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Abstract Recent investigations of neurological developmental disorders have revealed the Rho-family modulators such as Syde and its interactors as the candidate genes. Although the mammalian Syde proteins are reported to possess GTPase-accelerating activity for RhoA-family proteins, diverse species-specific substrate selectivities and binding partners have been described, presumably based on their evolutionary variance in the molecular organization. A comprehensive in silico analysis of Syde family proteins was performed to elucidate their molecular functions and neurodevelopmental networks. Predicted structural modeling of the RhoGAP domain may account for the molecular constraints to substrate specificity among Rho-family proteins. Deducing conserved binding motifs can extend the Syde interaction network and highlight diverse but Syde isoform-specific signaling pathways in neuronal homeostasis, differentiation, and synaptic plasticity from novel aspects of post-translational modification and proteolysis.

CD47 deficient mice are resistant to dextran sulfate sodium (DSS)-induced experimental colitis. The underlying mechanism, however, remains incompletely understood. In this study, we characterized the role of CD47 in modulating homeostasis of gastrointestinal tract. We found that CD47 expression in both human and mouse intestinal epithelium was upregulated in colitic condition compared to that under normal condition. In line with this, CD47 deficiency protected mice from DSS-induced colitis. Analysis based on both intestinal organoid and cultured cell assays showed that CD47 deficiency accelerated intestinal epithelial cell proliferation and migration. Mechanistically, western blot and functional assays indicated that CD47 deficiency promoting mouse intestinal epithelial cell proliferation and migration follow cell injury is likely through upregulating expression of four Yamanaka transcriptional factors Oct4, Sox2, Klf4 and c-Myc (OSKM in abbreviation). Our studies thus reveal CD47 as a negative regulator in intestinal epithelial cell renewal during colitis through downregulating OSKM transcriptional factors.

Shift metabolism profile from mitochondrial oxidative phosphorylation to aerobic glycolysis (Warburg effect) is a key for tumor cell growth and metastasis. Therefore, suppressing the tumor aerobic glycolysis shows a great promise in anti-tumor therapy. In the present study, we study the role of shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, in inhibiting tumor aerobic glycolysis and thus tumor growth. We found that shikonin dose-dependently inhibited glucose uptake and lactate production in Lewis lung carcinoma (LLC) and B16 melanoma cells, confirming the inhibitory effect of shikonin on tumor aerobic glycolysis. Treatment of shikonin also decreased tumor cell ATP production. Furthermore, pyruvate kinase M2 (PKM2) inhibitor or activator respectively altered the effect of shikonin on tumor cell aerobic glycolysis, suggesting that suppression of cell aerobic glycolysis by shikonin is through decreasing PKM2 activity. Western blot analysis confirmed that shikonin treatment reduced tumor cell PKM2 phosphorylation though did not reduce total cellular PKM2 level. In vitro assay also showed that shikonin treatment significantly promoted tumor cell apoptosis compared to untreated control cells. Finally, when mice implanted with B16 cells were administered with shikonin or control vehicle, only shikonin treatment significantly decreased B16 tumor cell growth. In conclusion, this study demonstrates that shikonin inhibits tumor growth in mice by suppressing PKM2-mediated aerobic glycolysis.

RNA-induced silencing complex (RISC) plays a critical role in small interfering RNA (siRNA) and microRNAs (miRNA) pathways. Accumulating evidence has demonstrated that the major RISC members (AGO, DICER, TRBP, PACT and GW182) represent expression discrepancies or multiple orthologues/paralogues in different species. To elucidate their evolutionary characteristics, an integrated evolutionary analysis was performed. Here, animal and plant AGOs were divided into three classes (multifunctional AGOs, siRNA-associated AGOs and piRNA-associated AGOs for animal AGOs and multifunctional AGOs, siRNA-associated AGOs and complementary functioning AGOs for plant AGOs). Animal and plant DICERs were grouped into one class (multifunctional DICERs) and two classes (multifunctional DICERs and siRNA-associated DICERs), respectively. Protista/fungi AGOs or DICERs were specifically associated with the siRNA pathway. Additionally, TRBP/PACT/GW182 were identified only in animals, and all of them functioned in the miRNA pathway. Mammalian AGOs, animal DICERs and chordate TRBP/PACT were found to be monophyletic. A large number of gene duplications were identified in AGO and DICER groups. Taken together, we provide a comprehensive evolutionary analysis, describe a phylogenetic tree-based classification of the major RISC members and quantify their gene duplication events. These findings are potentially useful for classifying RISCs, optimizing species-specific RISCs and developing research model organisms.

Salmonella can hijack host atypical miRNA processing machinery to cleave its small non-coding RNA into a ~22-nt RNA fragment, Sal-1, which facilitates Salmonella survival in the infected host. The mechanism through which Sal-1 promotes Salmonella survival, however, remains unknown. In the present study, we reported that Sal-1 targets cellular inducible nitric oxide synthase (iNOS) in a miRNA manner, leading to attenuation of host cell iNOS/NO-mediated anti-microbial capacity. First, depletion of Sal-1 in Salmonella-infected epithelial cells significantly increased the iNOS level but not the levels of various inflammatory cytokines. Bioinformatics analysis and mutagenesis strategies were consistent with the identification of mRNA of iNOS as a target of Sal-1 in both human and mice. Second, western blot and immunohistochemical analysis confirmed that Sal-1 suppressed iNOS expression in vitro and in vivo, thus reducing the production of NO. Finally, Sal-1 facilitating Salmonella survival through suppressing iNOS induction was confirmed in mouse model by expressing mutated iNOS that is not targeted by Sal-1 in mice colon. In conclusion, our study provides new insight into the pathogenic mechanism of intracellular bacteria to modulate host innate immune response.

The Warburg effect is a metabolic hallmark of cancer. Tumor cells rapidly adjust their energy source to glycolysis in order to efficiently proliferate in a hypoxic environment, but the mechanism underlying this switch remains incompletely understood. Here, we show that hypoxia potently induces the down-regulation of miR-125a expression in hepatocellular carcinoma (HCC) cells and tumors. Furthermore, we demonstrate that miR-125a could decrease the production of lactate, the uptake of glucose, and the levels of ATP and reactive oxygen species (ROS) in HCC cells. We investigated the molecular mechanism through which miR-125a inhibits HCC glycolysis and identified hexokinase II (HK2) as a direct target gene of miR-125a. Finally, we revealed that the miR-125a/HK2 axis is functionally important for regulating glycolysis of HCC cell and progression of cancer in vitro and in vivo. In summary, our findings demonstrate for the first time that hypoxia-down-regulated miR-125a regulated HCC glycolysis and carcinogenesis by targeting hexokinase HK2, a key glycolytic enzyme for the Warburg effect, and add a new dimension to hypoxia-mediated regulation of cancer metabolism.

Salmonella have developed a sophisticated machinery to evade immune clearance and promote survival in the infected cells. Previous studies were mostly focused on either bacteria itself or host cells, the interaction mechanism of host-pathogen awaits further exploration. In the present study, we show that Salmonella can exploit mammalian cell non-classical microRNA processing machinery to further process bacterial small non-coding RNAs into microRNA-like fragments. Sal-1, one such fragment with the highest copy number in the infected cells, is derived from Salmonella 5'-leader of the ribosomal RNA transcript and has a 'stem' structure-containing precursor. Processing of Sal-1 precursors to mature Sal-1 is dependent on host cell Argonaute 2 (AGO2) but not Dicer. Functionally, depleting cellular Sal-1 strongly renders the Salmonella bacteria less resistant to the host defenses both in vitro and in vivo. In conclusion, we demonstrate a novel strategy for Salmonella evading the host immune clearance, in which Salmonella produce microRNA-like functional RNA fragments to establish a microenvironment facilitating bacterial survival.

microRNAs (miRNAs) have emerged as major regulators of the initiation and progression of human cancers, including breast cancer. The aim of this study is to determine the expression pattern of miR-96 in breast cancer and to investigate its biological role during tumorigenesis. We showed that miR-96 was significantly upregulated in breast cancer. We then investigated its function and found that miR-96 significantly promoted cell proliferation, migration and invasion in vitro and enhanced tumor growth in vivo. Furthermore, we explored the molecular mechanisms by which miR-96 contributes to breast cancer progression and identified PTPN9 (protein tyrosine phosphatase, non-receptor type 9) as a direct target gene of miR-96. Finally, we showed that PTPN9 had opposite effects to those of miR-96 on breast cancer cells, suggesting that miR-96 may promote breast tumorigenesis by silencing PTPN9. Taken together, this study highlights an important role for miR-96 in the regulation of PTPN9 in breast cancer cells and may provide insight into the molecular mechanisms of breast carcinogenesis.

It is generally regarded that E-cadherin is downregulated during tumorigenesis via Snail/Slug-mediated E-cadherin transcriptional reduction. However, this transcriptional suppressive mechanism cannot explain the failure of producing E-cadherin protein in metastatic breast cancer cells after overexpressing E-cadherin mRNA. Here we reveal a novel mechanism that E-cadherin is post-transcriptionally regulated by Slug-promoted miR-221, which serves as an additional blocker for E-cadherin expression in metastatic tumor cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cells showed that miR-221 was abundantly expressed in breast tumor and metastatic MDA-MB-231 cells and its level was significantly higher in breast tumor or MDA-MB-231 cells than in distal non-tumor tissue and low-metastatic MCF-7 cells, respectively. MiR-221, which level inversely correlated with E-cadherin level in breast cancer cells, targeted E-cadherin mRNA open reading frame (ORF) and suppressed E-cadherin protein expression. Depleting or increasing miR-221 level in breast cancer cells induced or decreased E-cadherin protein level, leading to suppressing or promoting tumor cell progression, respectively. Moreover, miR-221 was specifically upregulated by Slug but not Snail. TGF-β treatment enhanced Slug activity and thus increased miR-221 level in MCF-7 cells. In summary, our results provide the first evidence that Slug-upregulated miR-221 promotes breast cancer progression via reducing E-cadherin expression.

Although piwi-interacting RNAs (piRNAs) play pivotal roles in spermatogenesis, little is known about piRNAs in the seminal plasma of infertile males. In this study, we systematically investigated the profiles of seminal plasma piRNAs in infertile males to identify piRNAs that are altered during infertility and evaluate their diagnostic value. Seminal plasma samples were obtained from 211 infertile patients (asthenozoospermia and azoospermia) and 91 fertile controls. High-throughput sequencing technology was employed to screen piRNA profiles in seminal plasma samples pooled from healthy controls and infertile patients. The results identified 61 markedly altered piRNAs in infertile patient groups compared with control group. Next, a quantitative RT-PCR assay was conducted in the training and validation sets to measure and confirm the concentrations of altered piRNAs. The results identified a panel of 5 piRNAs that were significantly decreased in seminal plasma of infertile patients compared with healthy controls. ROC curve analysis and risk score analysis revealed that the diagnostic potential of these 5 piRNAs to distinguish asthenozoospermic and azoospermic individuals from healthy controls was high. In summary, this study identifies a panel of piRNAs that can accurately distinguish fertile from infertile males. This finding may provide pathophysiological clues about the development of infertility.

Signal regulatory protein α (SIRPα) has been shown to operate as a negative regulator in cancer cell survival. The mechanism underneath such function, however, remains poorly defined. In the present study, we demonstrate that overexpression of SIRPα in acute promyelocytic leukemia (APL) cells results in apoptosis possibly via inhibiting the β-catenin signaling pathway and upregulating Foxo3a. Pharmacological activation of β-catenin signal pathway attenuates apoptosis caused by SIRPα. Interestingly, we also find that the pro-apoptotic effect of SIRPα plays an important role in arsenic trioxide (ATO)-induced apoptosis in APL cells. ATO treatment induces the SIRPα protein expression in APL cells and abrogation of SIRPα induction by lentivirus-mediated SIRPα shRNA significantly reduces the ATO-induced apoptosis. Mechanistic study further shows that induction of SIRPα protein in APL cells by ATO is mediated through suppression of c-Myc, resulting in reduction of three SIRPα-targeting microRNAs: miR-17, miR-20a and miR-106a. In summary, our results demonstrate that SIRPα inhibits tumor cell survival and significantly contributes to ATO-induced APL cell apoptosis.

The efficacy of interferon α (IFNα) therapy for chronic hepatitis B (CHB) patients is about 40% and often associates with adverse side-effects, thus identification of an easy accessible biomarker that can predict the outcome of IFNα treatment for individual CHB patients would be greatly helpful. Recent reports by us and others show that microRNAs encoded by human cytomegalovirus (HCMV) were readily detected in human serum and can interfere with lymphocyte responses required by IFNα therapeutic effect. We thus postulate that differential expression profile of serum HCMV miRNAs in CHB patients may serve as indicator to predict the efficacy of IFNα treatment for CHB patients. Blood was drawn from 56 individual CHB patients prior to IFNα treatment. By quantifying 13 HCMV miRNAs in serum samples, we found that the levels of HCMV-miR-US4-1 and HCMV-miR-UL-148D were significantly higher in IFNα-responsive group than in IFNα-non-responsive group. In a prospective study of 96 new CHB patients, serum level of HCMV-miR-US4-1 alone classified those who were and were not responsive to IFN-α treatment with correct rate of 84.00% and 71.74%, respectively. In conclusion, our results demonstrate that serum HCMV-miR-US4-1 can serve as a novel biomarker for predicting the outcome of IFNα treatment in CHB patients.

Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system.

We sought to identify a serum miRNA expression profile to improve disease surveillance and to predict post-operative disease recurrence for stage II/III colorectal cancer (CRC) patients. Using the TaqMan Low-Density Array (TLDA), we performed an initial survey to analyze 749 miRNAs in the pooled serum of 20 paired pre- and post-operative CRC patients and 20 matched normal subjects. Using individual RT-qPCR verification in 175 stage II/III CRC patients, we identified that miR-145, miR-106a and miR-17-3p were significantly differentially expressed between pre- and post-operative CRC patients and between pre-operative CRC patients and normal controls (P < 0.0001). The area under the ROC curve (AUC) for the three-miRNA panel was 0.886 (95% CI 0.850-0.921) for discriminating between pre-operative CRC patients and normal subjects and 0.850 (95% CI 0.809-0.891) for discriminating between pre- and post-operative CRC patients. Furthermore, using the Kaplan-Meier method and Cox proportional hazards analysis, we found that miR-17-3p and miR-106a were powerful and independent prognostic indicators and that high levels of these miRNAs were associated with shorter disease-free survival (DFS) (P < 0.0001 for miR-17-3p and P = 0.001 for miR-106a). The present study reveals novel serum-miRNA-based biomarkers for monitoring tumor dynamics as well as for predicting disease recurrence in patients with stage II/III CRC.

Although aberrant microRNA (miRNA) expression has frequently been observed in inflammatory bowel disease (IBD), its biological functions and targets remain largely unknown. Present study found that miR-19b was significantly downregulated in active Crohn's disease (CD). Using bioinformatics analysis, suppressor of cytokine signalling 3 (SOCS3), a physiological regulator of innate and adaptive immunity that controls several immuno-inflammatory diseases, was predicted to be a potential target of miR-19b. An inverse correlation between miR-19b and SOCS3 protein levels, but not mRNA, was identified in active-CD intestinal tissue samples. By overexpressing or knocking down miR-19b in Caco2 cells and HT29 cells, it was experimentally validated that miR-19b is a direct regulator of SOCS3. Using a luciferase reporter assay, it was confirmed that miR-19b directly recognizes the 3'-untranslated region (3'-UTR) of SOCS3. Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells. In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α. Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Taken together, our findings suggest that miR-19b suppresses the inflammatory response by inhibiting SOCS3 to modulate chemokine production in intestinal epithelial cells (IECs) and thereby prevents the pathogenesis of CD.

Circulating microRNAs (miRNAs) are emerging as clinically useful tools for cancer detection; however, little is known about their early diagnostic impact on RCC. The levels of 754 serum miRNAs were initially determined using a TaqMan Low Density Array in two pooled samples from 25 RCC and 25 noncancer controls. Markedly dysregulated miRNAs in RCC cases were subsequently validated individually by qRT-PCR in another 107 patients and 107 controls arranged in two sets. The serum levels of miR-193a-3p, miR-362 and miR-572 were significantly increased whereas the levels of miR-28-5p and miR-378 were markedly decreased in patients with RCC, even in those with stage I disease, compared with the noncancer controls (P < 0.01). The areas under the ROC curve (AUCs) for the 5 combined miRNAs were 0.807 (95% CI, 0.687-0.928) and 0.796 (95% CI, 0.724-0.867) for the training set and the validation set, respectively. Furthermore, the panel enabled the differentiation of stage I RCC from controls with AUC of 0.807 (95% CI, 0.731-0.871), a sensitivity of 80% and a specificity of 71%. This panel of 5 serum miRNA may have the potential to be used clinically as an auxiliary diagnostic tool for the early detection of RCC.