The Study On The Post-transcriptional Regulation Mechanism Of Heat Shock Induced NFKBIA Gene Expression

Backgrand: In the year of 1962, Ferruccio Ritossa observed that when the Drosophila expose to 37℃,its salivary gland can sythesis a kind of protein. With research ,this kind of protein have a wide spread of function from protecting cells to maintaining the stability of internal environment,so those proteins were named heat shock protein(HSP) and those genes expression and physical protective phenomenon were called heat shock reaction(HSR).Heat shock response, the most primitive and conservative reaction of all the stress response,can induce the synthesis of HSP,as a "molecplar chaperone" to maintain theproper folded state of proteins, protect cells against stress injury.including heat shock, hypoxia, ischemia-reperfusion injury, endotoxin, heavy metals,ethanol,Enhance the tolerance to damage of the cell, Maintain the normal function of cells and them etabolism, Improve the survival rate of the cells,with a strong protective effect.In the stress response, HSP gene expression firstly, the expression of other genes has been temporarily suppressed. In the high temperature, sodiumarsenite,prostaglandin-A1 induced HSR, the expression of HSP can inhibit that of the proinflammatory cytokines.The mainroleis to prevent excessive inflammatory response by inhibiting the activation of NF-κB in cell damage. The possible ways for the inhibition of NF-κB By HSP include: inhibition of IκBα phosphorylation and IKK activity, Enhance the mRNA expression of IκBα; Competitively inhibit NF-κB coming through nuclea pore complex into the nucleus.IκBα promoter contain heatshock element (Heat shock element, HSE), HSP gene transcription along with the NFKBIA gene, in recent years, HSR induce the NFKBIA gene expression at the post transcriptional level. In this study we want to find the mechanism of the NFKBIA gene expression induced by the HSR at the post-transcriptional level. NF-κB named after the discovery of IgK light chain transcriptional regulation in B cells In 1986, is a efficient eukaryotic nuclear transcription factor transmitted the signal from the cytoplasm to the nucleus and caused a corresponding gene expression, has an important regulatory function on the expression of many immuno modulatory genes the most important thing is to regulate gene transcription of cytokines, adhesion molecules and other immune media. The main activation of NF-κB is to increase the expression of proinflammatory mediators leading to acute inflammation.Proinflammatory cytokines such as TNF-a can activate NF-κB, Therefore, in the process of inflammatory response,it is difficult to distinguish a causal relationship between the two and interactions between them lead to the expansion of the inflammatory response.NF-κB is a group of transcription factor composed by a member of the Rel protein family in the form of homo- or hetero- dimers. In general sense NF-κB often refers to the p50/p65 dimers. In the resting state, κB inhibition factor (IκB) bind to NF-κB in the cytoplasm resulting the NF-κB inactive. Among the κB family,IκBα is the first founded and researched,weight 37 kD. When cells encounter stimulation of TNF-β,IL-18, endotoxin,NF-κB inducing kinase (NIK) activated the IκBs kinase complexes 1, 2 (IKK-1, 2), IKK-1, phosphorylate IκBα on the serine 32, 36 sites then ubiquitination, NF-κB free to the nucleus and then degraded by the proteasome, in the Nuclear, NF-κB bind to κB sequence in the gene, activate the transcription of downstream target genes. Meanwhile, the IκBα gene contains three NF-κB binding sites inits transcriptional promoter region,Thus,the expression of NF-κB induced IκBα activation constitute a negative feedback pathway of NF-κB activation.Organisms is a whole unit with organic functions, Various genes express procedurally and regularly according to body growth, development, the need for reproduction with the changes in the environment to play their normal physiological functions. The expression regulations of gene are at different levels: gene activation,transcription, post-transcriptional, translation, post translational. The transcription initiation of a gene is the main regulation form, but other forms can play a role through the way from DNA to protein to regulate the amount of gene products, to ensure the accuracy and precision regulation of the gene expression in vivo.The regulation of gene expression is a complex and orderly process including more than one level. A growing number of studies have shown that gene expression at post-transcriptional level plays an essential role mainly through influencing mRNA stability and translation. Post-transcriptional regulation of gene expression generally refers to the RNA polymerase binding to gene promoter and modifies a series of transcripts, including: early termination of transcription, splicing, mRNA going through the nuclear pore and the cytoplasm localization, RNA editing, translation initiation and translation efficiency, mRNA stability and many other aspects.The study found that components regulated the mRNA stability of the transcripts present in the 3’UTR, most of the post-transcriptional regulatory target site is located at the 3’UTR of the mRNA, it has been found that about 5-8% mRNA 3’UTR encoding by the human gene containing the ARE. ARE about 50-150nt rich in adenine and uridine has an close relation to mRNA stability.ARE can recruit many ARE-binding protein (ARE-BPs) which collaboratively or competitively binding to mRNA ARE domain to regulate mRNA stability and translation. NFKBIA mRNA contain ARE, our study focuses on the role of NFKBIA mRNA 3’UTR with heat shock treatment, using streptavidin-biotin RNA-pull down get the protein complexes binding to NFKBIA mRNA 3’UTR with heat shock treatment, separated by SDS-PAGE gel, silver staining, protein mass spectrum,identified hnRNP Q the interacting proteins of NFKBIA 3’UTR. hnRNP Q is a member of the heterogeneous nuclear ribonucleoprotein family (heterogeneoμs nuclear ribonucleo-protein, hnRNP), Mainly localized in the nucleus, can binding to the newly synthesized RNA (heterogeneous nuclear RNA, hnRNA) to regulate the mRNA precursors (pre-mRNA) splicing, mRNA nucleocytoplasmic transport,translation, and degradation, is an important RNA-binding protein. We want to explore the mechanisms hnRNP involved in the the functional regulation of NFKBIA mRNA 3’UTR upon heat shock treatment.Objective: research on the post-transcriptional regulation mechanism of the heat shock induced NFKBIA gene expression; focus on the regulation function of NFKBIA mRNA 3’UTR binding protein hnRNP Q.Content:1. IκBα protein expression and the changes of half life upon heat shock treatment.2. NFKBIA mRNA expression and he changes of half life upon heat shock treatment.3. Construct NFKBIA mRNA 3’UTR dual luciferase reporter plasmid, using the dual luciferase reporter gene system to study the role of NFKBIA mRNA 3’UTR upon heat shock treatment.4. To select and verify the binding protein of NFKBIA mRNA 3’UTR.5. hnRNP Q involved in the function regulation of NFKBIA mRNA 3’UTR upon heat shock treatment.6 . The influence of hnRNP Q on the expression of NFKBIA.7. The colocalization of NFKBIA mRNA 3’UTR with hnRNPQ, P body and S body upon heat shock treatment.Method: Fluorescent quantitative PCR to test the relative mRNA expression, WB to test the protein expression, Immunofluorescence to detect the protein localization, the dual luciferase reporter system indirectly reflect the function of NFKBIA mRNA 3’UTR. Firstly to test the homogeneity of variance with Levene’s, in this paper, the sample variance is homogeneity,then use One-way ANOVA,if there are differences between the overall group,Further to do the multiple comparisons, the test standards is a=0.05. The mapping software is Origin Pro 8.0.Results: The half-life of IxBa protein in the normal control group of RAW cell is about three h and extended to approximately 4 h upon heat shock treatment, The half-life of NFKBIA mRNA in the norrmal control group of RAW cell is about 40 rmin and extended to approximately 85 min upon heat shock treatment. Meanwhile detected NFKBIA mRNA and protein expression at different recovery time(return to normal training) points after heat shock,compared with control group,there is no difference of NFKBIA mRNA expression before 240 min, NFKBIA protein expression showing an decreased and then increased with a further reduction of the trend. It is not difficult to find throng above results that upon heat shock treatment for 1 h on the Raw cell, NFKBIA mRNA and protein expression levels showed inconsistent changes, indicating the exsistense of post-transcriptional regulatory mechanism. Construct the NFKBIA3’UTR after the luciferase reporter gene (referred pLuNB), detecting the luciferase reporter gene to indirectly reflect the function of NFKBIA mRNA 3’UTR with heat shock treatment. Transfected 3T3 cells 24 h to give treated with heat shock (43℃ for 1 h) after transfecting NIH 3T3 for 24 h to test the relative expression of luciferase (RLU). Compared with control group, the RLU were increased at all time points, we can conclude that the NFKBIA mRNA 3’UTR involved in the heat post-transcriptional regulation.With the overexpression of hnRNP Q1 for 48 h treated with heat shock 43 ℃ 1 h, return to a normal culture conditions, scratched the cell and test the RLU at different recovery time 0, 1, 2, 3 h after heat shock, compared with the control group,the RLU were lower. After transfect siRNA specifically targeted for hnRNP Q for 48 hours,interference efficiency is a about 47%,harvest cell at different recovery time 0,1, 2, 3 h after heat shock treatment and test RLU, the results are higher than that of control group at each time point. we can conclude that hnRNP Q can bind to NFKBIA mRNA 3’TUTR and participate in the post-transcriptinal regulatory function of the NFKBIA 3’UTR upon heat shock treatment. In other words,we can see that hnRNP Q exert a very important function for NFKBIA 3 ’UTR upon heat shock treatment.Overexpress hnRNP Q1 and hnRNP Q2 in NIH 3T3 and then treated the cells with heat shock to test the expression of NFKBIA mRNA and IκBα, we find that the expression of NFKBIA mRNA has little change before and after heat shock treatment compared with empty plasmid vector group, but the expression of IκBα was decreased.To further explore the role of hnRNP Q in the post-transcriptional regulation of the NFKBIA3’UTR upon heat shock treatmen, we introduced the MS2 vector system and construct pMS2-cherry expression vector express the protein specific binding to the stem-loop structure,when the NFKBIA mRNA 3’UTR plasmid with eight stem-loop structure express, they bind to each other to reflect the NFKBIA mRNA 3’UTR location in the cell.The hnRNP Q protein is dispersed in the cytoplasm and the nucleus untreated,significantly co-localized with NFKBIA 3’UTR mRNA, may be hnRNP Q involved in NFKBIA 3’UTR mRNA export from the nuclear, mature and localization, with heat shock treatment and recovery for 0-2 h, hnRNP Q forms granule in the cytoplasm and exist co-localization with NFKBIA 3’UTR mRNA, 4 h disappered.Experiment group of the NFKBIA 3’UTR mRNA co-localization with the protein hDcP1a the main component of P-body without stimulating,hDcP1 granule increased significantly when recover to normal culture condition and exist co-localization with NFKBIA 3’UTR mRNA and decreased gradually until recovery to 2 h. G3Bp1 protein is dispersed in the cytoplasm without co-localization with NFKBIA mRNA 3’UTR untreated, upon heat shock treatment G3Bpl forms significant granule in the cytoplasm and exist co-localization with NFKBIA mRNA 3’UTR and decreased when recovery for 4 h.