87 and U373.Methods (1) Twenty-nine cases of human glioma tissue samples and 6 normal brain tissues,collected in our hospital from January 2000 to January 2011,were chosen in our study; quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expressions ofmiR-16 and NF-κB1 in these tissues.(2) In vitro cultured U87,U373 and SHG44 cells were divided into blank-control group,nonsense sequence transfected group and miR-16 mimics transfected group; 48 h after the transfection,qRT-PCR was used to detect the expressions of miR-16 and NF-κB1; tmnswell assay was used to observe the cell invasion capability; 72 h after the transfection,Western blotting was employed to detect the protein expressions of NF-κB1,matrix metallopeptidase 9 (MMP-9) and MMP-2.(3) Luciferase reporter assay was used to detect the target regulating role of miR-16 in NF-κB1 gene.(4) U87 cells were used as negative control group,and U87 cells carried stably expressed miR-16 gene were implanted into intracranial and subcutaneous nude mice (U87-miR-16 group); immunofluorescence was used to detect the MMP-9 expression,and immunohistochemical staining was used to detect the protein expressions of Ki-67,NF-κ B1 and MMP-9; subcutaneous tumor volume was measured and the growth curve was drawn.Results (1) The qPCR results showed that the expression of miR-16 in human brain glioma tissues was significantly lower than that in normal brain tissues; and gradually decreased miR-16 expressions were noted in gliomas of graded Ⅰ,Ⅱ,Ⅲ and Ⅳ (P＜0.05); NF-κB1 expression in human brain glioma tissues was significantly higher than that in normal brain tissues; and gradually increased NF-κB1 expressions were noted in gliomas of graded Ⅰ,Ⅱ,Ⅲ and Ⅳ (P＜0.05).(2) As compared with those in the blank-control group and nonsense sequence transfected group,miR-16 mimics transfected group had significantly increased miR-16 expression,decreased NF-κB1 mRNA expression,decreased invasiveness,and decreased protein expressions of NF-κB1 and MMP-9 (P＜0.05).(3) Luciferase reporter assay showed that the fluorescence normalized ratio in the pMIR-NF-κB1 group was signfcaintly higher than that in the pMIR-NF-κB1+pre-miR-16 group (P＜0.05).(4) As compared with the negative control group,the U87-miR-16 group on the 24-42 d of implantation had significantly smaller volume of tumors (P＜0.05),and lower MMP9 expression,and NF-κB1,MMP-9 and Ki-67 expressions (the proliferation index of Ki-67:13.91％ and 32.98％).Conclusion MiR-16 inhibits glioma cell invasion and growth through down-mgulating NF-κB1 and MMP-9 expressions.

Expression changes in miR-16 and miR-146a in rat lungs after fast buoyancy ascent escape or diving decompression sickness / 军事医学

Objective To study the expression levels of microRNA (miR)-16 and miR-146a in rat lungs of decompres-sion sickness (DCS) caused by fast buoyancy ascent escape or diving .Methods At 0.5 h after fast buoyancy ascent es-cape or diving, the pathological changes in rat lungs and expression levels of miR-16,and miR-146a were detected by re-verse transcription-quantitive polymerase chain reaction and compared with normal control group .Results The pathological characteristics of lungs in two DCS groups were tissue damage .At 0.5 h after DCS caused by fast buoyancy ascent escape , the lung tissue expression levels of miR-16 and miR-146a did not significantly change compared with normal control and diving DCS groups ,but the rat lung tissue expression level of miR-146 a in diving DCS group was obviously increased , com-pared with normal control group .Conclusion miR-146a may play a role in post-transcriptional regulation in the process of diving DCS .

Effects of microRNA-16 on proliferation, invasion and cytokine secretion of synovial fibroblasts from rheumatoid arthritis patients / 中国病理生理杂志

AIM:To investigate the effect of microRNA-16 ( miR-16) on the proliferation, invasion and cyto-kine secretion of rheumatoid arthritis ( RA) synovial fibroblasts ( RASFs) from the RA patients.METHODS: miR-16 mimic and miR-16 inhibitor were synthesized, and then Transfected into RASFs isolated from RA patients with lipo-fectamine.MTT assay, Transwell chamber and flow cytometry were used to determine the effect of miR-16 on proliferation, invasion and apoptosis of RASFs.The expression of matrix metalloproteinase 3/13 ( MMP3/13) and interleukin 1β( IL-1β) was measured by RT-PCR and Western blotting.RESULTS: The proliferation and invasion of RASFs were signifi-cantly inhibited by miR-16 mimic.The result of flow cytometry demonstrated that miR-16 had no effect on apoptosis of RASFs.Furthermore, miR-16 down-regulated the expression of MMP3/13 and IL-1β.CONCLUSION:miR-16 plays an important role in the development of RA and may inhibit the proliferation and invasion of RASFs through down-regulating the expression of MMP3/13 and IL-1β.