| The mammalian genome transcribes numerous non-coding RNAs, most of whichhave not been functionally identified. The regulatory noncoding RNA mainly divides intotwo categories according to the length of RNA sequence, microRNA (miRNA) and longnoncoding RNA (lncRNA). miRNA exert functions mainly through targeting3’-untranslational region of mRNA to induce translation suppression or mRNAdegradation. It is well accepted that miRNA participate in many physiological andpathological process and the emerging roles of miRNA in immune development andresponse has been extensively explored in recent years. Innate immunity, mainly mediatedby innate immune cells, including monocytes/macrophages, dendritic cells (DC),granulocytes and NK cells, provides the first line of host defense against invadingpathogens. It has been proved that miRNA play an indispensable role in innate immuneresponses via regulating the development of monocytes/macrophages, DC, andgranulocytes and their responses to pathogens, TLR ligands, and viral infection. While NKcells are of vital importance in innate immune responses against viral infection,contributing to the first line of host antiviral defense，the function of intrinsic miRNA inNK cells remains elusive.After the application of second generation of sequencing, the study of lncRNAgradually attracted many attentions. The role of lncRNA has been explored in many fields,such as stem cell biology, biological development, X chromosome inactivation, neurologyand cancer biology. However, so far, in immunity system, few functional lncRNA has beendiscovered. Besides determining the function of lncRNA, the other major issue is to revealits molecular mechanism. Unlike the clear mechanism mode of microRNA through seedsequence base-pairing, lncRNA have diverse models, including through regulatingchromatin modification and transcription in nucleus and through affecting mRNA stabilityand translation in cytoplasm. Such diverse molecular mechanisms increase the difficulty of studying lncRNA. Moreover, whether there exist new other mechanism models requiresfurther elucidating.NK cells are a unique subset of innate immune cells, which exhibit cytotoxicityfunction like adoptive CTL, through a ligand-receptor-recognition manner. NK cellsmediate cytolysis through a variety of mechanisms, including perforin/granzymegranule-mediated exocytosis and activation of the TNF death receptor family members.Studies of gene-disrupted mice and individuals with inherited defect indicate that perforinand granzyme B are vital to the cytotoxicity function of NK cells, and they playindispensable roles in granzyme-mediated cellular apoptosis. During immune response, thecytotoxicity of NK cells can be enhanced by many activating cytokines, and type I IFN isone of the most potent activators of NK cells both in human and in mouse, while theunderlying mechanisms remain to be fully understood. Type I IFN was shown to stimulateaccessory DC to produce IL-15and then to activate NK cells, while type I IFNsignaling-induced granzyme B and perforin expression in murine NK cells was found to berequired for NK cell activation in response to vaccinia virus infection. It seems that type IIFN activates NK cells in both direct and indirect manners. It has been reported that type IIFN can modulate miRNA expression in macrophages, so, whether type I IFN couldregulate miRNA expression in NK cells and subsequently regulate NK cell activity needsto be explored.Dendritic cells (DC) are the most potent antigen-presenting cells in mammalianimmune system, whose maturation and functional state determines the outcome of immuneresponse. In normal immune response, DC can sense the invasion of pathogens, and thensecretes many cytokines, including IL-12and IL-10, to regulate other immunocytesactivity. At the same time, DC become mature and express more antigen presentingmolecules and co-stimulatory molecules, including HLAs, CD80, CD86and CD40, toactivate T cells and initiate adaptive immune response. Considering the important role ofDC between innate and adaptive immunology, lots of studies have been taken to reveal theunderlying regulatory mechanism of DC maturation and function. As to noncoding RNA,expression profile of miRNA and their functional roles in murine and human DC havebeen reported previously, while the expression and function of lncRNA in DC remains tobe elusive. Although many transcription factors, including STAT3and PU.1, have beenidentified to play important roles in the development and homeostasis of DC populations, DC, unlike different T cell subsets having specific transcription factors, have not beenidentified to have a specific molecule defining DC identity. Recently many works havedemonstrated that lncRNA could affect genome-wide transcription then to regulate cellulardifferentiation and function, such as somatic tissue differentiation, tumors malignanttransformation and metastasis. So, whether lncRNA play a role in DC differentiation andmaturation needs to be explored. We sought to identify a unique DC regulator at the levelof lncRNA.Our work divided into two parts.I, The role of miRNA in human NK cell activation and function and theunderlying mechanism.In order to investigate the roles of miRNA in human NK cell activation and function,we profiled small RNA components and established the miRNome of resting orIFN-α-activated human periphery blood NK cells by deep sequencing. Our bioinformaticsanalysis found that the majority of miRNA did not change much after activation, while afew miRNA decreased which probably contributed to the up-regulation of cytotoxicityduring NK activation by prediction. Among the twelve most abundant miRNA expressedin human NK cell, we found there are two miRNAs, miR-378and miR-30e, markedlydown-regulated after NK cell activation.As predicted by computational prediction via TargetScan (www.targetscan.org),human GZMB and PRF1mRNAs are potential targets of miR-378and miR-30e,respectively. To verify these predictions, we constructed luciferase reporter plasmid with3’UTR sequence of human GZMB or PRF1, or with their mutant3’UTR sequence.Overexpression and inhibition experiments using synthetic miRNA mimic or inhibitorrevealed that miR-378and miR-30e targeted the3’UTR of GZMB and PRF1preciously atthe indicated sites respectively.Next we investigated whether miR-378and miR-30e could suppress the expression ofintrinsic cytolytic molecules in human NK cells. Flow cytometry assay showed thatmiR-378mimic suppressed granzyme B expression and miR-30e mimic suppressedperforin expression in primary human NK cells, largely overcoming the effect of IFN-α ongranzyme B and perforin, respectively, indicating the decrease of miR-378and miR-30eplay a major role in IFN-α-induced cytolytic molecule expression.We wondered whether the enhancement of cytolytic molecule expression by decreasing miR-378and miR-30e was specific for type I IFN-mediated NK cell activationor a universal phenomenon for NK cell activation. We used cytokines IL-15, IL-12andIL-2to activate human NK cells, and then analyzed the expression of miRNAs andcytolytic molecules. We found that the expression of miR-378and miR-30e decreased tovarious degrees, meanwhile, flow cytometry assay and immune blot both confirmed theexpression of cytolytic molecules were enhanced by all these cytokines. Interestingly,IL-15stimulation enhanced granzyme B and perforin expression most markedly in NKcells, with most significantly decrease of miR-378and miR-30e. Also we freshly isolatedthe activated human NK cell subset (CD16+CD56dimCD3-CD69+) and resting NK cells(CD16+CD56dimCD3-CD69-) from the same donor sample to detect the expression level ofmiR-378and miR-30e. Indeed, the expression of these two miRNAs were lower in theactivated NK cell subset compared with that in resting NK cells from the same donor. Thisdata confirms that the negative regulatory relationship between miR378/miR-30e andcytolytic molecules also exist in physical process of human NK cell activation in vivo,together with other data further suggesting miR-378and miR-30e negatively regulategranzyme B and perforin expression in human NK cells in vivo.Finally, to confirm the roles of miR-378and miR-30e in the negative regulation ofNK cell cytotoxicity, we stably transfected human NK cell line NK92with miR-378ormiR-30e sponge plasmid, and confirmed the expressions of EGFP protein and spongemRNA. Obviously, increment of granzyme B in miR378sponge cell line and increment ofperforin in miR-30e sponge cell line were observed. Accordingly, the NK92cells stablytransfected with miR-378sponge plasmid or miR-30e sponge plasmid exhibited morepotent cytolytic activity compared with NK92cells stably transfected with control plasmid.Therefore, we demonstrated that miR-378and miR-30e significantly suppress cytotoxicityof human NK cells by targeting cytolytic molecules, granzyme B and perforin. This workprovides a new mechanistic explanation for the regulation of NK cell activation bymiRNAs and, as a regulator of cytotoxicity, miR-378and miR-30e may have potentialrelevance in clinic study.II, The role of long noncoding RNA in human dendritic cell maturation andfunction and the underlying mechanism.To identify the functional lncRNA in human DCs, we explored the expression profileof lncRNA during human DC differentiation from peripheral monocytes and DC maturation induced by lipopolysaccharide (LPS). Using microarray technique, weidentified76annotated lncRNAs with markedly altered expressions (fold>20) duringhuman DC differentiation and maturation. Among them, an intergenic lncRNA (lincRNA),named linc-DC, was most significantly increased in human mature DCs. Using antisenseRNA mediated knockdown (RNAi), we found that linc-DC depletion impaired theexpressions of functional molecules in mature DCs and attenuated IL12expression. Moreimportantly, linc-DC knockdown functionally impaired mature DCs to initiate allogeneicCD4+T cell proliferation and activation.By examining the expression profile of linc-DC in most of human immunocytes, wefound that linc-DC was only exclusively expressed in human DCs, indicating linc-DC ispossible a DC specific and DC crucial regulator. To explore mechanism of DC-specificexpression of linc-DC, we firstly identified the transcriptional starting site (TSS) andending site of linc-DC in human DC with rapid amplification of cloned cDNA ends(RACE) assay, and obtained its full-length transcript with417nucleotides verified to haveno coding potential. Next, using ChIP-qPCR assay，we found that histone modificationsH3K4me3and H3K27ac, which positively regulate transcription, were enhanced frommonocytes to mature DCs. Furthermore, chromatin accessibility gradually increased,indicating chromatin structure loosens or opens during the differentiation and maturationof DCs to facilitate linc-DC transcription. We believe that the acquired accessiblechromatin structure and high positive epigenetic histone modifications on linc-DC genelocus contribute to its specific expression in human mature DC. Also, we foundtranscription factor PU.1directs linc-DC expression through directly binding to thepromoter sequence, which adds new insight to PU.1function in DCs.We next investigated the underlying molecular mechanism of linc-DC promoting DCmaturation. RNA FISH and RT-PCR assay revealed linc-DC was a cytoplasmic RNA, andour RIP result and bioinformatics analysis found linc-DC did not function through reportedmechanisms. We performed RNA pull-down experiment using biotinylated linc-DCfollowed by gel separation and mass spectrometry identification. STAT3, a signaltransducer and transcription activator that mediates cellular responses to many importantcytokines in immunology and cancers, was identified to specifically interact with linc-DC,which is confirmed by independent immunoblot assay. The RIP assay further verified theirspecific interaction and confocal microscopy confirmed their physiological co-localization in human DC cytoplasm. Given that STAT3protein is not recognized as a RNA-bindingprotein, we carried out a modified RIP experiment known asPhotoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation(PAR-CLIP) to provide more conclusive evidence that STAT3directly associates withlinc-DC in the cytoplasm. Using different truncated version of linc-DC transcript, wefound3′-end segment of linc-DC (265~417nt) was sufficient to bind STAT3. Interestingly,RNA folding analyses of this region predicted a stable stem-loop structure, which mightprovide necessary spatial conformation for its interaction with STAT3. Comparingimmature and mature DCs, we found that the association of linc-DC with STAT3increasedduring DC maturation in both linc-DC RNA pull-down experiment and STAT3RIP assay.We then performed deletion-mapping experiments and showed that C-terminus ofSTAT3(583~770residues) of STAT3was sufficient to bind linc-DC. Since this portioncontains Tyr705(Y705) whose phosphorylation is crucial for STAT3activation (23), weask whether the binding of linc-DC affects STAT3phosphorylation status and thus STAT3signaling. Immunoblot results indeed showed that Y705phosphorylation of STAT3wassignificantly reduced after linc-DC knockdown in human DCs upon LPS treatment,together with decreased STAT3nuclear translocation. Consistently, overexpression oflinc-DC promoted STAT3phosphorylation in PMA-differentiated human THP1cells andSTAT3luciferase reporter activity in HEK293T cells. Additionally, we revealed thatlinc-DC knockdown in DCs did not affect the phosphorylation status of Jak1, Jak2, Tyk2,STAT1and STAT5, as well as MAPK, NF-κB and Akt/GSK-3signaling.We hypothesized that the interaction between linc-DC and STAT3affects theinteraction of STAT3with other protein kinases and phosphatases. So we examined theprotein interactome of STAT3via STAT3immunoprecipitation with or without linc-DCwhich let us to focus on tyrosine phosphatase SHP1. The immunoblot confirmed thatknockdown of linc-DC promoted the association of SHP1with STAT3in mature DCs andin immature DCs while overexpression of linc-DC attenuated their interaction.Furthermore, in vitro, phosphatase assay showed that linc-DC RNA protected STAT3fromdephosphorylation by recombinant human protein SHP1.Finally, to prove STAT3signaling is essential to human DC maturation, we used twoSTAT3pharmacological inhibitors, S3I-201and Stattic. Similar to the linc-DC knockdown,STAT3inhibitions exhibited impaired human DC maturation and function. So our data evidenced that linc-DC promotes human DC maturation through enhancing STAT3signaling.In this study, we identified a DC-specific lncRNA (linc-DC) that is indispensable forhuman DC maturation and function, together with the active histone marks and accessiblechromatin structure around its gene locus, contribute to defining human DC identity.Mechanistically, through binding to the C-terminus of STAT3with its stem-loop structureat3′end, linc-DC prevents SHP1-mediated dephosphorylation of STAT3on the criticalY705residue and thus strengthens STAT3signaling. Our work leads to a new mechanisticmodel that an lncRNA can affect cellular function through directly binding to a signalingmolecule in the cytoplasm and regulating posttranslational modification of the molecule.Whether other cytoplasmic lncRNA perform their functions in a similar manner as linc-DCand how many other signaling pathways can be regulated by lncRNA molecules need to befurther investigated. Furthermore, the identification of an endogenous functionalnon-coding RNA, linc-DC, as a master regulator of DC function and a regulator of STAT3signaling, may have potential relevance to clinical diseases involving DC dysfunction orabnormal STAT3activation, and may aid the design of DC vaccines with more potency toactivate T cell response in the immunotherapy of cancer and infectious diseases.Taking together, our work investigated the role of noncoding RNA in the functionalregulation of immunocytes and their underlying mechanism, established a techniquesystem for RNA study and laid the foundation for future research. We indentified twomiRNA and one lncRNA to be a regulator of human NK cell and DC function, respectively,which may have potential clinical implication. Meanwhile, we proposed a novel workingmodel for lncRNA function and gave a new light to cytoplasmic signaling regulation,through evidencing RNA directly regulating cytoplasmic signaling. |
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