TBK1-stabilized ZNF268a recruits SETD4 to methylate TBK1 for efficient interferon signaling.

Sufficient activation of interferon signaling is critical for the host to fight against invading viruses, in which post-translational modifications have been demonstrated to play a pivotal role. Here, we demonstrate that the human KRAB-zinc finger protein ZNF268a is essential for virus-induced interferon signaling. We find that cytoplasmic ZNF268a is constantly degraded by lysosome and thus remains low expressed in resting cell cytoplasm. Upon viral infection, TBK1 interacts with cytosolic ZNF268a to catalyze the phosphorylation of Serine 178 of ZNF268a, which prevents the degradation of ZNF268a, resulting in the stabilization and accumulation of ZNF268a in the cytoplasm. Furthermore, we provide evidence that stabilized ZNF268a recruits the lysine methyltransferase SETD4 to TBK1 to induce the mono-methylation of TBK1 on lysine 607, which is critical for the assembly of the TBK1 signaling complex. Notably, ZNF268 S178 is conserved among higher primates but absent in rodents. Meanwhile, rodent TBK1 607th aa happens to be replaced by arginine, possibly indicating a species-specific role of ZNF268a in regulating TBK1 during evolution. These findings reveal novel functions of ZNF268a and SETD4 in regulating antiviral interferon signaling.

RNF138 Downregulates Antiviral Innate Immunity by Inhibiting IRF3 Activation.

A viral infection activates the transcription factors IRF3 and NF-κB, which synergistically induces type I interferons (IFNs). Here, we identify the E3 ubiquitin ligase RNF138 as an important negative regulator of virus-triggered IRF3 activation and IFN-ß induction. The overexpression of RNF138 inhibited the virus-induced activation of IRF3 and the transcription of the IFNB1 gene, whereas the knockout of RNF138 promoted the virus-induced activation of IRF3 and transcription of the IFNB1 gene. We further found that RNF138 promotes the ubiquitination of PTEN and subsequently inhibits PTEN interactions with IRF3, which is essential for the PTEN-mediated nuclear translocation of IRF3, thereby inhibiting IRF3 import into the nucleus. Our findings suggest that RNF138 negatively regulates virus-triggered signaling by inhibiting the interaction of PTEN with IRF3, and these data provide new insights into the molecular mechanisms of cellular antiviral responses.

Effect of natural gums on pasting, rheological, structural and hydrolysis properties of kudzu starch.

Hydrocolloids have been widely used to adjust properties of natural starches, but related research on kudzu starch is still rare. In this study, we investigated the effects of gum arabic (AG), sodium alginate (SA), locust bean gum (LG), and guar gum (GG) on kudzu starch from the perspective of its particle size, pasting, texture, rheology, dehydration rate, thermal properties, microstructure, and sensitivity to amyloglucosidase. Results showed that GG significantly increased the particle size of starch. Addition of AG led to lower peak-, final- and holding-viscosity. SA increased the retention viscosity of kudzu starch, while LG and GG increased its peak viscosity. Addition of hydrocolloids increased the hardness, chewiness, and cohesiveness of starch-hydrocolloid complexes, and reduced the dehydration rate of complex gels. Dynamic rheological data showed that the energy storage modulus (G') was significantly higher than the loss modulus (G″). The magnitude of modulus increased with frequency, and elastic properties were better than viscous properties. Thermal analysis showed that hydrocolloids increased the starting temperature (To), and the final temperature (Tc). With addition of each of these four hydrocolloids, a more regular and porous thick-wall dense structure was formed, which effectively lowered kudzu starch's sensitivity to amyloglucosidase. It indicated that the binding of hydrocolloid to starch may slow down glucose release into blood during digestion. These results will help understand effects of natural hydrocolloid on kudzu starch, as well as expanding its application in food industry.

Engineering and Characterization of an Artificial Drug-Carrying Vesicles Nanoplatform for Enhanced Specifically Targeted Therapy of Glioblastoma.

Glioblastoma multiforme (GBM) treatment is hindered by complex pathologies and the need to cross the blood-brain barrier (BBB) during drug delivery. Although exosomes have great potential for GBM treatment, these alone cannot fully meet the therapeutic requirements, owing to their limitations in targeting and delivery. Herein, engineered artificial vesicles (EAVs), ANG-TRP-PK1@EAVs, which are constructed using a liposome extruder from HEK293T cells expressing ANG-TRP-PK1 peptides, is developed. ANG-TRP-PK1 is a fusion peptide of Angiopep-2 fused to the N-terminus of TRP-PK1, to present Angiopep-2 on the EAVs. ANG-TRP-PK1@EAVs have similar characteristics to the secreted exosomes, but a much higher yield. ANG-TRP-PK1@EAVs have efficient BBB-penetration and GBM-targeting abilities in a mock BBB model in in vitro and orthotopic GBM mouse models in vivo. Doxorubicin loading EAVs (ANG-TRP-PK1@DOX) do not alter the characteristics of the EAVs, which can cross the BBB, reach the GBM, and kill tumor cells in orthotopic GBM mouse models. These engineered drug-loaded artificial vesicles show better therapeutic effects on GBM than temozolomide in mice, with very few side effects. In conclusion, EAVs can be inserted into different targeting ligands and packed into different drugs, and they may serve as unique and efficient nanoplatforms for drug delivery and tumor promise therapy.

HBV X Protein Induces Degradation of UBXN7, a Novel Negative Regulator of NF-κB Signaling, to Promote HBV Replication.

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma. However, the function and mechanism of the effect of HBV on host protein ubiquitination remain largely unknown. We aimed at characterizing whether and how HBV promotes self-replication by affecting host protein ubiquitination. In this study, we identified UBXN7, a novel inhibitor for nuclear factor kappa B (NF-κB) signaling, was degraded via interaction with HBV X protein (HBx) to activate NF-κB signaling and autophagy, thereby affecting HBV replication. The expression of UBXN7 was analyzed by Western blot and quantitative reverse transcription polymerase chain reaction in HBV-transfected hepatoma cells and HBV-infected primary human hepatocytes (PHHs). The effects of UBXN7 on HBV replication were analyzed by using in vitro and in vivo assays, including stable isotope labeling by amino acids in cell culture (SILAC) analysis. Changes in HBV replication and the associated molecular mechanisms were analyzed in hepatoma cell lines. SILAC analyses showed that the ubiquitination of UBXN7 was significantly increased in HepG2.2.15 cells compared with control cells. After HBV infection, HBx protein interacted with UBXN7 to promote K48-linked ubiquitination of UBXN7 at K99, leading to UBXN7 degradation. On the other hand, UBXN7 interacted with the ULK domain of IκB kinase ß through its ubiquitin-associating domain to facilitate its degradation. This in turn reduced NF-κB signaling, leading to reduced autophagy and consequently decreased HBV replication.

Upregulation of miR-520c-3p via hepatitis B virus drives hepatocellular migration and invasion by the PTEN/AKT/NF-κB axis.

Hepatitis B virus (HBV) is a major risk factor for the development and progression of hepatocellular carcinoma (HCC). It has been reported that viral infection can interfere with the expression of cellular microRNA (miRNA) to affect oncogenesis. In this study, we showed that miR-520c-3p was upregulated in liver tumor specimens, and we revealed that HBV infection enhanced the expression of miR-520c-3p through the interaction of viral protein HBV X protein (HBx) with transcription factor CREB1. We further showed that miR-520c-3p induced by HBV transfection/infection caused epithelial-mesenchymal transition (EMT). Using the miRNA target prediction database miRBase and luciferase reporter assays, we identified PTEN as a novel target gene of miR-520c-3p and miR-520c-3p directly targeted PTEN's 3'-untranslated region. Moreover, we discovered that HBV promoted EMT via the miR-520c-3p-PTEN to activate AKT-NFκB signaling pathway, leading to increased HCC migration and invasion. Importantly, miR-520c-3p antagomir significantly represses invasiveness in HBx-induced hepatocellular xenograft models. Our findings indicate that miR-520c-3p is a novel regulator of HBV and plays an important role in HCC progression. It may serve as a new biomarker and molecular therapeutic target for HBV patients.

Selective sorting and secretion of hY4 RNA fragments into extracellular vesicles mediated by methylated YBX1 to promote lung cancer progression.

BACKGROUND: Extracellular vesicles (EVs) are emerging mediators of intercellular communication that have been shown to play important roles in tumor progression. YRNA fragments, a type of small non-coding RNA, are dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs, suggesting that they may be an effective biomarker for cancer diagnosis and treatment strategies. METHODS: Differentially expressed YRNA hY4 fragments (hY4F) in EVs from NSCLC cells and normal lung fibroblasts were isolated by differential ultra-centrifugation. RNA-binding proteins that interacted with hY4F were identified by screening with an RNA pulldown assay and mass spectrometry. The molecular mechanism of hY4F and the RNA-binding protein Y box binding protein 1 (YBX1) was demonstrated by qRT-PCR, western blot, RNA pulldown, and rescue experiments. Transcriptome sequencing, qRT-PCR validation, bioinformatics analysis and NF-κB pathway inhibitor assays elucidate the mechanism of YBX1 and hY4F inhibiting lung cancer. A peptide pulldown assay was performed to screen and identify a potential methyltransferase for YBX1. The roles of hY4F, YBX1, and SET domain containing 3 in biological functions, such as proliferation, migration, invasion, and apoptosis, in lung cancer cells were also examined by EdU incorporation assay, Transwell assay, flow cytometry, and other methods. Lastly, a mouse xenograft assay was used to assess the clinical relevance of YBX1 and hY4F in vivo. RESULTS: Our data demonstrate that hY4 RNA fragments were upregulated in lung cancer- derived EVs, hY4F inhibits tumor progression through downregulating MAPK/NF-κB signaling, and then the selective sorting and secretion of hY4F into lung cancer EVs is regulated by the RNA-binding protein YBX1. Furthermore, we identified lysine K264 within the YBX1 C-terminal domain as the necessary site for its interaction with hY4Fs. K264 is modified by methylation, which affects its binding to hY4F and subsequent selective sorting into EVs in lung cancer cells. CONCLUSION: Our findings demonstrate that hY4F acts as a tumor suppressor and is selectively sorted into lung cancer cell-derived EVs by interacting with methylated YBX1, which in turn promotes lung cancer progression. hY4F is a promising circulating biomarker for non-small cell lung cancer diagnosis and prognosis and an exceptional candidate for further therapeutic exploration.

Multiomics Analysis of Endocytosis upon HBV Infection and Identification of SCAMP1 as a Novel Host Restriction Factor against HBV Replication.

Hepatitis B virus (HBV) infection remains a major global health problem and the primary cause of cirrhosis and hepatocellular carcinoma (HCC). HBV intrusion into host cells is prompted by virus-receptor interactions in clathrin-mediated endocytosis. Here, we report a comprehensive view of the cellular endocytosis-associated transcriptome, proteome and ubiquitylome upon HBV infection. In this study, we quantified 273 genes in the transcriptome and 190 endocytosis-associated proteins in the proteome by performing multi-omics analysis. We further identified 221 Lys sites in 77 endocytosis-associated ubiquitinated proteins. A weak negative correlation was observed among endocytosis-associated transcriptome, proteome and ubiquitylome. We found 33 common differentially expressed genes (DEGs), differentially expressed proteins (DEPs), and Kub-sites. Notably, we reported the HBV-induced ubiquitination change of secretory carrier membrane protein (SCAMP1) for the first time, differentially expressed across all three omics data sets. Overexpression of SCAMP1 efficiently inhibited HBV RNAs/pgRNA and secreted viral proteins, whereas knockdown of SCAMP1 significantly increased viral production. Mechanistically, the EnhI/XP, SP1, and SP2 promoters were inhibited by SCAMP1, which accounts for HBV X and S mRNA inhibition. Overall, our study unveils the previously unknown role of SCAMP1 in viral replication and HBV pathogenesis and provides cumulative and novel information for a better understanding of endocytosis in response to HBV infection.

Piezoelectric energy harvester with double cantilever beam undergoing coupled bending-torsion vibrations by width-splitting method.

We propose piezoelectric energy harvester (PEH) with double-cantilever-beam (DCB) undergoing coupled bending-torsion vibrations by combining width-splitting method and asymmetric mass, in order that more ambient energy could be harvested from environmental vibration with multiple-frequency excitation. The geometrical dimensions are optimized for PEHDCB, when the maximum of output peak voltages Up-max and resonance frequency difference (Δf0) between the first and second modes are chosen as optimization objectives based on orthogonal test method. The energy harvesting efficiency is evaluated by the proportion of half-power bandwidth and quality factor, and the experimental and simulation results are compared to verify reliability. The Up-max1 and Pp-max1 are increased 25.2% and 57.3% for PEHDCB under the multi-frequency excitation, when the split-width method is applied into PEH with single-cantilever-beam (SCB) undergoing coupled bending-torsion vibrations. The deviations of Up-max1 and f0 are at the ranges of 4.9-14.2% and 2.2-2.5% for PEHDCB under the different mass ratios, and the measurement reliability is acceptable considering incomplete clamping, damping and inevitable assembly effects. The energy harvesting efficiency of PEHDCB presented is much higher than that of the conventional PEHSCB from environmental vibration with multiple-frequency excitation.

hnRNPA2B1-Mediated Extracellular Vesicles Sorting of miR-122-5p Potentially Promotes Lung Cancer Progression.

Extracellular vesicles (EVs) released by tumor cells play important roles on the remodeling of the tumor-stromal environment and on promoting tumor metastasis. Our earlier studies revealed that miR-122-5p, a type of small non-coding RNA, was dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs. In this study, we found that miR-122-5p was selectively sorted and secreted into lung cancer EVs through binding to RNA-binding protein hnRNPA2B1. In addition, we found that hnRNPA2B1 interacted with miR-122-5p through the EXO-motif. The delivering of lung cancer EVs-miR-122-5p promoted the migration of liver cells, which may play roles in establishing a pre-metastatic micro-environment and hepatic metastasis of lung cancer. Importantly, our findings revealed the molecular mechanism that RNA-binding protein controls the selective sorting of tumor-derived EV miR-122-5p, which potentially promotes lung cancer progression.

Global analysis of HBV-mediated host proteome and ubiquitylome change in HepG2.2.15 human hepatoblastoma cell line.

Hepatitis B virus (HBV) infection remains a major health issue worldwide and the leading cause of cirrhosis and hepatocellular carcinoma (HCC). It has been reported previously that HBV invasion can extensively alter transcriptome, the proteome of exosomes and host cell lipid rafts. The impact of HBV on host proteins through regulating their global post-translational modifications (PTMs), however, is not well studied. Viruses have been reported to exploit cellular processes by enhancing or inhibiting the ubiquitination of specific substrates. Nevertheless, host cell physiology in terms of global proteome and ubiquitylome has not been addressed yet. Here by using HBV-integrated HepG2.2.15 model cell line we first report that HBV significantly modify the host global ubiquitylome. As currently the most widely used HBV cell culture model, HepG2.2.15 can be cultivated for multiple generations for protein labeling, and can replicate HBV, express HBV proteins and secrete complete HBV Dane particles, which makes it a suitable cell line for ubiquitylome analysis to study HBV replication, hepatocyte immune response and HBV-related HCC progression. Our previous experimental results showed that the total ubiquitination level of HepG2.2.15 cell line was significantly higher than that of the corresponding parental HepG2 cell line. By performing a Ubiscan quantification analysis based on stable isotope labeling of amino acids in cell culture (SILAC) of HepG2.2.15 and HepG2 cell lines, we identified a total of 7188 proteins and the protein levels of nearly 19% of them were changed over 2-folds. We further identified 3798 ubiquitinated Lys sites in 1476 host proteins with altered ubiquitination in response to HBV. Our results also showed that the global proteome and ubiquitylome were negatively correlated, indicating that ubiquitination might be involved in the degradation of host proteins upon HBV integration. We first demonstrated the ubiquitination change of VAMP3, VAMP8, DNAJB6, RAB8A, LYN, VDAC2, OTULIN, SLC1A4, SLC1A5, HGS and TOLLIP. In addition, we described 5 novel host factors SLC1A4, SLC1A5, EIF4A1, TOLLIP and BRCC36 that efficiently reduced the amounts of secreted HBsAg and HBeAg. Overall, the HBV-mediated host proteome and ubiquitylome change we reported will provide a valuable resource for further investigation of HBV pathogenesis and host-virus interaction networks.

Genome editing of CCR5 by AsCpf1 renders CD4+T cells resistance to HIV-1 infection.

BACKGROUND: The chemokine receptor CCR5 is one of the co-receptor of HIV-1 infection. People with homozygous CCR5Δ32 deletion resist HIV-1 infection, which makes the CCR5 an important target for HIV-1 gene therapy. Although the CRISPR/Cas9 has ever been used for HIV-1 study, the newly developed CRISPR/AsCpf1 has never been utilized in HIV-1 co-receptor disruption. The CRISPR/Cpf1 system shows many advantages over CRISPR/Cas9, such as lower off-target, small size of nuclease, easy sgRNA design for multiplex gene editing, etc. Therefore, the CRISPR/Cpf1 mediated gene editing will confer a more specific and safe strategy in HIV-1 co-receptor disruption. RESULTS: Here, we demonstrated that CRISPR/AsCpf1 could ablate the main co-receptor of HIV-1 infection-CCR5 efficiently with two screened sgRNAs via different delivery strategies (lentivirus, adenovirus). The edited cells resisted R5-tropic HIV-1 infection but not X4-tropic HIV-1 infection compared with the control group in different cell types of HIV-1 study (TZM.bl, SupT1-R5, Primary CD4+T cells). Meanwhile, the edited cells exhibited selective advantage over unedited cells while under the pressure of R5-tropic HIV-1. Furthermore, we clarified that the predicted off-target sites of selected sgRNAs were very limited, which is much less than regular using sgRNAs for CRISPR/Cas9, and no evident off-target was observed. We also showed that the disruption of CCR5 by CRISPR/AsCpf1 took no effects on cell proliferation and apoptosis. CONCLUSIONS: Our study provides a basis for a possible application of CCR5-targeting gene editing by CRISPR/AsCpf1 with high specific sgRNAs against HIV-1 infection.

KRAB-Zinc Finger Protein ZNF268a Deficiency Attenuates the Virus-Induced Pro-Inflammatory Response by Preventing IKK Complex Assembly.

Despite progress in understanding how virus-induced, NF-κB-dependent pro-inflammatory cytokines are regulated, there are still factors and mechanisms that remain to be explored. We aimed to uncover the relationship between KRAB-zinc finger protein ZNF268a and NF-κB-mediated cytokine production in response to viral infection. To this end, we established a ZNF268a-knockout cell line using a pair of sgRNAs that simultaneously target exon 3 in the coding sequence of the ZNF268 gene in HEK293T. HEK293T cells lacking ZNF268a showed less cytokine expression at the transcription and protein levels in response to Sendai virus/vesicular stomatitis virus (SeV/VSV) infection than wild-type cells. Consistent with HEK293T, knock-down of ZNF268a by siRNAs in THP-1 cells significantly dampened the inflammatory response. Mechanistically, ZNF268a facilitated NF-κB activation by targeting IKKα, helping to maintain the IKK signaling complex and thus enabling proper p65 phosphorylation and nuclear translocation. Taken together, our data suggest that ZNF268a plays a positive role in the regulation of virus-induced pro-inflammatory cytokine production. By interacting with IKKα, ZNF268a promotes NF-κB signal transduction upon viral infection by helping to maintain the association between IKK complex subunits.

Hepatitis B virus-regulated growth of liver cancer cells occurs through the microRNA-340-5p-activating transcription factor 7-heat shock protein A member 1B axis.

Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis. Hepatitis B virus (HBV) is one of the leading causes of HCC, but the precise mechanisms by which this infection promotes cancer development are not fully understood. Recently, miR-340-5p, a microRNA (miRNA) that has been identified as a cancer suppressor gene, was found to inhibit the migration and invasion of liver cancer cells. However, the effect of miR-340-5p on cell proliferation and apoptosis in HBV-associated HCC remains unknown. In our study, we show that miR-340-5p plays an important role during HBV infection and hepatocellular carcinoma development. Specifically, this miRNA directly binds to the mRNA encoding activating transcription factor 7 (ATF7), a protein that both promotes cell proliferation and suppresses apoptosis through its interaction with heat shock protein A member 1B (HSPA1B). We further found that miR-340-5p is downregulated by HBV, which enhances ATF7 expression, leading to enhanced cell proliferation and inhibition of apoptosis. Notably, ATF7 is upregulated in HCC tissue, suggesting that HBV may target miR-340-5p in vivo to promote ATF7/HSPA1B-mediated proliferation and apoptosis and regulate liver cancer progression. This work helps to elucidate the complex interactions between HBV and host miRNAs and further suggests that miR-340-5p may represent a promising candidate for the development of improved therapeutic strategies for HCC.

Hepatitis B Virus Induces Autophagy to Promote its Replication by the Axis of miR-192-3p-XIAP Through NF kappa B Signaling.

Hepatitis B virus (HBV) is a major risk factor for the development and progression of hepatocellular carcinoma. It has been reported that viral infection can interfere with cellular microRNA (miRNA) expression and participate in the pathogenesis of oncogenicity. Here, we report that decreasing levels of the expression of the miRNA miR-192-3p is associated with rising levels of HBV DNA in the serum of HBV patients. We revealed that HBV infection repressed the expression of miR-192-3p through hepatitis B x protein interaction with c-myc. We further showed that miR-192-3p was repressed by HBV transfection in vitro and in a mouse model, leading to cellular autophagy. Using an miRNA target prediction database miRBase, we identified X-linked inhibitor of apoptosis protein (XIAP) as a target gene of miR-192-3p and demonstrated that miR-192-3p directly targeted the XIAP 3'-untranslated region of XIAP messenger RNA. Importantly, we discovered that HBV promoted autophagy through miR-192-3p-XIAP axis and that this process was important for HBV replication in vitro and in vivo. We demonstrated that miR-192-3p functioned through the nuclear factor kappa B signaling pathway to inhibit autophagy, thereby reducing HBV replication. Conclusions: Our findings indicate that miR-192-3p is a regulator of HBV infection and may play a potential role in hepatocellular carcinoma. It may also serve as a biomarker or therapeutic target for HBV patients.

miR-429 and miR-424-5p inhibit cell proliferation and Ca2+ influx by downregulating CaSR in pulmonary artery smooth muscle cells.

Cytosolic free Ca2+ concentration is a key factor in pulmonary vasoconstriction and vascular remodeling of pulmonary artery smooth muscle cells (PASMCs). These processes contribute to pulmonary arterial hypertension and are influenced by expression of calcium-sensing receptor (CaSR). Although regulation of CaSR expression is precisely controlled, the contribution of microRNAs (miR) is incompletely understood. Here, we demonstrate that miR-429, miR-424-5p, miR-200b-3p, and miR-200c-3p regulate CaSR by targeting specific 3'-untranslated region, suggesting that these miRNAs function as CaSR inhibitors in PASMCs. Moreover, miR-429 and miR-424-5p inhibit proliferation of PASMCs by downregulating CaSR, resulting in reduced Ca2+ influx under both normoxia and hypoxia. These findings indicate miR-429 and miR-424-5p target CaSR and may function as Ca2+ influx suppressors in pulmonary arterial hypertension-associated diseases.

Characterization and selective incorporation of small non-coding RNAs in non-small cell lung cancer extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) play important roles in intercellular communication through the delivery of their cargoes, which include proteins, lipids, and RNAs. Increasingly, multiple studies have reported the association between EV small non-coding RNAs and cancer, due to their regulatory functions in gene expression. Hence, analysis of the features of small non-coding RNA expression and their incorporation into EVs is important for cancer research. RESULTS: We performed deep sequencing to investigate the expression of small RNAs in plasma EVs from lung adenocarcinoma (ADC) patients, lung squamous cell carcinoma (SQCC) patients, and healthy controls. Then, eighteen differently expressed miRNAs in plasma EVs was validated by QRT-PCR. The small RNA expression profiles of plasma EVs were different among lung ADC, SQCC patients, and healthy controls. And many small RNAs, including 5' YRNA hY4-derived fragments, miR-451a, miR-122-5p, miR-20a-5p, miR-20b-5p, miR-30b-5p, and miR-665, were significantly upregulated in non-small cell lung cancer (NSCLC) EVs. And the cell viability assays indicated that hY4-derived fragments inhibited the proliferation of lung cancer cell A549. By comparing the cellular and EV expression levels of six miRNAs in NSCLC cells, we found that miR-451a and miR-122-5p were significantly downregulated in NSCLC cell lysates, while significantly upregulated in NSCLC EVs. CONCLUSIONS: The differently expressed EV small RNAs may serve as potential circulating biomarkers for the diagnosis of NSCLC. Particularly, YRNA hY4-derived fragments can serve as a novel class of biomarkers, which function as tumor suppressors in NSCLC. Additionally, miR-451a and miR-122-5p may be sorted into NSCLC EVs in a selective manner.

Genome editing of the HIV co-receptors CCR5 and CXCR4 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection.

BACKGROUND: The main approach to treat HIV-1 infection is combination antiretroviral therapy (cART). Although cART is effective in reducing HIV-1 viral load and controlling disease progression, it has many side effects, and is expensive for HIV-1 infected patients who must remain on lifetime treatment. HIV-1 gene therapy has drawn much attention as studies of genome editing tools have progressed. For example, zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 have been utilized to successfully disrupt the HIV-1 co-receptors CCR5 or CXCR4, thereby restricting HIV-1 infection. However, the effects of simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 in blocking HIV-1 infection in primary CD4+ T cells has been rarely reported. Furthermore, combination of different target sites of CXCR4 and CCR5 for disruption also need investigation. RESULTS: In this report, we designed two different gRNA combinations targeting both CXCR4 and CCR5, in a single vector. The CRISPR-sgRNAs-Cas9 could successfully induce editing of CXCR4 and CCR5 genes in various cell lines and primary CD4+ T cells. Using HIV-1 challenge assays, we demonstrated that CXCR4-tropic or CCR5-tropic HIV-1 infections were significantly reduced in CXCR4- and CCR5-modified cells, and the modified cells exhibited a selective advantage over unmodified cells during HIV-1 infection. The off-target analysis showed that no non-specific editing was identified in all predicted sites. In addition, apoptosis assays indicated that simultaneous disruption of CXCR4 and CCR5 in primary CD4+ T cells by CRISPR-Cas9 had no obvious cytotoxic effects on cell viability. CONCLUSIONS: Our results suggest that simultaneous genome editing of CXCR4 and CCR5 by CRISPR-Cas9 can potentially provide an effective and safe strategy towards a functional cure for HIV-1 infection.

Hepatitis B virus promotes cancer cell migration by downregulating miR-340-5p expression to induce STAT3 overexpression.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and infection with hepatitis B virus (HBV) is a leading cause of HCC. Previous studies have demonstrated that expression of the tumor inhibitor miR-340 is significantly downregulated in HCC tissues compared with normal liver tissues. However, the precise biological role of miR-340-5p in HBV-HCC and its molecular mechanism of action remain unknown. RESULTS: Expression of miR-340-5p was downregulated in HBV-associated HCC liver tissue and HBV-infected cells, facilitating migration of liver cancer cells. Signal transducer and activator of transcription (STAT)3 was found to be a direct functional target of miR-340-5p. The regulation of STAT3 expression by miR-340-5p was assessed using qRT-PCR and western blotting, and the effects of exogenous miR-340-5p and STAT3 on the migration of HBV-infected cells were evaluated in vitro using Transwell® and wound-healing assays. The expression of E-cadherin and vimentin, associated with epithelial-mesenchymal transition, was also assessed using Western blotting after transfection of miR-340-5p mimics and/or STAT3 expression vectors. Overexpression of STAT3 resulted in rescue of HBV effects, decreased E-cadherin expression, increased vimentin expression, and ultimately, enhanced cell migration. Re-introduction of the STAT3 CDS led to marked reversal of the inhibition of cell migration in HBV-infected cells mediated by miR-340-5p. CONCLUSIONS: Hepatitis B virus promotes the migration of liver cancer cells by downregulating miR-340-5p expression to induce STAT3 overexpression. Our results show that STAT3 plays a key role in regulating cell migration in HBV-HCC involving miR-340-5p.

The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription.

RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65.