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@#Objective To investigate the effect of aloperine(ALO)on interleukin-1β(IL-1β)-induced chondrocyte injury and its mechanism. Methods Chondrocytes were randomly divided into control(Con)group,IL-1 β group,IL-1β + ALO-L(25 mg/L)group,IL-1β + ALO-M(50 mg/L)group and IL-1 β + ALO-H(100 mg/L)group;Con group,IL-1βgroup,IL-1β + miR-NC group and IL-1β + miR-16-5p group;Con group,IL-1β group,IL-1β + si-NC group and IL-1β + siSOX5 group. Cells in IL-1β group were treated with 10 ng/mL IL-1β,while no treatment was given in Con group. The transcription levels of miR-16-5p and SOX5 mRNA in chondrocytes were detected by qRT-PCR;The contents of IL-6,TNF-αand IL-1β were detected by ELISA;The expression levels of Bcl-2,Bax and SOX5 protein were detected by Western blot and the apoptosis was detected by flow cytometry. Results Compared with IL-1 β group,the contents of IL-6,TNF-α and IL-1βin IL-1β + ALO-L group,IL-1β + ALO-M group and IL-1β + ALO-H group decreased significantly(t = 5. 002～20. 653,each P < 0. 001),the apoptosis rate decreased significantly(t = 5. 473～17. 371,each P < 0. 001),the expression level of Bcl-2 protein increased significantly(t = 7. 800～16. 100,each P < 0. 001),and the expression level of Bax protein decreased significantly(t = 4. 993～14. 311,each P < 0. 001);The mRNA transcription level of miR-16-5p gene increased significantly(t = 6. 688～16. 545,each P < 0. 001),while the mRNA transcription level and protein expression level of SOX5 gene decreased significantly(t = 4. 609～15. 393,each P < 0. 001). Compared with the IL-1β + miR-NC group,the mRNA transcription level of miR-16-5p in the IL-1β + miR-16-5p group increased significantly(t = 17. 106,P < 0. 001),the contents of IL-6,TNF-α and IL-1 β decreased significantly(t = 15. 030～20. 013,each P < 0. 001),the apoptosis rate decreased significantly(t = 12. 273,P < 0. 001),the expression level of Bcl-2 protein increased significantly(t = 15. 652,P < 0. 001),and the expression level of Bax protein decreased significantly(t = 12. 999,P < 0. 001). Compared with IL-1β +si-NC group,the expression level of SOX5(t = 13. 444,P < 0. 001),IL-6,TNF-α and IL-1β in IL-1β + si-SOX5 group decreased significantly(t = 14. 087～17. 103,each P < 0. 001),the apoptosis rate decreased significantly(t = 11. 991,P < 0. 001),the expression level of Bcl-2 protein increased significantly(t = 13. 864,P < 0. 001),and the expression level of Bax protein decreased significantly(t = 11. 818,P < 0. 001). Conclusion Alo inhibited the apoptosis of chondrocytes induced by IL-1β,thus reducing the injury of chondrocytes,of which the mechanism might be regulating the expression of miR-16-5p and SOX5 and the secretion of inflammatory factors in chondrocytes.
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Chronic lymphocytic leukemia (CLL) is a blood cancer characterized by the accumulation of clonal B-lymphocytes. This study evaluated the mRNA gene expression of miR-15a, miR-16- 1, ZAP-70, and Ang-2 by qPCR, as well as the plasma levels of Bcl-2 by Elisa immunoassay, in CLL patients and healthy controls. Significant differences were observed when comparing patients and controls regarding miR-15a (p < 0.001), miR-16-1 (p < 0.001) mRNA, Ang-2 gene expression, and Bcl-2 plasma levels (p < 0.001). When stratified by risk, differences were maintained with a significantly reduced expression in high-risk patients. A positive correlation was observed between miR-15a and platelets (R2 = 0.340; p = 0.009) as well as between Bcl-2 and leukocytes (R2 = 0.310; p = 0.019). Conversely, negative correlations were observed between ZAP-70 and platelets (R2 = - 0.334; p = 0.011), between miR-15a and lymphocytes (R2 = - 0.376; p = 0.004), as well as between miR-16-and lymphocytes (R2 = - 0.515; p = 0.00004). The data suggest that a reduction in miR-15a and miR-16-1 expressions, in addition to an overexpression of Bcl-2, are associated with the reduction in apoptosis and, consequently, to a longer survival of lymphocytes, thus contributing to lymphocyte accumulation and aggravation of the disease. By contrast, Ang-2 expression was significantly higher in A than in B + C Binet groups. This context leads to the speculation that this biomarker should be investigated in more robust studies within populations with a still relevantly indolent form of the disease in an attempt to identify those patients with a greater potential for an aggravation of the disease
Subject(s)
Humans , Male , Female , Biomarkers/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , ZAP-70 Protein-Tyrosine Kinase/analysis , Patients , Enzyme-Linked Immunosorbent Assay/instrumentation , Gene Expression , ApoptosisABSTRACT
【Objective】 To investigate the role of non-coding microRNA miR-16-5p in ZIKV replication and the underlying mechanism. 【Methods】 1×105/mL HeLa cells were seeded in 24-well plate and infected with ZIKV(MOI=5). RNAs were harvested, and miR-16-5p expression levels were measured by qRT-PCR at 24, 48 and 72 hour post infection, respectively. HeLa cells were transfected with 20nM miR-16-5p mimic and infected with ZIKV(MOI=5) at 24h post transfection. RNAs were extracted and ZIKV RNA and several inflammation factors expression were tested using qRT-PCR at 48h post infection. HeLa cells were co-transfected with 1μg NFκB-luc and 10ng pRL-TK with 20nM miR-16-5p mimic, and then infected with ZIKV(MOI=5) for 24h before the luciferase expression was tested at 48h post infection. HeLa cells were transfected with 20nM miR-16-5p mimic and infected with ZIKV(MOI=5) at 24h post transfection, and cell apoptosis was assayed through flow cytometry. 【Results】 Compared with uninfected control, miR-16-5p expression was significantly decreased at 24h, 48h and 72h following ZIKV infection. MiR-16-5p over-expression inhibited ZIKV replication, while upregulated NFκB activity and inflammation factors expression compared with the negative mimic-transfected cells. MiR-16-5p overexpression also promoted HeLa cell apoptosis. 【Conclusion】 ZIKV infection downregulated intracellular miR-16-5p expression. Overexpression of miR-16-5p suppressed ZIKV infection. MiR-16-5p inhibited ZIKV replication and promoted cell apoptosis probably by activating NFκB pathway and stimulating inflammation factors expression.
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This study is to investigate the effect of antidepressant medicine prescription Dingzhi Xiaowan (DZ) on miR-16 expression levels in vitro and in vivo, and to explore the mechanism of DZ elevated levels of 5-HT from the perspective of post transcriptional regulation. Firstly, a chronic unpredictable mild moderate stimulation (CUMS) combined with solitary rising depression rat model was established, the behavior changes were detected after different doses of DZ (600, 300, 150 mg·kg⁻¹) given for 3 weeks, high performance liquid chromatography (HPLC) was used to detect 5-HT level in hippocampal, PCR method was used to observe the effect of DZ on the expression of SERT mRNA and miR-16 in hippocampus of CUMS rat. The effects of DZ (10, 100, 200, 500 mg·L⁻¹) on the expression of miR-16 and SERT mRNA in the cell model induced by miR-16 silencing and corticosterone or glutamate injury were observed in primary cultured hippocampal neurons of rats in vitro. It was found that 300 mg·kg⁻¹ and 600 mg·kg⁻¹ DZ could significantly improve the behavioral score of CUMS rats, increase the level of 5-HT in hippocampus, and increase the expression of miR-16 and decrease the expression of SERT in the hippocampus of rats. At the same time, in primary cultured hippocampal neurons, 100, 200, 500 mg·L⁻¹ of DZ could significantly increase the expression level of miR-16 in miR-16 silencing and corticosterone or glutamate injury cell model, and decrease the expression level of SERT significantly. So DZ could inhibit the reuptake of 5-HT by inhibiting the expression of SERT by up regulating the expression level of miR-16, and finally increase the level of 5-HT in the brain and exert antidepressant effect.
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We investigated correlation between the level of miR-16 expression and the severity of Staphylococcus aureus sepsis,and further explored its potentially clinical significance.Blood samples were collected from 32 patients,including each 8 cases of septic shock,severe sepsis and general sepsis,as well as 8 cases of healthy volunteers.Blood samples from 24 cases of healthy subjects with different ages were measured,and additionally 8 cases of blood samples from gram negative bacteria sepsis were also determined in current study as a control.Trizol solution for the whole blood lysis was added into blood samples,and followed by the extraction of microRNA.The expression levels of miR-16 in different groups were measured by fluorescence quantitative PCR,in which 2-Delta Ct method was used.SPSS software (version 13.0) was used to analyze the statistical differences between the groups,and further analyze the correlation between miR-16 value and the corresponding CRP and PCT values.Results showed that the expression level of miR-16 was negatively correlated with the severity of Staphylococcus aureus sepsis.There were statistically significant differences in experimental groups when compared with the control (P＜0.001),and there was also a statistically significant difference between each experimental group (P＜0.01).We found that the expression level of miR-16 was negatively correlated with CRP and PCT,the correlation coefficients were-0.561 and-0.769 respectively,and trend analysis showed that there was a significantly negative correlation.A significantly negative correlation was found between the miR-16 expression level and severity of sepsis,suggesting that miR-16 may serve as a biomarker for the severity of Staphylococcus aureus sepsis.
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Objective To investigate the expression of miR-16 in spinal cord and the effects of miR-16 mimics on the pain behavior and the expression of its target gene BDNF during the development and maintenance of bone cancer pain.Methods The bone cancer pain model rats were developed by intra-tibia inoculation of Walker 256 mammary gland cells.Before inoculation and 3,5,7,10,14 d after inoculation,the samples of spinal cord L4～6 lumbar enlargement were collected to detect the expression of miR-16 using real-time PCR.Mice in group BM and group BN were intrathcal injected of miR-16 mimics and its negative control products on 10 d,11 d,12 d after inoculation.Paw withdrawal mechanical threshold (PWMT) was measured using von Frey hair mechanical stimulation.The expression of BDNF was detected using western-blot on 14 d after behavior tests.Results Compared with the base level and the level in group S,the significant decrease of miR-16 expression was observed at 5 d～14dingroupT (5d (0.91±0.04),7 d (0.77±0.01),10 d (0.73±0.03),14d (0.42±0.08)) (all P＜ 0.05).Compared with SH group,PWMT was significantly decreased in BC and BP groups at 5 d～ 14 d (P＜0.05),and in group BM at 5 d～ 10 d(P＜0.05).Compared with BP group,PWMT was significantly higher in BM group at 10～12 d(P＜0.05).Compared with SH group,the expression of BDNF was significantly increased in BN and BP groups((2.78±0.31),(2.34±0.23)) (all P＜0.01).Compared with BP group,the expression of BDNF was significantly decreased in BM group (1.42±0.16) (P＜0.01).Conclusion miR-16 is downregulated in spinal cord of bone cancer pain rats,while intrathcal injection of miR-16 mimics can attenuate the pain behaviors in a rat model of bone cancer pain and decrease the expression of BDNF.miR-16 may modulate bone cancer pain through BDNF.
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Objective To study whether miR-15a and miR-16-1 can enhance the sensitivity of Raji cells to cytarabine (Ara-C). Methods MiR-15a and miR-16-1 oligonucleotides were transfected into Raji cells with Lipofectamine™ 2000, and then the cells were treated with Ara-C. The IC50 values of Ara-C was detected by CCK8 assay. The growth of Raji cells was measured by trypan blue dye exclusion method. The apoptotic cells were observed by Hoechst dyeing; AnnexinV/PI double dyeing and glow cytometry(FCM) were used to examine the cell apoptotic rate. Results After transfection of miR-15a or miR-16-1 into Raji cells, the IC50 values of Ara-C were 10. 41 and 10. 86, respectively, which were significantly lower than that of the untransfected group(15.43)and scrambled oligonucleotides (SODN)transfection group(14. 92, P<0.05). Trypan blue dye exclusion assay showed that miR-15a/miR-16-1 transfection group had obviously decreased the cell growth compared to miR-15a, miR-16-1 group, untransfected group and SODN transfected group; Hoechst dyeing demonstrated plenty of apoptotic cells. AnnexinV/PI double dyeing assays by FCM indicated that the cell apoptotic rates in earlier period and late period were 20. 93% and 25. 27% in the miR-15a+Ara-C group, and 20. 69% and 23. 13% in the miR-16-1 + Ara-C group, which were obviously higher than those in miR-15a group (6. 99%, 10. 08%), miR-16-1 group(4. 73%, 10. 64%), Ara-C group (10. 88%, 11. 83%) and control group (14. 39%, 11. 93%). Conclusion MiR-15a and miR-16-1 oligonucleotides can enhance the sensitivity of Raji cells to Ara-C.