ABSTRACT
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
BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.
Subject(s)
Animals , Hair Follicle/cytology , Hair Follicle/metabolism , Dermis/cytology , Wnt3A Protein/metabolism , RNA, Long Noncoding/metabolism , Biological Assay/methods , Goats , RNA, Long Noncoding/genetics , Luciferases , MethylationABSTRACT
@#miRNA-15a(miR-15a)is a non-coding small molecule RNA located on 13q14 gene. It affects the growth, development, differentiation and apoptosis of all organs and cells of the whole body. As the study progressively deepened, it was found that the role of miR-15a in different tissues and cells was not entirely consistent. Sometimes it plays a role in suppressing cancer, and sometimes it promotes cancer. The signal pathways it affects are complex and diverse. With the deepening of biological research into cell signaling pathways, miRNA-15a has become a miRNA more extensively studied. But in the ophthalmology, the corresponding research is not much. In this article, we mainly focus on the mechanism of miR-15a and its current research situation in ophthalmic diseases, so as to provide a reference for further study and their treatment.
ABSTRACT
@#miRNA-15a(miR-15a)is a non-coding small molecule RNA located on 13q14 gene. It affects the growth, development, differentiation and apoptosis of all organs and cells of the whole body. As the study progressively deepened, it was found that the role of miR-15a in different tissues and cells was not entirely consistent. Sometimes it plays a role in suppressing cancer, and sometimes it promotes cancer. The signal pathways it affects are complex and diverse. With the deepening of biological research into cell signaling pathways, miRNA-15a has become a miRNA more extensively studied. But in the ophthalmology, the corresponding research is not much. In this article, we mainly focus on the mechanism of miR-15a and its current research situation in ophthalmic diseases, so as to provide a reference for further study and their treatment.
ABSTRACT
There is accumulating evidence that microRNAs are emerging as pivotal regulators in the development and progression of neuropathic pain. MicroRNA-15a/16 (miR-15a/16) have been reported to play an important role in various diseases and inflammation response processes. However, whether miR-15a/16 participates in the regulation of neuroinflammation and neuropathic pain development remains unknown. In this study, we established a mouse model of neuropathic pain by chronic constriction injury (CCI) of the sciatic nerves. Our results showed that both miR-15a and miR-16 expression was significantly upregulated in the spinal cord of CCI rats. Downregulation of the expression of miR-15a and miR-16 by intrathecal injection of a specific inhibitor significantly attenuated the mechanical allodynia and thermal hyperalgesia of CCI rats. Furthermore, inhibition of miR-15a and miR-16 downregulated the expression of interleukin-1β and tumor-necrosis factor-α in the spinal cord of CCI rats. Bioinformatic analysis predicted that G protein-coupled receptor kinase 2 (GRK2), an important regulator in neuropathic pain and inflammation, was a potential target gene of miR-15a and miR-16. Inhibition of miR-15a and miR-16 markedly increased the expression of GRK2 while downregulating the activation of p38 mitogen-activated protein kinase and NF-κB in CCI rats. Notably, the silencing of GRK2 significantly reversed the inhibitory effects of miR-15a/16 inhibition in neuropathic pain. In conclusion, our results suggest that inhibition of miR-15a/16 expression alleviates neuropathic pain development by targeting GRK2. These findings provide novel insights into the molecular pathogenesis of neuropathic pain and suggest potential therapeutic targets for preventing neuropathic pain development.
Subject(s)
Animals , Mice , Rats , Computational Biology , Constriction , Down-Regulation , Hyperalgesia , Inflammation , Injections, Spinal , MicroRNAs , Neuralgia , p38 Mitogen-Activated Protein Kinases , Phosphotransferases , Protein Kinases , Sciatic Nerve , Spinal Cord , Up-RegulationABSTRACT
AIM:To observe the influence of high expression of miR-15a-5p on the proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells.METHODS: The miR-15a-5p oligonucleotide , which was recon-structed with additional restriction sites of EcoRⅠand HindⅢ, was chemically synthesized and confirmed by sequencing . The miR-15a-5p eukaryotic expression system was constructed by pcDNA 6.2-GW/Em-GFP-pre-miR-15a-5p plasmid.The miR-15a-5p was transfected into the SMMC-7721 cells transiently by plasmid , and quantified by quantitative real-time PCR at the mRNA level.The cell viability was measured by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion .The migration ability of the SMMC-7721 cells with high expression of miR-15a-5p was de-tected by wound healing test .RESULTS: The sequence of miR-15a-5p oligonucleotide 100% matched the designed se-quence.Compared with control group , the miR-15a-5p expression was increased significantly (P<0.05).The viability, the living cell number and the migration ability of the SMMC-7721 cells were decreased in high expression of miR-15a-5p group with statistically significant difference (P<0.05).CONCLUSION: The abilities of proliferation and migration in human hepatocellular carcinoma SMMC-7721 cells are decreased by high expression of miR-15a-5p.
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OBJECTIVE: To synthesize cationic polymers of arginine-histidine (HRss) based on disulfide crosslink and construct novel nano-complex HRss/miR-15-a, then study its anti-prostate cancer effect in vitro. METHODS: 1H-NMR was used to characterize HRss2/miR-15-a. Zeta sizer was adopted to estimate the Zeta potential and particle size of the nano-complex. Gel electrophoresis was employed to determine the condensation capacity of HRssto miR-15-a. The cytotoxicity and transfection efficiency of HRss was evaluated using prostate cancer stem-like cells (RC-92a/hTERT). Transwell chambers were used to evaluate the influence of HRss/miR-15-a against the motility of RC-92a/hTERT. RESULTS: The RESULTS of cytotoxicity tests indicated that the carrier HRss2 had low toxicity in both normal cells and cancer cells, and the miR-15-a could be loaded in HRss2 to form stable nano-complex. The transfection efficiency and inhibited motility of HRss2/miR-15-a against RC-92a/hTERT were statistically higher than those of HRss1/miR-15-a and HRss3/miR-15-a. CONCLUSION: HRss may be useful for gene delivery, and HRss2, as the optimum polycationic carrier as shown by in vitro evaluation, has the potential to become a novel gene vector in the therapy of prostate cancer.
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Objective Several studies have indicated that miR-15a,miR-15b and miR-16 may be the important regulators of apoptosis.Since attenuate apoptosis could protect myocardium and reduce infarction size,the present study was aimed to find out whether these miRNAs participate in regulating myocardial ischemia reperfusion (I/R) injury.Methods Apoptosis in mice hearts subjected to I/R was detected by TUNEL assay in vivo,while flow cytometry analysis followed by Annexin V/PI double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia/reoxygenation (H/R).Taqman real-time quantitative PCR was used to confirm whether miR-15a/15b/16 were involved in the regulation of cardiac I/R and H/R.Results Compared to those of the controls,I/R or H/R induced apoptosis of cardiomyocytes was significantly iucreased both in vivo (24.4％ ± 9.4％ vs.2.2％ ± 1.9％,P ＜ 0.01,n =5) and in vitro (14.12％ ±0.92％ vs.2.22％ ± 0.08％).The expression of miR-15a and miR-15b,but not miR-16,was increased in the mice I/R model,and the results were consistent in the H/R model.Conclusions Our data indicate miR-15 and miR-15b are up-regulated in response to cardiac I/R injury,therefore,down-regulation of miR- 15a/b may be a promising strategy to reduce myocardial apoptosis induced by cardiac I/R injury.
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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.
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Objective To construct an eukaryotic expression vector of pre-miR-15a,and to investigate the inhibitory effect of pre-miR-15a to Raji cells. Methods The pGCSIL-GFP vector encoding pre-miR-15a nucleotides was transfected into the bacterial competent cells,and then confirmed by PCR and sequencing analysis. The identified vector was transfected into Raji cells with oligofectamine 2000. The cells were divided into 3 groups,blank,negative control and pre-miR-15a group. Semi-quantitative RT-PCR was used to detect the expression of Bcl-2 mRNA,and immunofluorescence indirect for Bcl-2 protein expression. The growth of Raji cells was assayed by trypan blue dye exclusion method. Results PCR and sequences analysis indicated that the recombinant clones was identical with target sequences. Many green fluorescent cells were observed under fluorescent microscopy. The levels of Bcl-2 mRNA at every group had no obviously difference. Bcl-2 protein expression was obviously decreased at pre-miR-15a group compared with the other groups. Trypan blue dye exclusion method showed the cell growth was inhibited at 48 h and 72 h post-transfection. Conclusion We successfully construct the eukaryotic expression vector of pre-miR-15a,and it can inhibit the growth of Raji cells.