| Cryptococcus neoformans is an environmental pathogen that can infect the lungs,skin,bones and other organs of the whole body in humans and many mammals.However,the invasion of the central nervous system is the most common(namely cryptococcus meningitis).It is estimated that in 2014 alone,there were 223,100 new cases of cryptococcus meningitis,among which 73% occurred in Africa [1].Different from the cryptococcal meningitis mainly occurring in the AIDS population in Europe,America,Africa and other regions,China's cryptococcal meningitis mainly occurs in the non-aids population without immunodeficiency,but the treatment is longer and more difficult than the immunocompromised patients [2].If patients with cryptococcal meningitis are not treated in time,86% of them will die within one year.Even with modern treatment,the mortality rate is still about 20%-30% [3].Therefore,how to effectively prevent and treat the central nervous system infection of cryptococcus neoformans is a problem that we urgently need to solve.THP-1 cells(mononuclear macrophage)is an important component of the innate immune system,under the stimulus external factors can be activated for classic activated macrophages(M1)and alternative activated macrophages(M2): M1 cells secreted tumor necrosis factor-?(TNF-?),interleukin-6(IL-6)and interleukins-1?(IL-1?),these inflammatory factors have very strong sterilization effect;M2-type macrophages secrete inflammatory factors such as transforming growth factor-?(TGF-?),arginase 1(Arg1)and interleukin-10(IL-10),which can inhibit the inflammatory response [4].In patients with cryptococcus neoformans meningitis,M1-type cells contribute to the clearance of pathogens,while M2-type cells mediate immune escape[5],so the promotion of THP-1 cell polarization toward M1-type cells contributes to the treatment of cryptococcus meningitis.Micro RNA(miRNA)is a class of small non-coding RNA molecules with a length of about 20 ~ 24 nt,which mainly regulates the expression of target genes at the posttranscriptional level and participates in a variety of life activities such as cell differentiation,signal transduction,immune response and tumorigenesis.Mi R-155-5p is located in the third exon of the b-cell Integration Cluster(bic)gene on human chromosome 21,and can perform its function by targeting and degrading the target gene m RNA.Some studies have found that it is highly expressed in human lung cancer,thyroid cancer,cervical cancer,pancreatic cancer and other solid tumors,and is related to the proliferation and differentiation of tumor cells [6].Other studies have shown that miR-155-5p is involved in the development and differentiation of immune cells,and is significantly expressed in activated macrophages,T lymphocytes,B lymphocytes and dendritic cells,and plays an important role in the immune response [7].IKBKE,a gene encoding serine/threonine protein kinase,is a newly discovered member of the I?B kinase family(IKKs)and is involved in tumor development and the activation,proliferation,differentiation and apoptosis of immune cells [8].Studies have found that it plays an important role in the innate immune system by regulating the nuclear transcription factor-?B(NF-?B)signal transduction pathway [9].Through previous mirnada website prediction,we found that miR-155-5p and IKBKE genes have binding sites,and both miR-155-5p and IKBKE genes are involved in cellular inflammatory response,so we speculated that miR-155-5p plays a role in THP-1 cell anti-cryptococcus neoformans immune response by targeting degradation of IKBKE m RNA.PART I Changes of mir155-5p,IKBKE,TNF-?,IL-1?and IL-6 expression in thp-1 cells induced by cryptococcus neoformansReplacement cell SAP,adjust good state of THP 1 cells growth,join PMA will cells differentiation into macrophages,with inactivated cryptococcus in accordance with the number of 5:1 in cell culture bottles,intervention respectively 0 h,3 h,6 h,9 h,12 h,each point in time and on cell,save to-80 ? refrigerator.The supernatant was thawed and the contents of TNF-?,IL-1? and IL-6 in the supernatant were determined by Elisa.Cell RNA was extracted by Trizol method,and the expression multiples of miR-155-5p and IKBKE were measured by real-time fluorescence quantitative polymerase chain reaction(q PCR).In our study,we found that under the intervention of cryptococcus neoformans,the expression of miR-155-5p in THP-1 cells increased,which reached the peak at 6h,the content of IKBKE decreased,and there were statistical differences at 3h,6h and 9h.The expression levels of TNF-?,IL-1?and IL-6 increased significantly with time.PART? The dual luciferase reporting system verified the targeting relationship between miR-155-5p and IKBKEAccording to the predicted results of mirnada website,miR-155-5p can target to degrade IKBKE m RNA.In order to further verify the targeting relationship,we conducted a double-luciferase reporting experiment: The experiment was divided into 6 groups,respectively: sea renin luciferase(p RL)plasmid +mimics NC,p RL plasmid + mir-155-5p,p RL plasmid +IKBKE 3' UTR+mimics NC,p RL plasmid +IKBKE 3' UTR+ mir-155-5p,p RL plasmid +IKBKE 3' UTR+mimics NC,p RL plasmid +IKBKE 3' UTR+mimics NC,p RL plasmid +IKBKE 3' UTR+mimics NC.The well-grown cells were cultured on a 24-well culture plate,and the plasmid transfection experiment was conducted according to the groups designed in the experiment.24 hours after the transfection fluorescence microscope fluorescent marker gene expression in cells,and then use the "Dual-Luciferase ? Reporter Assay System(promega E1910)" kit handle cells,and detection of Luciferase expression.Our study found that the fluorescence value of the experimental group was 30% lower than that of the control group,indicating that miR-155-5p could target the degradation of IKBKE m RNA 3'utr,and the activity of IKBKE 3'utr mutation was 15% higher than that of the wild type,indicating that the binding site was more important for the function of miR-155-5p and IKBKE.PART? Verify the regulatory effect of mir-155-5p on the expressions of IKBKE, TNF-?,IL-1? and IL-6The cell fluid was changed to adjust the growth state of THP-1 cells,and PMA was added to induce THP-1 cells to differentiate into macrophages.The experiment was divided into four groups: the control group at 0h,the control group at 6h,the mimics group at 0h and the mimics group at 6h.Lipofectamine 2000 kits were used and operated according to the operation steps of the kits.Heat inactivated cryptococcus neoformans was added to each group at a ratio of 5:1,at the same time,the experimental group was transfected with miR-155-5p mimics and the control group with miR-155-5p mimics control.The transfection efficiency was detected.The RNA and supernatant were extracted for q PCR and Elisa experiments,and the expressions of IKBKE,TNF-?,IL-1? and IL-6 were detected.Our study found that the expression of miR-155-5p was increased in both the experimental group and the control group at 6h,and the increase was more obvious in the experimental group,with statistical difference,indicating successful transfection.The expression of IKIBKE decreased at 6h,especially in the experimental group,with statistical difference,indicating that miR-155-5p could target the degradation of IKBKE m RNA.The expressions of TNF-?,IL-1? and IL-6 were increased in each group at 6h,and the increase was more obvious in the experimental group,with statistical difference,indicating that the up-regulated expression of miR-155-5p could promote the expression of TNF-?,IL-1? and IL-6.PART ? Changes in TNF-?,IL-1? and IL-6 expression after transfection with IKBKE si RNA into THP-1 cellsThe cell fluid was changed to adjust the growth state of the cells.PMA was added to induce thp-1 cells to differentiate into macrophages.The IKBKE si RNA was diluted with the culture medium without antibiotics or serum.Heat inactivated cryptococcus neoformans was added to each group at a ratio of 5:1,at the same time,the experimental group was transfected with IKBKE si RNA.The control group was transfected with IKBKE si RNA,and the control group was transfected with its control material.The RNA and supernatant were extracted for q PCR and Elisa experiments,and the expressions of IKBKE,TNF-?,IL-1? and IL-6 were detected.In our study,we found that the expression of IKBKE in each group decreased at 6h,and the decrease was more obvious in the experimental group,and it had statistically significant difference,indicating that the transfection of IKBKE si RNA was successful.The expression of TNF-?,IL-1? and IL-6 increased at 6h,and the increase was more obvious in the experimental group,the difference was statistically significant,indicating that the reduced expression of IKBKE can promote the expression of TNF-?,IL-1? and IL-6.Based on the above studies,we found that miR-155-5p has binding sites with IKBKE and can target the degradation of IKBKE m RNA to promote the expression of TNF-?,IL-1? and IL-6.According to the existing research results show that M1 macrophages secrete inflammatory factor such as TNF-?,IL-1? and IL-6,the cells as the proinflammatory type of macrophage,have the characteristics of potent bactericidal and promote helper T cells involved in the immune response of type 1(Th1 immune response),it plays an anti-infection and bactericidal role in the course of cryptococcal meningitis [5].Therefore,miR-155-5p can be used as a potential therapeutic target for cryptococcal meningitis and plays an important role in clinical practice. |
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