A host cell long noncoding RNA NR_033736 regulates type I interferon-mediated gene transcription and modulates intestinal epithelial anti-Cryptosporidium defense.

The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection.

A novel long intergenic non-coding RNA, Nostrill, regulates iNOS gene transcription and neurotoxicity in microglia.

BACKGROUND: Microglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity. METHODS: Microglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity. RESULTS: We report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity. CONCLUSIONS: Our data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.

Induction of a Long Noncoding RNA Transcript, NR_045064, Promotes Defense Gene Transcription and Facilitates Intestinal Epithelial Cell Responses against Cryptosporidium Infection.

Cryptosporidium is an important opportunistic intestinal pathogen for immunocompromised individuals and a common cause of diarrhea in young children in developing countries. Gastrointestinal epithelial cells play a central role in activating and orchestrating host immune responses against Cryptosporidium infection, but underlying molecular mechanisms are not fully understood. We report in this paper that C. parvum infection causes significant alterations in long noncoding RNA (lncRNA) expression profiles in murine intestinal epithelial cells. Transcription of a panel of lncRNA genes, including NR_045064, in infected cells is controlled by the NF-κB signaling. Functionally, inhibition of NR_045064 induction increases parasite burden in intestinal epithelial cells. Induction of NR_045064 enhances the transcription of selected defense genes in host cells following C. parvum infection. Epigenetic histone modifications are involved in NR_045064-mediated transcription of associated defense genes in infected host cells. Moreover, the p300/MLL-associated chromatin remodeling is involved in NR_045064-mediated transcription of associated defense genes in intestinal epithelial cells following C. parvum infection. Expression of NR_045064 and associated genes is also identified in intestinal epithelium in C57BL/6J mice following phosphorothioate oligodeoxynucleotide or LPS stimulation. Our data demonstrate that lncRNAs, such as NR_045064, play a role in regulating epithelial defense against microbial infection.

Induction of Inflammatory Responses in Splenocytes by Exosomes Released from Intestinal Epithelial Cells following Cryptosporidium parvum Infection.

Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. This parasite is referred to as a "minimally invasive" mucosal pathogen, and epithelial cells play a central role in activating and orchestrating host immune responses. We previously demonstrated that Cryptosporidium parvum infection stimulates host epithelial cells to release exosomes, and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. In this study, we detected the upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the exosomes released from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells by shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.

Attenuation of Intestinal Epithelial Cell Migration During Cryptosporidium parvum Infection Involves Parasite Cdg7_FLc_1030 RNA-Mediated Induction and Release of Dickkopf-1.

Intestinal infection by Cryptosporidium is known to cause epithelial cell migration disorder but the underlying mechanisms are unclear. Previous studies demonstrated that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using multiple models of intestinal cryptosporidiosis, we report here that C. parvum infection induces expression and release of the dickkopf protein 1 (Dkk1) from intestinal epithelial cells. Delivery of parasite Cdg7_FLc_1030 RNA to intestinal epithelial cells triggers transactivation of host Dkk1 gene during C. parvum infection. Release of Dkk1 is involved in C. parvum-induced inhibition of cell migration of epithelial cells, including noninfected bystander cells. Moreover, Dkk1-mediated suppression of host cell migration during C. parvum infection involves inhibition of Cdc42/Par6 signaling. Our data support the hypothesis that attenuation of intestinal epithelial cell migration during Cryptosporidium infection involves parasite Cdg7_FLc_1030 RNA-mediated induction and release of Dkk1 from infected cells.

A long noncoding RNA, lincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. We report here that the lincRNA gene lincRNA-Tnfaip3, located at mouse chromosome 10 proximal to the tumor necrosis factor α-induced protein 3 (Tnfaip3) gene, is an early-primary response gene controlled by nuclear factor-κB (NF-κB) signaling in murine macrophages. Functionally, lincRNA- Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA-Tnfaip3 is required for the transactivation of NF-κB-regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA-Tnfaip3 physically interacts with the high-mobility group box 1 (Hmgb1), assembling a NF-κB/Hmgb1/lincRNA-Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF-κB/Hmgb1/lincRNA-Tnfaip3 complex can modulate Hmgb1-associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-κB-induced lincRNA-Tnfaip3 to act as a coactivator of NF-κB for the transcription of inflammatory genes in innate immune cells through modulation of epigenetic chromatin remodeling.-Ma, S., Ming, Z., Gong, A.-Y., Wang, Y., Chen, X., Hu, G., Zhou, R., Shibata, A., Swanson, P. C., Chen, X.-M. A long noncoding RNA, LincRNA-Tnfaip3, acts as a coregulator of NF-κB to modulate inflammatory gene transcription in mouse macrophages.

Delivery of parasite Cdg7_Flc_0990 RNA transcript into intestinal epithelial cells during Cryptosporidium parvum infection suppresses host cell gene transcription through epigenetic mechanisms.

Cryptosporidial infection causes dysregulated transcription of host genes key to intestinal epithelial homeostasis, but the underlying mechanisms remain obscure. Previous studies demonstrate that several Cryptosporidium parvum (C. parvum) RNA transcripts are selectively delivered into epithelial cells during host cell invasion and may modulate gene transcription in infected cells. We report here that C. parvum infection suppresses the transcription of LRP5, SLC7A8, and IL33 genes in infected intestinal epithelium. Trans-suppression of these genes in infected host cells is associated with promoter enrichment of suppressive epigenetic markers (i.e., H3K9me3). Cdg7_FLc_0990, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected epithelial cells, is recruited to the promoter regions of LRP5, SLC7A8, and IL33 genes. Cdg7_FLc_0990 appears to be recruited to their promoter regions together with G9a, a histone methyltransferase for H3K9 methylation. The PR domain zinc finger protein 1, a G9a-interacting protein, is required for the assembly of Cdg7_FLc_0990 to the G9a complex and gene-specific enrichment of H3K9 methylation. Our data demonstrate that cryptosporidial infection induces epigenetic histone methylations in infected cells through nuclear transfer of parasite Cdg7_Flc_0990 RNA transcript, resulting in transcriptional suppression of the LRP5, SLC7A8, and IL33 genes.

LincRNA-Cox2 Promotes Late Inflammatory Gene Transcription in Macrophages through Modulating SWI/SNF-Mediated Chromatin Remodeling.

Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. One of the most highly induced lincRNAs in macrophages upon TLR ligation is lincRNA-Cox2, which was recently shown to mediate the activation and repression of distinct classes of immune genes in innate immune cells. We report that lincRNA-Cox2, located at chromosome 1 proximal to the PG-endoperoxide synthase 2 (Ptgs2/Cox2) gene, is an early-primary inflammatory gene controlled by NF-κB signaling in murine macrophages. Functionally, lincRNA-Cox2 is required for the transcription of NF-κB-regulated late-primary inflammatory response genes stimulated by bacterial LPS. Specifically, lincRNA-Cox2 is assembled into the switch/sucrose nonfermentable (SWI/SNF) complex in cells after LPS stimulation. This resulting lincRNA-Cox2/SWI/SNF complex can modulate the assembly of NF-κB subunits to the SWI/SNF complex, and ultimately, SWI/SNF-associated chromatin remodeling and transactivation of the late-primary inflammatory-response genes in macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role for NF-κB-induced lincRNA-Cox2 as a coactivator of NF-κB for the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling.

Trans-suppression of defense DEFB1 gene in intestinal epithelial cells following Cryptosporidium parvum infection is associated with host delivery of parasite Cdg7_FLc_1000 RNA.

To counteract host immunity, Cryptosporidium parvum has evolved multiple strategies to suppress host antimicrobial defense. One such strategy is to reduce the production of the antimicrobial peptide beta-defensin 1 (DEFB1) by host epithelial cells but the underlying mechanisms remain unclear. Recent studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the expression profile of host beta-defensin genes in host cells following infection. We found that C. parvum infection caused a significant downregulation of the DEFB1 gene. Interestingly, downregulation of DEFB1 gene was associated with host delivery of Cdg7_FLc_1000 RNA transcript, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected host cells. Knockdown of Cdg7_FLc_1000 in host cells could attenuate the trans-suppression of host DEFB1 gene and decreased the parasite burden. Therefore, our data suggest that trans-suppression of DEFB1 gene in intestinal epithelial cells following C. parvum infection involves host delivery of parasite Cdg7_FLc_1000 RNA, a process that may be relevant to the epithelial defense evasion by C. parvum at the early stage of infection.

Involvement of Cryptosporidium parvum Cdg7_FLc_1000 RNA in the Attenuation of Intestinal Epithelial Cell Migration via Trans-Suppression of Host Cell SMPD3.

Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3.

Delivery of Parasite RNA Transcripts Into Infected Epithelial Cells During Cryptosporidium Infection and Its Potential Impact on Host Gene Transcription.

Cryptosporidium parvum is an important opportunistic parasite pathogen for immunocompromised individuals and a common cause of diarrhea in young children. Previous studies have identified a panel of RNA transcripts of very low protein-coding potential in C. parvum. Using an in vitro model of human intestinal cryptosporidiosis, we report here that some of these C. parvum RNA transcripts were selectively delivered into the nuclei of host epithelial cells during C. parvum infection. Nuclear delivery of several such parasitic RNAs, including Cdg7_FLc_0990, involved heat-shock protein 70-mediated nuclear importing mechanism. Overexpression of Cdg7_FLc_0990 in intestinal epithelial cells resulted in significant changes in expression levels of specific genes, with significant overlapping with alterations in gene expression profile detected in host cells after C. parvum infection. Our data demonstrate that C. parvum transcripts of low protein-coding potential are selectively delivered into epithelial cells during infection and may modulate gene transcription in infected host cells.

LincRNA-Cox2 modulates TNF-α-induced transcription of Il12b gene in intestinal epithelial cells through regulation of Mi-2/NuRD-mediated epigenetic histone modifications.

Long intergenic noncoding RNAs (lincRNAs) can regulate the transcription of inflammatory genes and thus may represent a new group of inflammatory mediators with a potential pathogenic role in inflammatory diseases. Here, our genome-wide transcriptomic data show that TNF-α stimulation caused up-regulation of 171 lincRNAs and down-regulation of 196 lincRNAs in murine intestinal epithelial cells in culture. One of the up-regulated lincRNAs, lincRNA-Cox2, is an early-responsive lincRNA induced by TNF-α through activation of the NF-ĸB signaling pathway. Knockdown of lincRNA-Cox2 resulted in reprogramming of the gene expression profile in intestinal epithelial cells in response to TNF-α stimulation. Specifically, lincRNA-Cox2 silencing significantly (P < 0.05) enhanced the transcription of Il12b, a secondary late-responsive gene induced by TNF-α. Mechanistically, lincRNA-Cox2 promoted the recruitment of the Mi-2/nucleosome remodeling and deacetylase (Mi-2/NuRD) repressor complex to the Il12b promoter region. Recruitment of the Mi-2/NuRD complex was associated with decreased H3K27 acetylation and increased H3K27 dimethylation at the Il12b promoter region, which might contribute to Il12b trans-suppression by lincRNA-Cox2. Thus, our data demonstrate a novel mechanism of epigenetic modulation by lincRNA-Cox2 on Il12b transcription, supporting an important role for lincRNAs in the regulation of intestinal epithelial inflammatory responses.

Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

miR-27b targets KSRP to coordinate TLR4-mediated epithelial defense against Cryptosporidium parvum infection.

Cryptosporidium is a protozoan parasite that infects the gastrointestinal epithelium and causes a diarrheal disease. Toll-like receptor (TLR)- and NF-κB-mediated immune responses from epithelial cells, such as production of antimicrobial peptides and generation of reactive nitrogen species, are important components of the host's defense against cryptosporidial infection. Here we report data demonstrating a role for miR-27b in the regulation of TLR4/NF-κB-mediated epithelial anti-Cryptosporidium parvum responses. We found that C. parvum infection induced nitric oxide (NO) production in host epithelial cells in a TLR4/NF-κB-dependent manner, with the involvement of the stabilization of inducible NO synthase (iNOS) mRNA. C. parvum infection of epithelial cells activated NF-κB signaling to increase transcription of the miR-27b gene. Meanwhile, downregulation of KH-type splicing regulatory protein (KSRP) was detected in epithelial cells following C. parvum infection. Importantly, miR-27b targeted the 3'-untranslated region of KSRP, resulting in translational suppression. C. parvum infection decreased KSRP expression through upregulating miR-27b. Functional manipulation of KSRP or miR-27b caused reciprocal alterations in iNOS mRNA stability in infected cells. Forced expression of KSRP and inhibition of miR-27b resulted in an increased burden of C. parvum infection. Downregulation of KSRP through upregulating miR-27b was also detected in epithelial cells following LPS stimulation. These data suggest that miR-27b targets KSRP and modulates iNOS mRNA stability following C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.

Downregulation of PCAF by miR-181a/b provides feedback regulation to TNF-α-induced transcription of proinflammatory genes in liver epithelial cells.

Aberrant cellular responses to proinflammatory cytokines, such as TNF-α, are pathogenic features in most chronic inflammatory diseases. A variety of extracellular and intracellular feedback pathways has evolved to prevent an inappropriate cellular reaction to these proinflammatory cytokines. In this study, we report that TNF-α treatment of human and mouse cholangiocytes and hepatocytes downregulated expression of p300/CBP-associated factor (PCAF), a coactivator and an acetyltransferase that promotes histone acetylation and gene transcription. Of these upregulated microRNAs in TNF-α-treated cells, miR-181a/b (miR-181a and miR-181b) suppressed translation of PCAF mRNA. Functional manipulation of miR-181a/b caused reciprocal alterations in PCAF protein expression in cultured cholangiocytes and hepatocytes. Inhibition of miR-181a/b function with anti-miRs blocked TNF-α-induced suppression of PCAF expression. Promoter recruitment of PCAF was shown to be associated with TNF-α-induced transcription of inflammatory genes. Intriguingly, pretreatment of cells with TNF-α inhibited transcription of inflammatory genes in response to subsequent TNF-α stimulation. Overexpression of PCAF or inhibition of miR-181a/b function with anti-miRs attenuated the inhibitory effects of TNF-α pretreatment on epithelial inflammatory response to subsequent TNF-α stimulation. Downregulation of PCAF and the inhibitory effects of TNF-α pretreatment on liver epithelial inflammatory response were further confirmed in a mouse model of TNF-α i.p. injection. These data suggest that PCAF is a target for miR-181a/b, and downregulation of PCAF by TNF-α provides negative feedback regulation to inflammatory reactions in liver epithelial cells, a process that may be relevant to the epigenetic fine-tuning of epithelial inflammatory processes in general.

Cryptosporidium parvum hijacks a host's long noncoding RNA U90926 to evade intestinal epithelial cell-autonomous antiparasitic defense.

Cryptosporidium is a zoonotic apicomplexan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans. It is an important opportunistic pathogen in AIDS patients and a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. The intestinal epithelial cells provide the first line of defense against Cryptosporidium infection and play a central role in activating and regulating the host's antiparasitic response. Increasing evidence suggests that long noncoding RNAs (lncRNAs) participate in host-pathogen interactions and play a regulatory role in the pathogenesis of diseases but the underlying molecular mechanisms are not fully understood. We previously identified a panel of host lncRNAs that are upregulated in murine intestinal epithelial cells following Cryptosporidium infection, including U90926. We demonstrate here that U90926 is acting in a pro-parasitic manner in regulating intestinal epithelial cell-autonomous antiparasitic defense. Inhibition of U90926 resulted in a decreased infection burden of the parasite while overexpression of U90926 showed an increase in infection burden in cultured murine intestinal epithelial cells. Induction of U90926 suppressed transcription of epithelial defense genes involved in controlling Cryptosporidium infection through epigenetic mechanisms. Specifically, transcription of Aebp1, which encodes the Aebp1 protein, a potent modulator of inflammation and NF-κB signaling, was suppressed by U90926. Gain- or loss-of-function of Aebp1 in the host's epithelial cells caused reciprocal alterations in the infection burden of the parasite. Interestingly, Cryptosporidium carries the Cryptosporidium virus 1 (CSpV1), a double-stranded (ds) RNA virus coding two dsRNA fragments, CSpV1-dsRdRp and CSpV1-dsCA. Both CSpV1-dsRdRp and CSpV1-dsCA can be delivered into infected cells as previously reported. We found that cells transfected with in vitro transcribed CSpV1-dsCA or CSpV1-dsRdRp displayed an increased level of U90926, suggesting that CSpV1 is involved in the upregulation of U90926 during Cryptosporidium infection. Our study highlights a new strategy by Cryptosporidium to hijack a host lncRNA to suppress epithelial cell-autonomous antiparasitic defense and allow for a robust infection.

Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.

Cryptosporidium infects gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. There are no vaccines and no fully effective therapy available for the infection. Type II and III interferon (IFN) responses are important determinants of susceptibility to infection but the role for type I IFN response remains obscure. Cryptosporidium parvum virus 1 (CSpV1) is a double-stranded RNA (dsRNA) virus harbored by Cryptosporidium spp. Here we show that intestinal epithelial conditional Ifnar1-/- mice (deficient in type I IFN receptor) are resistant to C. parvum infection. CSpV1-dsRNAs are delivered into host cells and trigger type I IFN response in infected cells. Whereas C. parvum infection attenuates epithelial response to IFN-γ, loss of type I IFN signaling or inhibition of CSpV1-dsRNA delivery can restore IFN-γ-mediated protective response. Our findings demonstrate that type I IFN signaling in intestinal epithelial cells is detrimental to intestinal anti-C. parvum defense and Cryptosporidium uses CSpV1 to activate type I IFN signaling to evade epithelial antiparasitic response.

miR-141 modulates androgen receptor transcriptional activity in human prostate cancer cells through targeting the small heterodimer partner protein.

BACKGROUND: Aberrant expressions of microRNAs, including upregulation of miR-141, are closely associated with the tumorigenesis of prostate cancer (PCa). The orphan receptor small heterodimer partner (Shp) is a co-repressor to androgen receptor (AR) and represses AR-regulated transcriptional activity. METHODS: Here, we investigated the correlation of Shp expression with the cellular level of miR-141 and its effects on AR transcriptional activity in non-malignant and malignant human prostate epithelial cell lines. RESULTS: We found that Shp was downregulated in multiple PCa cell lines. The mature form of miR-141 was upregulated in PCa cells. miR-141 could target 3'-untranslated region of Shp mRNA resulting in translational suppression and RNA degradation. Moreover, enforced expression of Shp or inhibition of miR-141 function by anti-miR-141 attenuated AR-regulated transcriptional activity in AR-responsive LNCaP cells. Phenethyl isothiocyanate, a natural constituent of many edible cruciferous vegetables, increased Shp expression, downregulated miR-141, and inhibited AR transcriptional activity in LNCaP cells. CONCLUSIONS: Shp is a target for miR-141 and it is downregulated in cultured human PCa cells with the involvement of upregulation of miR-141, which promotes AR transcriptional activity. Moreover, Shp and miR-141 could be targets for chemoprevention for PCa.

miR-17-5p targets the p300/CBP-associated factor and modulates androgen receptor transcriptional activity in cultured prostate cancer cells.

BACKGROUND: Androgen receptor (AR) signalling is critical to the initiation and progression of prostate cancer (PCa). Transcriptional activity of AR involves chromatin recruitment of co-activators, including the p300/CBP-associated factor (PCAF). Distinct miRNA expression profiles have been identified in PCa cells during the development and progression of the disease. Whether miRNAs regulate PCAF expression in PCa cells to regulate AR transcriptional activity is still unclear. METHODS: Expression of PCAF was investigated in several PCa cell lines by qRT-PCR, Western blot, and immunocytochemistry. The effects of PCAF expression on AR-regulated transcriptional activity and cell growth in PCa cells were determined by chromatin immunoprecipitation, reporter gene construct analysis, and MTS assay. Targeting of PCAF by miR-17-5p was evaluated using the luciferase reporter assay. RESULTS: PCAF was upregulated in several PCa cell lines. Upregulation of PCAF promoted AR transcriptional activation and cell growth in cultured PCa cells. Expression of PCAF in PCa cells was associated with the downregulation of miR-17-5p. Targeting of the 3'-untranslated region of PCAF mRNA by miR-17-5p caused translational suppression and RNA degradation, and, consequently, modulation of AR transcriptional activity in PCa cells. CONCLUSIONS: PCAF is upregulated in cultured PCa cells, and upregulation of PCAF is associated with the downregulation of miR-17-5p. Targeting of PCAF by miR-17-5p modulates AR transcriptional activity and cell growth in cultured PCa cells.

Binding of NF-kappaB p65 subunit to the promoter elements is involved in LPS-induced transactivation of miRNA genes in human biliary epithelial cells.

The majority of human miRNA genes is transcribed by polymerase II and can be classified as class II genes similar to protein-coding genes. Whereas current research on miRNAs has focused on the physiological and pathological functions, the molecular mechanisms underlying their transcriptional regulation are largely unknown. We recently reported that lipopolysaccharide (LPS) alters mature miRNA expression profile in human biliary epithelial cells. In this study, we tested the role of transcription factor NF-kappaB in LPS-induced transcription of select miRNA genes. Of the majority of LPS-up-regulated mature miRNAs in cultured human biliary epithelial cells, potential NF-kappaB binding sites were identified in the putative promoter elements of their corresponding genes. Inhibition of NF-kappaB activation by SC-514, an IKK2 inhibitor, blocked LPS-induced up-regulation of a subset of pri-miRNAs, including pri-miR-17-92, pri-miR-125b-1, pri-miR-21, pri-miR-23b-27b-24-1, pri-miR-30b, pri-miR-130a and pri-miR-29a. Moreover, direct binding of NF-kappaB p65 subunit to the promoter elements of mir-17-92, mir-125b-1, mir-21, mir-23b-27b-24-1, mir-30b and mir-130a genes was identified by chromatin immunoprecipitation analysis and confirmed by the luciferase reporter assay. Thus, a subset of miRNA genes is regulated in human biliary epithelial cells through NF-kappaB activation induced by LPS, suggesting a role of the NF-kappaB pathway in the transcriptional regulation of miRNA genes.