N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA.

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.

Epigenetic regulation of asymmetric cell division by the LIBR-BRD4 axis.

Asymmetric cell division (ACD) is a mechanism used by stem cells to maintain the number of progeny. However, the epigenetic mechanisms regulating ACD remain elusive. Here we show that BRD4, a BET domain protein that binds to acetylated histone, is segregated in daughter cells together with H3K56Ac and regulates ACD. ITGB1 is regulated by BRD4 to regulate ACD. A long noncoding RNA (lncRNA), LIBR (LncRNA Inhibiting BRD4), decreases the percentage of stem cells going through ACD through interacting with the BRD4 mRNAs. LIBR inhibits the translation of BRD4 through recruiting a translation repressor, RCK, and inhibiting the binding of BRD4 mRNAs to polysomes. These results identify the epigenetic regulatory modules (BRD4, lncRNA LIBR) that regulate ACD. The regulation of ACD by BRD4 suggests the therapeutic limitation of using BRD4 inhibitors to treat cancer due to the ability of these inhibitors to promote symmetric cell division that may lead to tumor progression and treatment resistance.

Interplay between lncRNA RP11-367G18.1 variant 2 and YY1 plays a vital role in hypoxia-mediated gene expression and tumorigenesis.

BACKGROUND: The hypoxia-responsive long non-coding RNA, RP11-367G18.1, has recently been reported to induce histone 4 lysine 16 acetylation (H4K16Ac) through its variant 2; however, the underlying molecular mechanism remains poorly understood. METHODS: RNA pull-down assay and liquid chromatography-tandem mass spectrometry were performed to identify RP11-367G18.1 variant 2-binding partner. The molecular events were examined utilizing western blot analysis, real-time PCR, luciferase reporter assay, chromatin immunoprecipitation, and chromatin isolation by RNA purification assays. The migration, invasion, soft agar colony formation, and in vivo xenograft experiments were conducted to evaluate the impact of RP11-367G18.1 variant 2-YY1 complex on tumor progression. RESULTS: In this study, RNA sequencing data revealed that hypoxia and RP11-367G18.1 variant 2 co-regulated genes were enriched in tumor-related pathways. YY1 was identified as an RP11-367G18.1 variant 2-binding partner that activates the H4K16Ac mark. YY1 was upregulated under hypoxic conditions and served as a target gene for hypoxia-inducible factor-1α. RP11-367G18.1 variant 2 colocalized with YY1 and H4K16Ac in the nucleus under hypoxic conditions. Head and neck cancer tissues had higher levels of RP11-367G18.1 and YY1 which were associated with poor patient outcomes. RP11-367G18.1 variant 2-YY1 complex contributes to hypoxia-induced epithelial-mesenchymal transition, cell migration, invasion, and tumorigenicity. YY1 regulated hypoxia-induced genes dependent on RP11-367G18.1 variant 2. CONCLUSIONS: RP11-367G18.1 variant 2-YY1 complex mediates the tumor-promoting effects of hypoxia, suggesting that this complex can be targeted as a novel therapeutic strategy for cancer treatment.

Secretome profiling of primary cells reveals that THBS2 is a salivary biomarker of oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC), which is a leading cause of cancer-related death worldwide, is frequently associated with poor prognosis and mortality. The discovery of body fluid-accessible biomarkers may help improve the detection of OSCC. In the present work, we established primary cell cultures derived from OSCC and adjacent noncancerous epithelium and performed comparative profiling of their secretomes. Using spectral counting-based label-free quantification, we found that 64 proteins were significantly higher in primary OSCC cells compared with primary adjacent noncancerous cells. We then retrieved the mRNA expression levels of these 64 proteins in oral cavity tumor and noncancerous tissues from public domain array-based transcriptome data sets and used this information to prioritize the biomarker candidates. We identified 19 candidates; among them, the protein levels of THBS2, UFD1L, and DNAJB11 were found to be elevated in OSCC tissues compared with adjacent noncancerous epithelia. Importantly, higher levels of THBS2 in OSCC tissues were associated with a higher overall pathological stage, positive perineural invasion, and a poorer prognosis. Moreover, the salivary levels of THBS2 in OSCC patients were elevated compared to those of noncancer controls. Our results collectively indicate that analysis of the primary cell secretome is a feasible strategy for biomarker identification, and that THBS2 is a potentially useful salivary marker for the detection of OSCC.

PLK1 and its substrate MISP facilitate intrahepatic cholangiocarcinoma progression by promoting lymphatic invasion and impairing E-cadherin adherens junctions.

Intrahepatic cholangiocarcinoma (iCCA) is a subtype of CCA and has a high mortality rate and a relatively poor prognosis. However, studies focusing on increased cell motility and loss of epithelial integrity during iCCA progression remain relatively scarce. We collected seven fresh tumor samples from four patients to perform RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to determine the transcriptome profile and chromatin accessibility of iCCA. The increased expression of cell cycle regulators, including PLK1 and its substrate MISP, was identified. Ninety-one iCCA patients were used to validate the clinical significance of PLK1 and MISP. The upregulation of PLK1 and MISP was determined in iCCA tissues. Increased expression of PLK1 and MISP was significantly correlated with tumor number, N stage, and lymphatic invasion in an iCCA cohort. Knockdown of PLK1 or MISP reduced trans-lymphatic endothelial migration and wound healing and affected focal adhesions in vitro. In cell‒cell junctions, MISP localized to adherens junctions and suppressed E-cadherin dimerization. PLK1 disrupted adherens junctions in a myosin-dependent manner. Furthermore, PLK1 and MISP promoted cell proliferation in vitro and tumorigenesis in vivo. In iCCA, PLK1 and MISP promote aggressiveness by increasing lymphatic invasion, tumor growth, and motility through the repression of E-cadherin adherens junctions.

Integration of the cancer cell secretome and transcriptome reveals potential noninvasive diagnostic markers for bladder cancer.

PURPOSE: Bladder cancer (BLCA) is a major cancer of the genitourinary system. Although cystoscopy is the standard protocol for diagnosing BLCA clinically, this procedure is invasive and expensive. Several urine-based markers for BLCA have been identified and investigated, but none has shown sufficient sensitivity and specificity. These observations underscore the importance of discovering novel BLCA biomarkers and developing a noninvasive method for detection of BLCA. Exploring the cancer secretome is a good starting point for the development of noninvasive biomarkers for cancer diagnosis. EXPERIMENTAL DESIGN: In this study, we established a comprehensive secretome dataset of five representative BLCA cell lines, BFTC905, TSGH8301, 5637, MGH-U1, and MGH-U4, by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Expression of BLCA-specific secreted proteins at the transcription level was evaluated using the Oncomine cancer microarray database. RESULTS: The expressions of four candidates-COMT, EWSR1, FUSIP1, and TNPO2-were further validated in clinical human specimens. Immunohistochemical analyses confirmed that transportin-2 was highly expressed in tumor cells relative to adjacent noncancerous cells in clinical tissue specimens from BLCA patients, and was significantly elevated in BLCA urine compared with that in urine samples from aged-matched hernia patients (controls). CONCLUSIONS: Collectively, our findings suggest TNPO2 as a potential noninvasive tumor-stage or grade discriminator for BLCA management.

RP11-367G18.1 V2 enhances clear cell renal cell carcinoma progression via induction of epithelial-mesenchymal transition.

PURPOSE: Metastasis is the end stage of renal cell carcinoma (RCC), and clear cell renal cell carcinoma (ccRCC) is the most common malignant subtype. The hypoxic microenvironment is a common feature in ccRCC and plays an essential role in the regulation of epithelial-mesenchymal transition (EMT). Accumulating evidence manifests that long non-coding RNAs (lncRNAs) participate in RCC tumorigenesis and regulate hypoxia-induced EMT. Here, we identified a lncRNA RP11-367G18.1 induced by hypoxia, that was overexpressed in ccRCC tissues. METHODS: A total of 216 specimens, including 149 ccRCC tumor samples and 67 related normal kidney parenchyma tissue samples, were collected. To investigate the biological fucntions of RP11.367G18.1 in ccRCC, migration, invasion, soft agar colony formation, xenograft tumorigenicity assays, and tail vein and orthotopic metastatic mouse models were performed. The relationship between RP11-367G18.1 and downstream signaling was analyzed utilizing reporter assay, RNA pull-down, chromatin immunopreciptation, and chromatin isolation by RNA purification assays. RESULTS: Hypoxic conditions and overexpression of HIF-1α increased the level of RP11-367G18.1. RP11-367G18.1 induced EMT and enhanced cell migration and invasion through variant 2. Inhibition of RP11-367G18.1 variant 2 reversed hypoxia-induced EMT phenotypes. An in vivo study revealed that RP11-367G18.1 variant 2 was required for hypoxia-induced tumor growth and metastasis in ccRCC. Mechanistically, RP11-367G18.1 variant 2 interacted with p300 histone acetyltransferase to regulate lysine 16 acetylation on histone 4 (H4K16Ac), thus contributing to hypoxia-regulated gene expression. Clinically, RP11-367G18.1 variant 2 was upregulated in ccRCC tissues, particularly metastatic ccRCC tissues, and it is linked to poor overall survival. CONCLUSION: These findings demonstrate the prognostic value and EMT-promoting role of RP11-367G18.1 and indicate that this lncRNA may provide a therapeutic target for ccRCC.

Biliverdin modulates the long non-coding RNA H19/microRNA-181b-5p/endothelial cell specific molecule 1 axis to alleviate cerebral ischemia reperfusion injury.

Injuries caused by cerebral ischemia reperfusion (CIR) can worsen neurological outcomes, Biliverdin (BV) is an antioxidant and anti-apoptotic agent that was shown to affect CIR, although the underlying mechanisms remain unclear. In this study, we investigated the role of BV and its potential underlying mechanism in CIR injury. CIR rat models and primary cortical neurons were established and treated with and without BV. Additionally, adenovirus vectors that could overexpress LncRNA H19 and overexpress or knock-down miR-181b-5p and Esm1 were created to investigate their regulation of molecular expression. Our findings showed that BV could significantly improve CIR injury, both in vivo and in vitro, decrease LncRNA H19 and Esm1 expression, and increase miR-181b-5p expression. Overexpression of LncRNA H19 inhibited the anti-injury effects of BV. Further, the down-regulation of miR-181b-5p or up-regulation of Esm1 expression weakened the in vitro protective effect of BV. RNA immunoprecipitation assay and dual luciferase reporter gene assay further confirmed that LncRNA H19 could sponge miR-181b-5p, and Esm1 was the target of miR-181b-5p. Rescue experiments confirmed that BV could regulate the LncRNA H19/miR-181b-5p/Esm1 molecular axis. Lastly, proteomic and bioinformatic analyzes revealed that Esm1 upregulation in BV-treated neurons resulted in the differential expression of 16 proteins, including 9 upregulated and 7 downregulated proteins. In conclusion, this study found that BV could ameliorate CIR injury by regulating the LncRNA H19/miR-181b-5p/Esm1 axis.

METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.

BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive. RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α. CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.

Using bioinformatics approaches to identify survival-related oncomiRs as potential targets of miRNA-based treatments for lung adenocarcinoma.

Lung cancer is a major cause of cancer-associated deaths worldwide, and lung adenocarcinoma (LUAD) is the most common lung cancer subtype. Micro RNAs (miRNAs) regulate the pattern of gene expression in multiple cancer types and have been explored as potential drug development targets. To develop an oncomiR-based panel, we identified miRNA candidates that show differential expression patterns and are relevant to the worse 5-year overall survival outcomes in LUAD patient samples. We further evaluated various combinations of miRNA candidates for association with 5-year overall survival and identified a four-miRNA panel: miR-9-5p, miR-1246, miR-31-3p, and miR-3136-5p. The combination of these four miRNAs outperformed any single miRNA for predicting 5-year overall survival (hazard ratio [HR]: 3.47, log-rank p-value = 0.000271). Experiments were performed on lung cancer cell lines and animal models to validate the effects of these miRNAs. The results showed that singly transfected antagomiRs largely inhibited cell growth, migration, and invasion, and the combination of all four antagomiRs considerably reduced cell numbers, which is twice as effective as any single miRNA-targeted transfected. The in vivo studies revealed that antagomiR-mediated knockdown of all four miRNAs significantly reduced tumor growth and metastatic ability of lung cancer cells compared to the negative control group. The success of these in vivo and in vitro experiments suggested that these four identified oncomiRs may have therapeutic potential.

Liquid-liquid phase separation (LLPS) in cellular physiology and tumor biology.

Liquid-liquid phase separation (LLPS) has emerged as a mechanism that has been used to explain the formation of known organelles (e.g. nucleoli, promyelocytic leukemia nuclear bodies (PML NBs), etc) as well as other membraneless condensates (e.g. nucleosome arrays, DNA damage foci, X-chromosome inactivation (XCI) center, paraspeckles, stress granules, proteasomes, autophagosomes, etc). The formation of membraneless condensates could be triggered by proteins containing modular domains or intrinsically disordered regions (IDRs) and nucleic acids. Multiple biological processes including transcription, chromatin organization, X-chromosome inactivation (XCI), DNA damage, tumorigenesis, autophagy, etc have been shown to utilize the principle of LLPS to facilitate these processes. This review will summarize the principle and components of LLPS, and describe how LLPS regulate these numerous biological processes and disruption of LLPS would cause disease formation. The role of LLPS in regulating normal cellular physiology and contributing to tumorigenesis will be discussed.

The role of hypoxia-induced long noncoding RNAs (lncRNAs) in tumorigenesis and metastasis.

Long noncoding RNAs (lncRNAs) are noncoding RNAs with length greater than 200 nt. The biological roles and mechanisms mediated by lncRNAs have been extensively investigated. Hypoxia is a proven microenvironmental factor that promotes solid tumor metastasis. Epithelial-mesenchymal transition (EMT) is one of the major mechanisms induced by hypoxia to contribute to metastasis. Many lncRNAs have been shown to be induced by hypoxia and their roles have been delineated. In this review, we focus on the hypoxia-inducible lncRNAs that interact with protein/protein complex and chromatin/epigenetic factors, and the mechanisms that contribute to metastasis. The role of a recently discovered lncRNA RP11-390F4.3 in hypoxia-induced EMT is discussed. Whole genome approaches to delineating the association between lncRNAs and histone modifications are discussed. Other topics related to hypoxia-induced tumor progression but require further investigation are also mentioned. The clinical significance and treatment strategy targeted against lncRNAs are discussed. The review aims to identify suitable lncRNA targets that may provide feasible therapeutic venues for hypoxia-involved cancers.

Biliverdin Protects Against Cerebral Ischemia/Reperfusion Injury by Regulating the miR-27a-3p/Rgs1 Axis.

BACKGROUND: We have previously demonstrated that biliverdin has neuroprotective effects that ameliorate cerebral ischemia/reperfusion (I/R) injury in rats. However, the underlying mechanism is unknown. This study aimed at elucidating on the modulatory role of miR-27a-3p on Rgs1 as a mechanism by which biliverdin affects cerebral I/R injury. METHODS: Middle cerebral artery occlusion/reperfusion (MCAO/R) was used to establish I/R rat models while oxygen glucose deprivation/reoxygenation (OGD/R) was used to induce hippocampal neurons to establish I/R models in vitro. Infarct volume was assessed by TTC staining. Apoptotic analyses of ischemic cortical neurons and cells were performed by TUNEL staining and flow cytometry, respectively. Cell viability was assessed by the CCK-8 assay while the target of miR-27a-3p was determined by double luciferase reporter assay. Relative expression levels of miR-27a-3p and Rgs1 (in vivo and in vitro) as well as markers of inflammation and apoptosis (in vitro) were detected by RT-qPCR. Then, Elisa and western blot were used to assess protein expression levels of inflammatory and apoptotic markers in vitro. RESULTS: Biliverdin suppressed inflammation and apoptosis in hippocampal neurons upon OGD/R, and reduced cerebral infarction volume as well as apoptosis in the MCAO/R rat model. Furthermore, biliverdin upregulated miR-27a-3p and downregulated hippocampal neuron Rgs1 after OGD/R as well as in rat brain tissues after cerebral I/R. Bioinformatic analysis revealed an miR-27a-3p docking site in the 3'-UTR region of Rgs1. Luciferase reporter assays showed that Rgs1 is an miR-27a-3p target. Moreover, miR-27a-3p upregulation inhibited OGD/R-triggered inflammation and suppressed neuronal apoptosis. Rgs1 knockdown suppressed OGD/R-triggered inflammation and decreased neuronal apoptosis while miR-27a-3p downregulation reversed the protective effect of Rgs1 knockdown. Moreover, miR-27a-3p overexpression and Rgs1 silencing suppressed NF-κB (p65) expression. CONCLUSION: Biliverdin protects against cerebral I/R injury by regulating the miR-27a-3p/Rgs1 axis, thereby inhibiting inflammation and apoptosis.

Chromatin accessibility analysis identifies GSTM1 as a prognostic marker in human glioblastoma patients.

BACKGROUND: Glioblastoma (GBM) is a malignant human brain tumor that has an extremely poor prognosis. Classic mutations such as IDH (isocitrate dehydrogenase) mutations, EGFR (epidermal growth factor receptor) alternations, and MGMT (O6-methylguanine-methyltransferase) promoter hypermethylation have been used to stratify patients and provide prognostic significance. Epigenetic perturbations have been demonstrated in glioblastoma tumorigenesis. Despite the genetic markers used in the management of glioblastoma patients, new biomarkers that could predict patient survival independent of known biomarkers remain to be identified. METHODS: ATAC-seq (assay for transposase accessible chromatin followed by sequencing) and RNA-seq have been used to profile chromatin accessible regions using glioblastoma patient samples with short-survival versus long-survival. Cell viability, cell cycle, and Western blot analysis were used to characterize the cellular phenotypes and identify signaling pathways. RESULTS: Analysis of chromatin accessibility by ATAC-seq coupled with RNA-seq methods identified the GSTM1 (glutathione S-transferase mu-1) gene, which featured higher chromatin accessibility in GBM tumors with short survival. GSTM1 was confirmed to be a significant prognostic marker to predict survival using a different GBM patient cohort. Knockdown of GSTM1 decreased cell viability, caused cell cycle arrest, and decreased the phosphorylation levels of the NF-kB (nuclear factor kappa B) p65 subunit and STAT3 (signal transducer and activator of transcription 3) (pSer727). CONCLUSIONS: This report demonstrates the use of ATAC-seq coupled with RNA-seq to identify GSTM1 as a prognostic marker of GBM patient survival. Activation of phosphorylation levels of NF-kB p65 and STAT3 (pSer727) by GSTM1 is shown. Analysis of chromatin accessibility in patient samples could generate an independent biomarker that can be used to predict patient survival.

Induction of epithelial-mesenchymal transition (EMT) by hypoxia-induced lncRNA RP11-367G18.1 through regulating the histone 4 lysine 16 acetylation (H4K16Ac) mark.

Hypoxia activates various long noncoding RNAs (lncRNAs) to induce the epithelial-mesenchymal transition (EMT) and tumor metastasis. The hypoxia/HIF-1α-regulated lncRNAs that also regulate a specific histone mark and promote EMT and metastasis have not been identified. We performed RNA-sequencing dataset analysis to search for such lncRNAs and lncRNA RP11-367G18.1 was the hypoxia-induced lncRNA with the highest hazard ratio. High expression of lncRNA RP11-367G18.1 is correlated with a worse survival of head and neck cancer patients. We further showed that lncRNA RP11-367G18.1 is induced by hypoxia and directly regulated by HIF-1α in cell lines. Overexpression of lncRNA RP11-367G18.1 induces the EMT and increases the in vitro migration and invasion and in vivo metastatic activity. Knockdown experiments showed that lncRNA RP11-367G18.1 plays an essential role in hypoxia-induced EMT. LncRNA RP11-367G18.1 specifically regulates the histone 4 lysine 16 acetylation (H4K16Ac) mark that is located on the promoters of two "core" EMT regulators, Twist1 and SLUG, and VEGF genes. These results indicate that lncRNA RP11-367G18.1 regulates the deposition of H4K16Ac on the promoters of target genes to activate their expression. This report identifies lncRNA RP11-367G18.1 as a key player in regulating the histone mark H4K16Ac through which activates downstream target genes to mediate hypoxia-induced EMT.

Scutellarin Reduces Cerebral Ischemia Reperfusion Injury Involving in Vascular Endothelium Protection and PKG Signal.

Flavonoid glycoside scutellarin (SCU) has been widely applied in the treatment of cerebral ischemic diseases in China. In this article, we conducted research on the working mechanisms of SCU in hypoxia reoxygenation (HR) injury of isolated cerebral basilar artery (BA) and erebral ischemia reperfusion (CIR) injury in rat models. In isolated rat BA rings, HR causes endothelial dysfunction (ED) and acetylcholine (ACh) induces endothelium-dependent vasodilation. The myography result showed that SCU (100 µM) was able to significantly improve the endothelium-dependent vasodilation induced by Ach. However, SCU did not affect the ACh-induced relaxation in normal BA. Further studies suggested that SCU (10-1000 µM) dose-dependently induced relaxation in isolated BA rings which were significantly blocked by the cGMP dependent protein kinase (PKG) inhibitor Rp-8-Br-cGMPs (PKGI-rp, 4 µM). Pre-incubation with SCU (500 µM) reversed the impairment of endothelium-dependent vasodilation induced by HR, but the reversing effect was blocked if PKGI-rp (4 µM) was added. The brain slice staining test in rats' model of middle cerebral artery occlusion (MCAO) induced CIR proved that the administration of SCU (45, 90 mg/kg, iv) significantly reduced the area of cerebral infarction. The Western blot assay result showed that SCU (45 mg/kg, iv) increased brain PKG activity and PKG protein level after CIR surgery. In conclusion, our findings suggested that SCU possesses the ability of protecting brain cells against CIR injury through vascular endothelium protection and PKG signal.

Isoforskolin, an adenylyl cyclase activator, attenuates cigarette smoke-induced COPD in rats.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by limited airflow due to pulmonary and alveolar abnormalities from exposure to cigarette smoke (CS). Current therapeutic drugs are limited and the development of novel treatments to prevent disease progression is challenging. Isoforskolin (ISOF) from the plant Coleus forskohlii is an effective activator of adenylyl cyclase (AC) isoforms. Previously we found ISOF could attenuate acute lung injury in animal models, while the effect of ISOF on COPD has not been elucidated. PURPOSE: In this study, we aimed to evaluate the efficacy of ISOF on COPD and reveal its potential mechanisms. METHODS: A rat model of COPD was established by long-term exposure to CS, then the rats were orally administered with ISOF (0.5, 1 and 2 mg/kg). The pulmonary function, lung morphology, inflammatory cells and cytokines in serum or bronchoalveolar lavage fluid (BALF) were evaluated. Transcriptomics, proteomics and network pharmacology analysis were utilized to identify potential mechanisms of ISOF. Droplet digital PCR was used to detect the mRNA expression of AC1-10 in donor lung tissues. AC activation was determined in recombinant human embryonic kidney 293 (HEK293) cells stably expressing human AC isoforms. In addition, ISOF caused trachea relaxation ex vivo were assessed in isolated trachea rings from guinea pigs. RESULTS: ISOF significantly ameliorated pathological damage of lung tissue and improved pulmonary function in COPD rats. ISOF treatment decreased the number of inflammatory cells in peripheral blood, and also the levels of pro-inflammatory cytokines in serum and BALF. Consistent with omics-based analyses, ISOF markedly downregulated the mTOR level in lung tissue. Flow cytometry analysis revealed that ISOF treatment reduced the ratio of Th17/Treg cells in peripheral blood. Furthermore, the expression levels of AC1 and AC2 are relatively higher than other AC isoforms in normal lung tissues, and ISOF could potently activate AC1 and AC2 in vitro and significantly relax isolated guinea pig trachea. CONCLUSION: Collectively, our studies suggest that ISOF exerts its anti-COPD effect by improving lung function, anti-inflammation and trachea relaxation, which may be related to AC activation, mTOR signaling and Th17/Treg balance.

Identification of candidate nasopharyngeal carcinoma serum biomarkers by cancer cell secretome and tissue transcriptome analysis: potential usage of cystatin A for predicting nodal stage and poor prognosis.

Nasopharyngeal carcinoma (NPC) is usually diagnosed at advanced clinical stages, resulting in poor outcomes. To discover serum biomarkers for improved NPC diagnosis and/or management, we simultaneously analyzed the NPC cell secretome and tissue transcriptome to identify candidate genes/proteins that are highly upregulated in NPC tissues and also secreted/released from NPC cells. Among the 30 candidates identified, 11 proteins were chosen for further validation using the serum samples from NPC patients and healthy controls, including cystatin A, cathepsin B, manganese superoxide dismutase and matrix metalloproteinase 2. The results showed that serum levels of all the four proteins were indeed higher in NPC patients versus healthy controls and that the use of a three-marker panel (cystatin A, manganese superoxide dismutase and matrix metalloproteinase 2) can contribute to a better NPC detection than each marker alone. In addition, a higher pretreated serum level of cystatin A was found to be associated with a higher nodal stage and poorer prognosis of NPC patients and cystatin A could modulate the migration and invasion of NPC cells in vitro. Altogether, our results indicate that analysis of both the cancer cell secretome and tissue transcriptome is a feasible strategy for efficient identification of novel NPC serum marker panel.

Hypoxia-induced lncRNA RP11-390F4.3 promotes epithelial-mesenchymal transition (EMT) and metastasis through upregulating EMT regulators.

Hypoxia-induced long noncoding RNAs (lncRNAs) have been shown to induce tumor metastasis. However, lncRNAs that are regulated by hypoxia/HIF-1α and subsequently control the expression of multiple epithelial-mesenchymal transition (EMT) regulators have not been identified. To identify such lncRNAs, analysis of RNA-sequencing datasets was performed. The lncRNA RP11-390F4.3 was shown to be induced by hypoxia and directly activated by HIF-1α. Overexpression of lncRNA RP11-390F4.3 induced EMT and metastasis. LncRNA RP11-390F4.3 was essential for hypoxia-induced EMT and metastasis. LncRNA RP11-390F4.3 overexpression induced the expression of multiple EMT regulators. This report demonstrates that LncRNA RP11-390F4.3 is induced by hypoxia/HIF-1α and is essential for hypoxia-induced EMT and metastasis via the activation of multiple EMT regulators.

Targeting interleukin-17 receptor B enhances gemcitabine sensitivity through downregulation of mucins in pancreatic cancer.

Pancreatic cancer is the fourth leading cause of death worldwide due to its poorest prognoses with a 7% 5-year survival rate. Eighty percent of pancreatic cancer patients relapse after chemotherapy and develop early metastasis and drug resistance. Resistance to nucleoside analog gemcitabine frequently used in first-line therapy is an urgent issue in pancreatic cancer treatment. Expression of mucin (MUC) glycoproteins has been shown to enhance chemoresistance via increased cell stemness. Here we show interlukine-17 receptor B (IL-17RB) expression is positively correlated with MUC1 and MUC4 expression in pancreatic cancer cells and tumor tissue. Moreover, IL-17RB transcriptionally up-regulates expression of MUC1 and MUC4 to enhance cancer stem-like properties and resistance to gemcitabine. These results suggest IL-17RB can be a potential target for pancreatic cancer therapy. Indeed, treatment with IL-17RB-neutralizing antibody has a synergistic effect in combination with gemcitabine for killing pancreatic cancer cells. Altogether, these findings provide feasible applications for IL-17RB-targeting therapy in pancreatic cancer treatment.