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Objective:To investigate the expression and significance of microRNA-21 (miRNA-21) and microRNA-181b (miRNA-181b) in the peripheral blood of patients with schizophrenia.Methods:A total of 100 patients with schizophrenia who received treatment in Shaoxing 7 th People's Hospital from March 2020 to March 2022 were included in the study group. An additional 30 healthy controls who concurrently underwent physical examination were included in the control group. The expression of miRNA-21 and miRNA-181b in peripheral blood was compared between the two groups. The 100 patients with schizophrenia received standardized clinical treatment. Their mental symptoms were evaluated with the Positive and Negative Symptom Scale (PANSS). miRNA-21 and miRNA-181b expression and PANSS scores before and 1, 4, 8, and 12 weeks after treatment were collected and compared between the two groups. The receiver operating characteristic curve was plotted to analyze the value of miRNA-21 and miRNA-181b expression in the diagnosis of schizophrenia. Results:Serum miRNA-21 and miRNA-181b expression in the study group were (2.41 ± 1.12) and (15.62 ± 2.26), respectively, which were significantly higher than (0.73 ± 0.37) and (8.11 ± 0.98) in the control group ( t = 8.07,17.67, both P < 0.05). With the prolongation of treatment time, serum miRNA-21 and miRNA-181b expression and PANSS score in the study group gradually decreased (all P < 0.001). The area under the receiver operating characteristic curve plotted for evaluating the value of miRNA-21 and miRNA-181b expression in the diagnosis of schizophrenia was 0.616 and 0.683, respectively. The area under the receiver operating characteristic curve plotted for evaluating the value of miRNA-21 combined with miRNA-181b expression in the diagnosis of schizophrenia was 0.788, which was markedly higher than that for the detection of miRNA-21 or miRNA-181b expression alone. Conclusion:miRNA-21 and miRNA-181b are abnormally highly expressed in the peripheral blood of patients with schizophrenia. Both of them can be used as objective and effective indicators for early diagnosis of schizophrenia. Combined detection of miRNA-21 and miRNA-181b provides higher accuracy in the diagnosis of schizophrenia than the detection of miRNA-21 or miRNA-181b alone.
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【Objective】 To explore the effects of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) on the invasion and migration of prostate cancer cells (PC3 cells) by regulating microRNA-181b-5p (miR-181b-5p)/tissue inhibitor of metalloproteinase 3 (TIMP3). 【Methods】 The prostate cancer tissues and adjacent tissues were collected from 20 prostate cancer patients treated in our hospital during Dec.2020 and Dec.2021. The expressions of MEG3 and miR-181b-5p in tissues were detected with quantitative real-time PCR (qRT-PCR). P3 cells were randomly divided into control group (untreated), pcDNA3.1-NC (transfected with pcDNA3.1-NC), pcDNA3.1-MEG3 group (transfected with pcDNA3.1-MEG), pcDNA3.1-MEG3+miR-NC group (pcDNA3.1-MEG3 co-transfected with miR-NC), pcDNA3.1-MEG3+miR-181b-5p mimic group (pcDNA3.1-MEG3 co-transfected with miR-181b-5p mimic). The expressions of MEG3 and miR-181b-5p in PC3 cells were detected with qRT-PCR. The cell viability, invasion and migration ability were determined with MTT assay, Transwell assay and scratch assay. The protein expressions of TIMP3, matrix metalloproteinase (MMP)9 and MMP2 in PC3 cells were detected with Western blot. The targeting relationship of MEG3, miR-181b-5p and TIMP3 was analyzed with dual luciferase assay. 【Results】 The expressions of MEG3 in prostate cancer tissues ( 0.37±0.05 vs. 1.00±0.04) and cells (0.31±0.06 vs. 1.00±0.01) were significantly decreased (P<0.05). Compared with the control group, the pcDNA3.1-MEG3 group had significantly decreased expression of miR-181b-5p (0.26±0.04 vs.1.00±0.02 ), cell survival rate (53.60±5.22 vs.100.00±0.00), number of invasive cells (62.33±9.85 vs.162.34±21.30), cell migration rate (32.85±3.80 vs.75.22±5.96), expressions of MMP9 (0.61±0.08 vs.1.62±0.23) and MMP2 (0.73±0.10 vs.1.20±0.16), but significantly higher expressions of MEG3 (2.31±0.36 vs. 1.00±0.01) and TIMP3 (1.32±0.24 vs. 0.53±0.08) (P<0.05). Overexpression of miR-181b-5p reversed the above changes (P<0.05). MiR-181b-5p had a targeting relationship with MEG3 and TIMP3. 【Conclusion】 Overexpression of lncRNA MEG3 can inhibit miR-181b-5p to promote the expression of TIMP3, thereby inhibiting invasion and migration of PC3 cells.
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Objective To investigate the protective effect of microRNA-181b (miR-181b) on aged rats with sepsis-induced hippocampus injury in vivo. Methods Seventy-five male healthy old Sprague-Dawley (SD) rats were randomly divided into five groups (n = 15) using a random number table: sham operation group (Sham group), sepsis group [cecal ligation and puncture (CLP) group], miR-181b Agomir+CLP group (Ag+CLP group), miR-181b Antagomir+CLP group (An+CLP group) and normal saline (NS) control group (NS+CLP group). Rats sepsis model was reproduced by CLP, and in Sham group, the cecum of rats was separated only after abdominal operation without ligation or perforation. The rats in Ag+CLP group were given miR-181b Agomir 10 μL via lateral ventricle at 24 hours before CLP, the rats in An+CLP group were given 10 μL miR-181b Antagomir, and those in NS+CLP group were given 10 μL NS. At 6, 12, 24 hours after CLP, 5 rats of each group were sacrificed randomly, and hippocampus were harvested. The expression of miR-181b in hippocampus was determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression of nuclear factor-κB p65 (NF-κB p65) was determined by Western Blot. The contents of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Results Compared with Sham group, the expression of miR-181b in hippocampus of CLP group was significantly decreased at 6 hours after CLP (2-ΔΔCT: 0.70±0.12 vs. 0.98±0.06, P < 0.05), and the expressions of NF-κB p65, IL-1β and TNF-α were significantly increased [NF-κB p65/Histone H3:0.30±0.03 vs. 0.07±0.01, IL-1β (ng/L): 120.39±8.02 vs. 50.55±11.12, TNF-α (ng/L): 59.48±4.60 vs. 40.31±3.96, all P < 0.05], this trend was continued till 24 hours, and these results indicated that there was obvious inflammation in hippocampus of sepsis rats. There was no statistical difference in the expression of miR-181b, NF-κB p65, IL-1β or TNF-α in hippocampus between NS+CLP group and CLP group, which indicated that injection of NS into the rat lateral ventricle, had not aggravated the damage degree of hippocampus. Compared with CLP group, the expression of miR-181b in hippocampus of Ag+CLP group was significantly increased at 6 hours after CLP (2-ΔΔCT: 1.87±0.25 vs. 0.70±0.12, P < 0.05), and the expressions of NF-κB p65, IL-1β and TNF-α were significantly lowered [NF-κB p65/Histone H3:0.16±0.03 vs. 0.30±0.03, IL-1β (ng/L): 73.76±8.17 vs. 120.39±8.02, TNF-α (ng/L): 49.52±4.77 vs. 59.48±4.60, all P < 0.05]. There was no statistical difference in the expression of miR-181b in hippocampus between An+CLP group and CLP group (2-ΔΔCT: 0.80±0.08 vs. 0.70±0.12 at 6 hours, 0.48±0.03 vs. 0.46±0.05 at 12 hours, 0.61±0.09 vs. 0.63±0.07 at 24 hours, all P > 0.05), but the expressions of NF-κB p65, IL-1β and TNF-α in hippocampus at 6 hours after CLP of An+CLP group were significantly higher than those of CLP group [NF-κB p65/Histone H3: 0.44±0.02 vs. 0.30±0.03, IL-1β (ng/L): 134.21±5.78 vs. 120.39±8.02, TNF-α (ng/L): 67.62±5.86 vs. 59.48±4.60, all P < 0.05], this trend was continued till 24 hours after CLP. The above results showed that overexpression of miR-181b might attenuate the inflammation of hippocampus through down-regulation of NF-κB, IL-1β and TNF-α. Conclusions The expression of hippocampal miR-181b was significantly decreased in septic rats. Up-regulation of miR-181b could inhibit the activation of NF-κB signal pathway and the release of the inflammatory cytokine IL-1β and TNF-α stimulated by sepsis, and alleviate the inflammatory reaction and hippocampus injury in rat with sepsis.
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Objective To verify whether early growth response-3(EGR3) gene is targeted by microRNA-181b using molecular biology methods so as to provide guidance for the subsequent study on microRNA-181b`s role in the molecular mechanisms of schizophrenia.Methods Bioinformatic methods predicted that EGR3 gene is targeted by microRNA-181b.PCR methods amplified the fragment in EGR3 gene 3`UTR including the putative microRNA-181b binding site.Then the sequence was cloned into the pmirGLO luciferase vector.The DNA sequences of the amplified fragments were identified by restriction enzyme digestion and sequencing, and were consistent with the reference sequence from UCSC.This constructed vector was marked as pmirGLO-EGR3 vector.Finally, the pmirGLO vector, the pmirGLO-EGR3 vector, microRNA-181b mimics and negative control (NC) were divided into 5 groups and transfected into HEK393T cells;the luciferase activity was tested by dual luciferase reporter gene assay.Results The results of restriction enzyme digestion and sequencing demonstrated that the PCR fragmentwas successfully cloned into pmirGLO vector.The transfection results showed that the recombinant plasmid was successful transfected into HEK293T under the fluorescence microscope, with transfection efficiency being about 90％.The results of dual luciferase activity assay demonstrated that microRNA-181b significantly decreased the reporter gene`s activity compared with the NC.Conclusion At the cellular level, the schizophrenia susceptibility gene EGR3 was verified to be targeted by micorRNA-181b, which provides a new clue for the subsequent study on microRNA-181b`s role in the molecular mechanisms of schizophrenia.
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Objective To explore the role of microRNA-181b (miR-181b) in cerebral ischemic injury in vivo and its mechanism. Methods Using middle cerebral artery occlusion (MCAO) model to mimic ischemic injury in vivo, the heat shock protein A5(HSPA5) protein level was determined by using Western blotting. The extent of neural cell loss in ischemic cortex after MCAO was assessed by Nissl staining. Neurological score was performed to evaluate the degree of cerebral ischemic injury after MCAO. Results We found that miR-181b antagomir down-regulated miR-181b expression levels in cerebral ischemic cortex of mice after MCA0(P <0.05, n =3). MiR-181 b antagomir improved neurological deficit of mice at 24 hours after transient MCAO (P < 0.05, n =6). HSPA5 protein levels were significantly up-regulated in ischemic cortex of mice after MCAO, and miR-181b antagomir further up-regulated HSPA5 (P < 0.05, n=3). Consequently, miR-181b antagonists attenuated neural cell loss in ischemic cortex after MCAO (P <0.05, n=3). Conclusion MiR-181 b plays an important role in ischemic injury of mice through regulating HSPA5 protein level.
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AIM:To explore the role of microRNA-181b (miR-181b) in ischemic injury and autophagy pro-tein 5 (Atg5) levels of mice .METHODS:Oxygen-glucose depletion (OGD) model in N2A cells to mimic ischemic in-jury in vitro was established .A middle cerebral artery occlusion ( MCAO) model to mimic ischemic injury in vivo was also induced in mice.The N2A cell apoptosis after OGD was assessed by in situ cell death detection kit.The Atg5 and caspase-9 expressions were determined by Western blotting .Luciferase reporter assay was performed to identify the direct binding of miR-181b with 3’-UTR of Atg5 mRNA.RESULTS:The alteration of miR-181b expression level by transfection with pre-miR-181b or anti-miR-181b significantly affected N2A cell apoptosis (P<0.05).Accordingly, the changes of miR-181b levels significantly altered the protein level of Atg 5 ( P<0.05 ) .Co-transfection of the luciferase reporters with pre-miR-181b or anti-miR-181b resulted in the inhibition or enhancement of the luciferase activities of luciferase expressing plasmid containing 3’-UTR of Atg5 mRNA (P<0.05).In addition, the miR-181b antagonist significantly reduced the cleaved caspase-9 levels in cerebral ischemic cortex of the mice after MCAO ( P<0.05 ) .CONCLUSION: Down-regulation of miR-181b plays an important role in ischemic injury of mice through regulating Atg 5 protein level.