Agomir Group Research Articles

MicroRNA Let-7b-5p Induces Electroacupuncture Tolerance by Downregulating the MKP-1 Gene in Rats Subjected to CFA-induced Inflammatory Nociception

Electroacupuncture (EA) treatment has proved to significantly decrease nociception in inflammatory nociception model by suppressing the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). However, repeated EA treatment results in gradual attenuation of its analgesic effects, which was defined as “EA tolerance.” Recent studies have shown that let-7b-5p microRNA (miRNA) contributes to the EA tolerance. The present study aimed to explore the function of let-7b-5p in p38MAPK pathway and the development of EA tolerance in the inflammatory nociception. Dual luciferase reporter gene experiments were used in cortical neurons to determine the target gene locus of let-7b-5p. The threshold of nociception was assessed by tail flick latency (TFL) and paw withdrawal threshold (PWT). Western blots were used to measure the expression of mitogen-activated protein kinase phosphatase 1 (MKP-1) and phosphorylation level of p38MAPK after intracerebroventricular (ICV) injections of let-7b-5p agomir, antagomir, and controls. In vitro dual luciferase experiments demonstrated that the MKP-1-3′ untranslated region (UTR) is a target of let-7b-5p. In vivo experiment, rat with repeated EA treatment exhibits gradual decrease in TFL and PWT, which showed formation of EA tolerance. This trend was delayed after IVC injection of let-7b-5p antagomir and facilitated after IVC injection of let-7b-5p agomir. The protein levels of MKP-1 in the EA+let-7b-5p antagomir group were significantly higher than in the EA + let-7b-5p agomir group. However, P-p38MAPK in the EA+let-7b-5p antagomir group was significantly lower than in the EA+let-7b-5p agomir group. By upregulating the p38MAPK pathway through the inactivation of the MKP-1 gene, let-7b-5p contributes to EA tolerance in complete Freund’s adjuvant (CFA)-induced inflammatory nociception rats. Our work revealed the mechanism of EA tolerance and indicated that let-7b-5p could be targeted to improve the long-term effects of EA.

Effect of miR-9 on Chondrocyte Apoptosis in Rats with Osteoarthritis by Regulating Notch1

Objective: Notch1 is an important receptor in Notch signaling pathway. Abnormal Notch1 expression or function is associated with the pathogenesis of OA. There is evidence that the abnormal expression of miR-9 is related to the pathogenesis of OA. Bioinformatics analysis showed that there was a targeting relationship between miR-9 and Notch1’s 3′-UTR, suggesting that there might be a mutual regulation between them. In this study, we established an OA rat model to investigate whether miR-9 plays a role in regulating Notch1 expression, affecting cartilage matrix degradation and chondrocyte apoptosis in OA. Methods: OA model rats were divided into three groups: OA group, OA + agomir group, OA + miR-9 agomir group. The content of Hyp in articular fluid was measured by ELISA, the activity of caspase-3 was detected by kit, the mRNA expressions of miR-9, COL2A1, Notch1 and Hes5 was detected by qRT-PCR, and the protein expressions of Notch1 and Hes5 in articular cartilage was detected by Western blot. Chondrocytes were separated into control group, IL-1β +miR-NC group, IL-1β +miR-9 mimic group followed by analysis of mRNA expressions of miR-9, COL2A1, Notch1 and Hes5 were detected by qRT-PCR, protein expressions of Notch1 and Hes5 by Western blot, and cell apoptosis by flow cytometry. Results: Compared with Sham group, the levels of IL-1β , Hyp, Notch1, Hes5 and Caspase-3 in the synovial fluid of rats in OA group were increased significantly, while the mRNA expressions of miR-9, COL2A1 and Notch1 and Hes5 level in cartilage tissue were decreased significantly. Intra-articular injection of miR-9 agomir could significantly reduce the content of Hyp, the mRNA expressions of Notch1 and Hes5 and the activity of caspase-3 in cartilage tissue of OA rats, increase the mRNA expressions of miR-9 and COL2A1, and decrease the protein expressions of Notch1 and Hes5. After treated with IL-1β , the expressions of Notch1 and Hes5 were increased, the mRNA expressions of miR-9 and COL2A1 were decreased, and apoptosis of chondrocyte was increased in chondrocyte. After miR-9 mimic transfection, miR-9 and COL2A1 mRNA expressions were increased, Notch1 and Hes5 expressions were decreased, and apoptosis of chondrocyte was decreased under IL-1β treatment. Conclusion: Reduced miR-9 expression upregulates Notch1 expression and promotes the pathogenesis of OA. Increased miR-9 expression can inhibit Notch1 expression, reduce cartilage matrix degradation and inhibit chondrocyte apoptosis.

MiR-195 alleviates ulcerative colitis in rats via MAPK signaling pathway.

To study the effect of micro ribonucleic acid (miR)-195 on the inflammatory response of ulcerative colitis (UC) model rats and to explore its regulatory mechanism, thus providing a new scheme for the clinical treatment of UC. A rat model of UC was prepared by 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)/ethanol assay, and the rats were randomly divided into Control group, Model group, and miR-195 mimic (miR-195 agomir) group. The disease activity index (DAI) in each group was observed. Hematoxylin and eosin (H&E) staining was utilized to detect the pathological changes in the rat colon tissues in each group. The levels of interleukin-6 (IL-6) and IL-1β in the colon tissues of the rats in each group were detected by enzyme-linked immunosorbent assay (ELISA). In addition, the messenger RNA (mRNA) and protein levels of p38 mitogen-activated protein kinase (p38 MAPK) and tumor necrosis factor-alpha (TNF-α) in the colon tissues of each group of rats were examined via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, respectively. Compared with those in Control group, the rats in Model group had an increased DAI score, severely pathologically damaged colon tissues, raised levels of IL-6 and IL-1β in the colon tissues and significantly elevated mRNA and protein levels of p38 MAPK and TNF-α. In comparison with those in Model group, the DAI score was decreased, the pathological damage to the rat colon tissues was improved, the levels of IL-6 and IL-1β in the rat colon tissues were reduced, and the mRNA and protein levels of p38 MAPK and TNF-α were notably lowered in miR-195 agomir group. MiR-195 mimics can alleviate the pathological damage to the colon and inflammatory responses in UC model rats, and its mechanism may be related to the inhibition on the p38 MAPK signaling pathway.

Overexpression of miR-146a inhibits the apoptosis of hippocampal neurons of rats with cerebral hemorrhage by regulating autophagy.

In this study, to investigate the effect of overexpression of miR-146a on autophagy of hippocampal neurons in rats with intracerebral hemorrhage (ICH), 72 Sprague-Dawley rats were randomly divided into the sham, ICH, miR-146a agomir, and miR-146a agomir control groups. The ICH model was constructed by injection of collagenase VII. The apoptosis of hippocampal neurons was measured by TUNEL assay. The levels of LC3 and Beclin 1 were analyzed by immunohistochemistry. Mitochondrial autophagy was examined by transmission electron microscopy. The levels of LC3A, LC3B, Beclin 1, Bax, Bcl-2, and cleaved caspase 3 were examined by Western blot. Western blot was also used to evaluate the expression of nuclear factor κB signaling pathway-related factors. To examine the effect of autophagy inhibitor (3-methyladenine (3-MA)) on miR-146a-regulated apoptotic protein expression, 30 rats were further divided into the sham, ICH, miR-146a agomir, 3-MA, and miR-146a + 3-MA groups. The levels of Bax, Bcl-2, and cleaved caspase 3 were examined by Western blot. Compared with the sham group, the nerve function scores, brain water content, the percentage of apoptotic cells, and the expression levels of LC3, Beclin 1, Bax, cleaved caspase 3, and p-P65 in the hippocampus of rats in the ICH group were all significantly increased (p < 0.05), whereas the expression levels of miR-146a, Bcl-2, and p-IκBα were markedly decreased (p < 0.05). Mitochondrial autophagy was also evident. Furthermore, compared with the ICH group, the results of the abovementioned tests in the miR-146a agomir group were reversed. The overexpression of miR-146a inhibited the autophagy of hippocampal neurons in rats with ICH.

Protective Effects of miR-126 Specifically Regulates Nrf2 Through Ischemic Postconditioning on Renal Ischemia/Reperfusion Injury in Mice

The protective effects of ischemic postconditioning on renal ischemia/reperfusion injury in mice and the role of miR-126 and Nrf2 signaling pathway in the process were the focus of this study. Mice were classified into 5 groups: sham-operation, I/R6h, I/R24h, postconditioning (POC), and miR-126 agomir. Serum creatinine, renal histopathology changes, and oxidative stress were examined. The expression of miR-126 and Nrf2 were detected. We also treated NRK52E cells with hypoxia reoxygenation. The I/R group showed significant renal injury and increased generation of oxidative stress. However, the severity of renal injury and oxidative stress were markedly attenuated in the POC group. MiR-126 was downregulated, and Nrf2 was upregulated in NRK52E cells subjected to hypoxia/reoxygenation (H/R) and in mouse kidneys subjected to POC. Oxidative stress was higher and Nrf2 was lower in the mouse miR-126 agomir group; apoptosis were higher in H/R-treated NRK52E cells transfected with Nrf2 siRNA and lower in anti-miR-126. These findings demonstrate that after renal ischemic postconditioning, miR-126 inhibits oxidative stress by inducing Nrf2 and suppresses injury.

MiR-203a-3p regulates the cellular processes of esophageal cancer cells via targeting CtBP2.

BackgroundMicroRNAs (miRNA) (small noncoding RNAs) are vital modulators of gene expression by mRNA degradation and translational silencing. However, the definite mechanism and character of miR-203a-3p in regulating esophageal carcinoma cells remain unexplained. Here we further investigate the effect and the latent target gene of miR-203a-3p on the progression of esophageal squamous cell cancer (ESCC) tissues and cells.MethodsThe expressions of miR-203a-3p in ESCC tissues and peri-neoplastic tissues were further measured by RT-quantitative-PCR (RT-qPCR). Luciferase assay was applied to confirm that C-terminal-binding protein 2 (CtBP2) was the potential target gene of miR-203a-3p. miRNA mimic was transfected into ECA109 cells to up-regulate the miR-203a-3p expression, and its and CtBP2 expression were tested using RT-qPCR and Western blot. In vitro, MTT, transwell, wound healing, TUNEL and flow cytometry (FCM) assay were used to explore the role of miR-203a-3p on the cellular processes of ECA109 cells via targeting CtBP2. Furthermore, we designed rescue experiments by using CtBP2 stable over-expression ECA109 cells.ResultsWe found the miR-203a-3p expressions in ESCC tissues and cells were significantly raised. miR-203a-3p negatively regulated the CtBP2 expression, and caused to inhibiting proliferation, migration and invasion, and promoting apoptosis in ECA109 cell. In addition, proteins involved in epithelial-mesenchymal transition (EMT) were measured by Western blot in ECA109 cells. miR-203a-3p enhanced the E-cadherin and β-catenin expression, while reduced vimentin expression in ECA109 cells. In vivo, Xenograft tumor model demonstrated that tumor volume in miR-203a-3p agomir group was remarkably decreased.ConclusionsmiR-203a-3p plays a vital role in the metastasis of ESCC cell by targeting CtBP2, and offers a promising therapeutic target for ESCC treatment.

Regulatorg Mchanism of MiR-152 on Proliferation, Metastasis and Tumorigenicity of SHI-1 Cells

To investigate the regulatory mechanism of miR-152 on proliferation, metastasis and tumorigenesis of human acute monocytic leukemia SHI-1 cells. The purchased SHI-1 cell line was treated with miR-152 over-expression (miR-152 agomir group) or miR-152 inhibition (miR-152 antagomir group), and the negative control (NC) group was set up. The cell proliferation of each group was detected by CCK-8 assay. Scratch healing assay was employed to determine the migration of cells. Transwell assay was used to measure the invasion of cells. The expressions of Cyclin D1, Caspase-3, MMP-2, TIMP-2, E-cadherin and N-cadherin were detected by Western blot. The flow cyronetry with annexin V-FITC/PI double staining was applied to detect the cell apoptosis. The tumorigenesis of cells was examined by tumor formation experiment in nude mice. Compared with the NC group, the cell proliferation, migration and invasion ability in miR-152 agomir group were significantly decreased (P＜0.05), while that in miR-152 antagomir were significantly up-regulated (P＜0.05) . Compared with the NC group, the protein expression of Cyclin D1, MMP-2, N-cadherin were down-regulated in miR-152 agomir group, but the protein expression of Caspase-3, TIMP-2 and E-cadherin were all up-regulated siginificantly. At the same time, the apoptosis were enhanced, but the timorigenicity in nude mice were significantly decreased (all P＜0.05). The protein expression of Cyclin D1, MMP-2, N-cadherin in miR-152 antagomir group, showed high levels but Caspase-3, TIMP-2 and E-cadherin protein showed low levels in comparison with NC group. At the same time, the apoptosis was decreased but the timorigenicity in nude mice was significantly enhanced (all P＜0.05) . miR-152 can inhibit the proliferation, metastasis and tumorigenesis of SHI-1 cell line, at the same time induce cell apoptosis, thus providing a theoretical basis for the treatment of acute lymphoblastic leukemia.

MiR-1906 Attenuates Neuropathic Pain in Rats by Regulating the TLR4/mTOR/ Akt Signaling Pathway.

BackgroundThis study determined the role of miR-1906 in neuropathic pain and proliferation in neuronal cells using a chronic constriction injury (CCI)-induced neuropathic pain (NP) rat model.MethodologyNP was induced by CCI. Animals were divided into a sham group, an NP group, and a miR-1906 mimic group, which received 500 nmol/kg of a miR-1906 mimic intrathecally for 10 consecutive days following surgery. The effect of miR-1906 agomir was determined by estimating the thermal and mechanical withdrawal latency; an enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of proinflammatory mediators. Western blotting and reverse-transcription polymerase chain reaction (RT-PCR) were used to determine protein expression in the spinal tissues of the CCI-induced neuropathic pain rat model.ResultsAdministration of miR-1906 agomir increased the mechanical and thermal withdrawal latency period and the levels of inflammatory mediators compared with the NP group. Western blotting showed that treatment with miR-1906 agomir attenuated the levels of Akt, mTOR, TLR-4, and PI3K proteins in the spinal tissues of the CCI-induced neuropathic pain model. TLR-4 and NF-κB gene expression was lower in the miR-1906 agomir group than in the NP group.ConclusionmiR-1906 gene stimulation reduced neuropathic pain by enhancing Akt/nTOR/PI3K and TLR-4/NF-κB pathway regulation.

Effect of miR-26a on diabetic rats with myocardial injury by targeting PTEN.

To investigate the effect of micro ribonucleic acid (miR)-26a on diabetes-induced myocardial injury in rats by targeting the gene of phosphate and tension homology detected on chromosome ten (PTEN). Male Wistar rats aged 8-9 weeks old were divided into the control group (n=10), GK group (n=10), and miR-26a agomir group (n=10) according to the body weight. MiRanda and TargetScan target gene prediction software were used to predict and analyze the target gene of miR-26a-5p. The expressions of miR-26a and PTEN in the myocardial tissues of the diabetic rats were detected by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Hematoxylin-eosin (HE) staining was adopted to observe the pathological changes in the myocardial tissues. In addition, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was conducted to detect myocardial apoptosis, while the expression of PTEN protein was detected via immunohistochemistry, and the protein expressions of PTEN, b-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteinyl aspartate specific proteinase-3 (Caspase-3) were tested by Western blotting. TargetScan database analysis results showed that miR-26a-5p and PTEN 3'UTR had 6 pairs of complementary bases with the same sequence. Compared with those in the control group, the messenger RNA (mRNA) expression of PTEN in the GK group was notably increased (p<0.05), while that of miR-26a was substantially reduced (p<0.05). In comparison with those in the GK group, the mRNA expression of PTEN was significantly decreased, but that of miR-26a was significantly raised in miR-26a agomir group (p<0.05). Through observation under an optical microscope, it was manifested that in the control group, the myocardial fibers were intact with clear texture but no fracture, and the solid necrosis did not appear in myocardial cells. In the GK group, the myocardial fibers were disorderedly arranged and incomplete with an unclear edge and burrs. The myocardial fibers in the miR-26a agomir group were more regular, with less breakage and solid necrosis. According to TUNEL staining results, the TUNEL-stained brown granules in rats in the GK group were remarkably increased, relative to the control group (p<0.05). Compared with the GK group, miR-26a agomir group had markedly decreased the TUNEL-stained brown particles (p<0.05). It was found in immunohistochemical results that PTEN protein was in lighter color after staining in the control group, with a clear myocardial cell stripe structure. Compared with that in control group, PTEN protein in the GK group was in deeper color after staining, and in comparison with that in the GK group, the color of PTEN protein in miR-26a agomir group became significantly lighter. Moreover, the Western blotting results demonstrated that, compared with those in the control group, the Caspase-3 and Bax protein expressions in the GK group were significantly raised, while Bcl-2 protein expression was notably reduced (p<0.05). Besides, in comparison with the GK group, miR-26a agomir group evidently elevated Caspase-3 and Bax protein expressions and a notably increased Bcl-2 protein expression (p<0.05). We showed that miR-26a can protect against myocardial injury in diabetic rats by regulating PTEN.

Catalpol may improve axonal growth via regulating miR-124 regulated PI3K/AKT/mTOR pathway in neurons after ischemia.

MicroRNA-124 (miR-124) is a brain-specific miRNA molecule, the highest expression in the cortex and is associated with neuronal protection after stroke. This study aimed to investigate whether catalpol could affect miR-124 to regulate PI3K/AKT/mTOR pathway, promoting axonal growth in stroke rats. Cells were divided into three groups: control group, miRNA124 agomir group, and miRNA124 antagomir group. To explore the mechanism, cells were divided into seven groups: control group, OGD group (OGD/R), miRNA124 agomir group, miRNA124 agomir plus catalpol group, miRNA124 antagomir group, miRNA124 antagomir plus catalpol group, and catalpol group. Before OGD/R, miRNA124 antagomir and microRNA124 agomir were transfected into neurons for 6 h by using ribo FECT nd Consumablesn/reper transfection kit. Cell survival and cell death were detected by MTT and LDH assay. Axonal growth was assessed by MAP-2 immunofluorescence staining. Western blotting and qPCR were used to detect the expression of molecules in the PI3K/AKT/mTOR pathway. Inhibition of miR-124 activated PI3K/AKT/mTOR pathway and promoted neuronal survival and axonal growth. The expression of miR-124 increased after OGD/R, and catalpol could inhibit miR-124 to activate PI3K/AKT/mTOR pathway to further promote axonal growth. It is concluded that catalpol may inhibit miR-124 to activate PI3K/AKT/mTOR pathway, promoting axonal growth.

MicroRNA-497 inhibition mitigates myocardial infarction via enhancing wingless/integrated signal pathway in bone marrow mesenchymal stem cells.

High association between microRNA-497 (miR-497) inhibition and the improvement of myocardial infarction (MI) has been proved. Bone marrow mesenchymal stem cells (BMSCs) therapy is regarded as a highly promising approach to MI treatment. We studied the functional role of miR-497 inhibition in the transplantation of BMSCs for MI treatment. BMSCs were isolated from 10 to 14 days old male Sprague-Dawley (SD) rats for in vitro and in vivo experiments. First, flow cytometry was used for BMSCs identification. miR-497 antagomir and agomir were transfected into BMSCs, and the migratory capacity was detected by wound healing assay. Protein levels were analyzed by Western blot analysis. Second, rat MI models were constructed and injected with each experimental group BMSCs. Four weeks later, the cellular morphology of cardiomyocyte and infarcted size was observed after histopathologic evaluation (HE) and Masson's trichrome staining. Moreover, WNT3A siRNA (siWNT3A) was used for further investigating the involvement of Wnt/β-catenin pathway. BMSCs were confirmed to be CD90+ CD45- CD11b/c- cells. The number of rats with wound closure increased more in miR-497 inhibitor group than that in agomir group, the number markedly decreased in agomir group ( P < 0.01). As the miR-497 decreased, the protein levels of WNT3A, matrix metalloproteinase-9 and β-catenin were notably increased. The injection of BMSCs inhibiting miR-497 repaired almost all infarcted zones. siWNT3A, on the contrary, could decrease the wound closure rate and relative protein levels and inhibit MI treatment. miR-497 antagomir contributes to BMSCs transplantation for MI treatment by Wnt/β-catenin activation, and Wnt/β-catenin pathway is essential for the functional effects of miR-497 antagomir.

Effect of intracerebroventricular injection of miRNA-22 on expression of P2X7 receptor and inflammatory factors in hippocampus of rats with epilepsy

Objective To investigate the effect of microRNA-22 (miRNA-22) on the expression of P2X7 receptor and inflammatory factors in hippocampus of rats with epilepsy. Methods Healthy SD male rats were intraperitoneal injected with lithium chloride and pilocarpine to induce epilepsy. Three days later, 45 epileptic rats were randomly divided into three groups: epilepsy group(EP group), miRNA-22 agomir group (EF+ agomir group)and miRNA-22 agomir control group(EF+ agomir control group). Another 15 healthy rats were selected as control group(N group). The expression of P2X7 protein was detected by Western blot and the levels of miRNA-22, P2X7 mRNA, NF-κB mRNA , IL-1β mRNA were detected by qRT-PCR. Nissl staining was used to observe the damage of Nissl bodies. Results Western blot result showed that compared with the N group(0.91±0.10), the level of P2X7 protein in EP group (1.17±0.052) increased, and the difference was statistically significant (t=-4.11, P=0.02). Compared with the EP+ agomir control group(0.94±0.14), the expression of P2X7 protein in EP+ agomir group (0.66±0.06) decreased and the difference was significant (t=-3.10, P=0.04). And the qRT-PCR results showed that compared with N group, the levels of P2X7mRNA (9.08±0.94), NF-κB mRNA (20.10±2.15) and IL-1β mRNA (50.64±5.42) in EP group increased(t=-14.96, P<0.05; t=-15.38, P<0.05; t=-15.87, P<0.05). The expression of P2X7mRNA (1.31±0.64), NF-κB mRNA (2.28±1.10) and IL-1β mRNA (2.12±1.20) in EF+ agomir group decreased compared with EP group((9.08±0.94), (20.10±2.15), (50.64±5.42)) and EF+ agomir control group((7.03 ±1.90), (18.72±1.76), (47.39±6.16)), and the differences were statistically significant(F=29.77, P<0.01; F=98.99, P<0.05; F=96.29, P<0.01). Nissl staining results showed that a large number of morphologically abnormal and disintegrated Nissl bodies could be observed in the hippocampal CA1 and CA3 regions of EP group, which showed a smaller size, irregular morphology, chromatin pyknosis, boundary blur between nucleus and cytoplasm. Compared with the normal group, the difference was significant (P<0.05). While in miRNA-22 agomir group, the disintegration of Nissl bodies was improved and the number of Nissl bodies increased. Conclusion Intraventricular injection of miRNA-22 agomir can down-regulate the expression of P2X7 receptor and related inflammatory factors in hippocampus of epileptic rats, thus inhibiting seizures. Key words: miRNA-22; P2X7 receptor; Epileptogenic zone; Inflammatory factor; Neuroprotection

MiR-128b Promotes Cerebral Infarction by Regulating the Expressions of BCL-2 and CAPASE3

To investigate the effect of miR-128b on apoptosis and BCL-2 and CAPASE3 expression in a rat middle cerebral artery occlusion (MCAO) model. The MCAO model was established by the thread embolism method. miR-128b agomir and antagomir were injected into the ventricle of MCAO rats by stereotaxic intracerebral injection. Then the rats were divided into a sham group, model group, miR-128b agomir group, and miR-128b antagomir group. Zea Longa was used to score the modeling rats. The area of cerebral infarction was assessed by 2,3,5-triphenyltetrazolium chloride staining. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The miR-128b relative expression was detected by real-time polymerase chain reaction. The expressions of BCL-2 and CAPASE3 were detected by immunohistochemistry and Western blotting. The MCAO model was constructed successfully. The expressions of miR-128b in the MCAO groups were higher than that of the sham group (P < 0.05). Compared with the model group, the cerebral infarction area in the miR-128b agomir group was significantly bigger and that of the miR-128b antagomir group was smaller (P < 0.05). The number of apoptotic cells in the miR-128b agomir group was more and that of miR-128b antagomir group was less (P < 0.05 vs. model group). The BCL-2 expression reduced and CAPASE3 expression increased in the MCAO groups (P < 0.05 vs. sham group). Compared with the model group, the Bcl-2 expression decreased and Caspase 3 expression increased in the miR-128b agomir group, and those in the miR-128b antagomir group were opposite. miR-128b promoted cerebral infarction in MCAO rats by regulating Bcl-2 and Caspase 3 expression.

Effect of exogenous miR-181b on postoperative cognitive dysfunction in mice

Objective To evaluate the effect of exogenous miR-181b on postoperative cognitive dysfunction (POCD) in mice. Methods A total of 108 clean-grade healthy male C57BL/6 mice, weighing 25-30 g, were divided into 4 groups (n=27 each) using a random number table method: control group (group C), POCD group, miR-181b agonist agomir group (group Ag), and agomir negative control group (group AC). POCD was induced by open tibial fracture in 2.1% isoflurane-anesthetized mice.A total volume of miR-181b agomir 4 μl (0.25 nmol/μl) was injected into bilateral hippocampi at 2 days before establishing the model in group Ag.Agomir negative control solution 4 μl was given at 2 days before establishing the model in group AC.Eighteen mice were selected at days 1, 3 and 7 after surgery, and contextual fear conditioning test was performed to assess cognitive function.Nine mice in each group were sacrificed at 6, 12 and 24 h after surgery (3 mice at each time point), and the hippocampus was isolated for determination of miR-181b expression (by quantitative real-time polymerase chain reaction) and interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) contents (by enzyme-linked immunosorbent assay). Results Compared with group C, the percentage of time spent freezing in the contextual fear conditioning test was significantly decreased at 1, 3 and 7 days after surgery, and the expression of miR-181b was down-regulated and the contents of IL-1β and TNF-α were increased at 6, 12 and 24 h after surgery in POCD and AC groups (P<0.05). Compared with POCD and AC groups, the percentage of time spent freezing in the contextual fear conditioning test was significantly increased 1, 3 and 7 days after surgery, and the expression of miR-181b was up-regulated and the contents of IL-1β and TNF-α were decreased at 6, 12 and 24 h after surgery in group Ag (P<0.05). Conclusion Exogenous miR-181b can mitigate POCD, and the mechanism may be related to inhibiting inflammatory responses in the hippocampus of mice. Key words: MicroRNAs; Cognition disorders; Hippocampus; Neuroinflammation

Role of miR-34c in the cognitive function of epileptic rats induced by pentylenetetrazol.

Studies suggest that microRNA (miR)-34c may serve a role in cognitive function in rodent and primate groups. A previous study demonstrated an increase in miR-34c expression in chronic epileptic rats with memory disorders, induced by pentylenetetrazol (PTZ). However, the mechanism underlying the effects of miR-34c on cognitive function in epileptic rats remains unclear. Therefore, the present study investigated alterations in cognitive function in temporal lobe epileptic rats, induced by repeated injections of PTZ, following treatment with an miR-34c agomir compared with a scramble group. Increased expression of miR-34c was observed in the agomir group, in addition to an increased deficit in learning and memory function in the Morris water maze test. Glutamate receptor ionotropic N-methyl-D-aspartate (NMDA) 2B (NR2B), phosphorylated (p)-reduced nicotinamide-adenine dinucleotide phosphate-dependent diflavin oxidoreductase 1 (NR1) and p-glutamate receptor 1 (GluR1) protein expression was detected in the hippocampus using western blotting. Additionally, the downregulation of NR2B, p-NR1 and p-GluR1 in the miR-34c agomir group demonstrated that miR-34c may serve a negative role in cognitive function in epileptic seizures, by dysregulating NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, which are associated with long-term potentiation.

MiR-24 reduces serum lipid levels and inhibits brain tissue cells apoptosis of rats with cerebral infarction.

To study miR-24 effects on cerebral infarction in rats. A rat middle cerebral artery occlusion model (MCAO) was constructed. Intracerebroventricular stereotactic injection of miR-24 agomir/antagomir was performed in the rat MCAO model. According to different experiences, rats were divided into normal, sham, MCAO, miR-24 agomir and miR-24 antagomir groups. Serum TCH, HDL and TG levels were detected. Comparing the normal and sham groups, we observed decreased relative miR-24 expression (P 0.05) and increased cerebral infarction area percentage, apoptotic cells and relative caspase-3 protein expression (P 0.05) in the MCAO, miR-24 agomir and miR-24 antagomir groups. TC, TG and HDL-C levels of the MCAO and miR-24 antagomir groups were higher than those of normal and sham groups (P 0.05). Compared with the MCAO group, increased relative miR-24 expression (P 0.05) and decreased TC, TG and HDL-C levels, cerebral infarction area percentage, number of apoptotic cells and caspase-3 expression (P 0.05) were found in the miR-24 agomir group, contrasting with the observations from the miR-24 antagomir group. miR-24 reduced serum TCH, HDL and TG levels and inhibited brain tissue cell apoptosis in rats with cerebral infarction.

MiR-24 Reduces Serum Lipid Levels and Inhibits Brain Tissue Cells Apoptosis of Rats with Cerebral Infarction.

To study miR-24 effects on cerebral infarction in rats. A rat middle cerebral artery occlusion model (MCAO) was constructed. Intracerebroventricular stereotactic injection of miR-24 agomir/antagomir was performed in the rat MCAO model. According to different experiences, rats were divided into normal, sham, MCAO, miR-24 agomir and miR-24 antagomir groups. Serum TCH, HDL and TG levels were detected. Comparingthe normal and sham groups, we observed decreased relative miR-24 expression (p < 0.05) and increased cerebral infarction area percentage, apoptotic cells and relative caspase-3 protein expression (p < 0.05) in theMCAO, miR-24 agomirand miR-24 antagomir groups. TC, TG and HDL-C levels of the MCAO and miR-24 antagomir groups were higher than those of normal and sham groups (p < 0.05).Compared with the MCAO group, increased relative miR-24 expression (p < 0.05) and decreased TC, TG and HDL-C levels,cerebral infarction area percentage, number of apoptotic cells and caspase-3 expression (p < 0.05) were found in themiR-24 agomir group, contrasting with theobservations from the miR-24 antagomir group. miR-24 reduced serum TCH, HDL and TG levels and inhibited brain tissue cell apoptosis in rats with cerebral infarction.

Effects of miR-23b on hypoxia-induced cardiomyocytes apoptosis

ObjectivesThe aim of this study was to investigate the role of miR-23b in hypoxic cardiomyocytes and the potential mechanism. MethodsMyocardial samples of patients with cyanotic or acyanotic congenital heart disease (CHD) were collected to evaluate miR-23b expression. Agomir or antagomir of miR-23b was transfected into H9C2 cells. MTT, LDH assay and TUNEL staining were used to determine the cell proliferation and apoptosis under hypoxic conditions. Besides, the expression levels of cleaved-caspase-3, cleaved-PARP, Bad, Bcl-2 and Bax in hypoxic H9C2 cells were determined by western blot and qRT-PCR, respectively. ResultsHigher miR-23b expression levels were found in the patients with cyanotic CHD compared with the patients with acyanotic CHD. In addition, the expression of miR-23b was gradually up-regulated with prolonged hypoxia time in the H9C2 cells. Using MTT and LDH assays, cell growth was significantly decreased in the agomir group than that in the agomir-negative control (NC) group, while antagomir increased the cell growth. Using TUNEL staining and flow cytometry analysis, miR-23b promoted hypoxia-induced apoptosis. The expression levels of pro-apoptotic proteins, such as cleaved-caspase-3, cleaved-PARP and Bad, were significantly increased in the agomir group, while the Bcl-2 levels and Bcl-2/Bax ratio were decreased. Opposite tendency was observed in the antagomir group. Dual luciferase reporter assay and western blot analysis confirmed that Smad3 was a direct target of miR-23b. ConclusionOver-expression of miR-23b may increase cardiomyocyte apoptosis and reduce cell growth under hypoxic conditions.

MiR-449a regulates caprine endometrial stromal cell apoptosis and endometrial receptivity

In this study, an RT-qPCR analysis showed that the expression levels of miR-449a in the pre-receptive endometrium were lower compared to the receptive endometrium, which is consistent with previous sequencing data (previous investigations). To detect the role of miR-449a in endometrial receptivity, we transfected caprine endometrial stromal cells (ESCs) cultured in vitro with miR-449a mimics. The results revealed that miR-449a decreased the mRNA and protein levels of LGR4 by targeting its 3′-untranslated region. The miR-449a mimics significantly reduced the G1 cell population from 52.56% (mimic NC) to 42.19% with a concordant increase in the G2 and S cell populations from 47.44% (mimic NC) to 57.81%, suggesting that miR-449a caused ESC cell cycle arrest. In addition, the number of apoptotic cells was significantly higher in ESCs transfected with miR-449a mimics (P < 0.05) than in ESCs transfected with mimic NC. In vivo, rich pinopodes were observed on the endometrial surface in the miR-449a agomir group compared with the miR-449a antagomir group. The results of hematoxylin-eosin staining showed that endometrial thickness was significantly increased in the miR-449a agomir group compared with the miR-449a antagomir group. These results suggest that miR-449a could enhance endometrial receptivity.

MiR125b-5p protects endothelial cells from apoptosis under oxidative stress

Endothelial cell damage, such as apoptosis and necrosis, is involved in many cardiovascular diseases. In recent years, the crucial role of microRNAs in controlling tissue homeostasis and disease in the epithelium has become widely recognized. In the present study, human umbilical vein endothelial cells were transfected with a miRNA agomir and a SMAD4 expression vector. The expression of miR125b-5p was determined by using quantitative real-time polymerase chain reaction. Cell apoptosis and necrosis were measured with flow cytometry. The expression of SMAD4 was evaluated with Western blotting. Here, we demonstrated that the rates of apoptosis and necrosis were significantly decreased in the miR125b-5p agomir group of HUVECs under H2O2-induced oxidative stress compared with the miR125b-5p antagomir group. Further experiments revealed that the expression of SMAD4 is negatively regulated by miR125b-5p. Moreover, we identified that the rates of apoptosis and necrosis were increased when SMAD4 and miR125b-5p were both overexpressed compared with miR125b-5p overexpression alone. The present study demonstrates for the first time that the overexpression of miR125b-5p can reduce H2O2-induced oxidative damage via SMAD4, suggesting that miR125b-5p has therapeutic potential for preventing oxidative stress-related diseases.