Circ6834 suppresses non-small cell lung cancer progression by destabilizing ANHAK and regulating miR-873-5p/TXNIP axis.

BACKGROUND: Circular RNAs (circRNAs) play important roles in cancer progression and metastasis. However, the expression profiles and biological roles of circRNAs in non-small cell lung cancer (NSCLC) remain unclear. METHODS: In this study, we identified a novel circRNA, hsa_circ_0006834 (termed circ6834), in NSCLC by RNA-seq and investigated the biological role of circ6834 in NSCLC progression in vitro and in vivo. Finally, the molecular mechanism of circ6834 was revealed by tagged RNA affinity purification (TRAP), western blot, RNA immunoprecipitation, dual luciferase reporter gene assays and rescue experiments. RESULTS: Our results showed that circ6834 was downregulated in NSCLC tumor tissues and cell lines. Circ6834 overexpression inhibited NSCLC cell growth and metastasis both in vitro and in vivo, while circ6834 knockdown had the opposite effect. We found that TGF-ß treatment decreased circ6834 expression, which was associated with the QKI reduction in NSCLC cells and circ6834 antagonized TGF-ß-induced EMT and metastasis in NSCLC cells. Mechanistically, circ6834 bound to AHNAK protein, a key regulator of TGF-ß/Smad signaling, and inhibited its stability by enhancing TRIM25-mediated ubiquitination and degradation. In addition, circ6834 acted as a miRNA sponge for miR-873-5p and upregulated TXNIP gene expression, which together inactivated the TGF-ß/Smad signaling pathway in NSCLC cells. CONCLUSION: In conclusion, circ6834 is a tumor-suppressive circRNA that inhibits NSCLC progression by forming a negative regulatory feedback loop with the TGF-ß/Smad signaling pathway and represents a novel therapeutic target for NSCLC.

Parkin-mediated ubiquitination inhibits BAK apoptotic activity by blocking its canonical hydrophobic groove.

BAK permeabilizes the mitochondrial outer membrane, causing apoptosis. This apoptotic activity of BAK is stimulated by binding prodeath activators within its canonical hydrophobic groove. Parkin, an E3 ubiquitin (Ub) ligase, can ubiquitinate BAK, which inhibits BAK apoptotic activity. However, the molecular mechanism underlying the inhibition of ubiquitination remains structurally uncharacterized. Here, we utilize truncated and soluble BAK to construct a mimetic of K113-ubiquitinated BAK (disulfide-linked UbG76C ~ BAKK113C) and further present its NMR-derived structure model. The classical L8-I44-H68-V70 hydrophobic patch of the conjugated Ub subunit binds within the canonical hydrophobic groove of BAK. This Ub occludes the binding of prodeath BID activators in the groove and impairs BID-triggered BAK activation and membrane permeabilization. Reduced interaction between Ub and BAK subunits allows BID to activate K113-ubiquitinated BAK. These mechanistic insights suggest a nonsignaling function of Ub in that it directly antagonizes stimuli targeting Ub-modified proteins rather than by recruiting downstream partners for cellular messaging.

Solid-state NMR structure determination of a membrane protein in E. coli cellular inner membrane.

Structure determination of membrane proteins in native cellular membranes is critical to precisely reveal their structures in physiological conditions. However, it remains challenging for solid-state nuclear magnetic resonance (ssNMR) due to the low sensitivity and high complexity of ssNMR spectra of cellular membranes. Here, we present the structure determination of aquaporin Z (AqpZ) by ssNMR in Escherichia coli inner membranes. To enhance the signal sensitivity of AqpZ, we optimized protein overexpression and removed outer membrane components. To suppress the interference of background proteins, we used a "dual-media" expression approach and antibiotic treatment. Using 1017 distance restraints obtained from two-dimensional 13C-13C spectra based on the complete chemical shift assignments, the 1.7-Å ssNMR structure of AqpZ is determined in E. coli inner membranes. This cellular ssNMR structure determination paves the way for analyzing the atomic structural details for membrane proteins in native cellular membranes.

LINC02159 promotes non-small cell lung cancer progression via ALYREF/YAP1 signaling.

Lung cancer is the leading cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have emerged as key regulators of cancer development and progression, and as promising biomarkers for the diagnosis and prognosis of cancer. In this study, we identified a new lncRNA (LINC02159) that was upregulated in the tumor tissues and serum of non-small cell lung cancer (NSCLC) patients. We demonstrated that knockdown of LINC02159 inhibited NSCLC cell proliferation, migration, and invasion, but induced cell apoptosis and cell cycle arrest in vitro and retarded tumor growth in vivo, while overexpression of LINC02159 led to the opposite effect. We discovered that LINC02159 was highly correlated with cancer growth and metastasis-related pathways by using transcriptomic analysis and that YAP1 was a potential target gene of LINC02159. Mechanistically, LINC02159 bound to the Aly/REF export factor (ALYREF) to enhance the stability of YAP1 messenger RNA (mRNA) via m5C modification, which led to the overexpression of YAP1 and the activation of the Hippo and ß-catenin signaling pathways in NSCLC cells. Rescue experiments showed that LINC01259 promoted NSCLC progression in a YAP1- and ALYREF-dependent manner. In conclusion, LINC02159 plays an oncogenic role in NSCLC progression by regulating ALYREF/YAP1 signaling, and it has the potential to be utilized as a diagnostic marker and therapeutic target for NSCLC.

IGF2BP3-EGFR-AKT axis promotes breast cancer MDA-MB-231 cell growth.

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an emerging prognostic indicator, and its elevated expression correlates with malignancy in a broad spectrum of cancers. However, its regulatory networks have not yet been reported. In this study, we identified the regulatory targets of IGF2BP3 in breast cancer MDA-MB-231 cells using RNA immunoprecipitation sequencing (RIP-seq) and high-throughput RNA-sequencing (RNA-seq). We discovered that these targets were enriched in the inflammatory response, endoplasmic reticulum stress, cell cycle, and cancer-related pathways, providing a new perspective for better understanding the functional mechanisms of IGF2BP3. Moreover, we identified that the epidermal growth factor receptor (EGFR), a downstream target, is regulated by IGF2BP3. IGF2BP3 binds to and protects EGFR mRNA from degradation and facilitates cell proliferation via the EGFR/AKT pathway in MDA-MB-231 cells. In addition, IGF2BP3 expression was robust and could not be altered by stimulation with EGF and anti-EGFR siRNA or EGFR signaling pathway inhibitors (gefitinib, LY294002 and SL-327). These results demonstrate that IGF2BP3, as a stubborn oncogene, promotes triple-negative breast cancer MDA-MB-231 cell proliferation by strengthening the role of the EGFR-AKT axis.

miR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

BACKGROUND: MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. METHODS: Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). RESULTS: The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. CONCLUSIONS: The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

MicroRNA-17 Family Targets RUNX3 to Increase Proliferation and Migration of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one common cancer in the world. Previous studies have shown that miR-17 family members are elevated in most tumors and promote tumor progression. However, there is no comprehensive analysis of the expression and functional mechanism of the microRNA-17 (miR-17) family in HCC. The aim of this study is to comprehensively analyze the function of the miR-17 family in HCC and the molecular mechanism of its role. Bioinfoimatics analysis of the miR-17 family expression profile and its relationship to clinical significance using The Cancer Genome Atlas (TCGA) database, and this result was confirmed using quantitative real-time polymerase chain reaction. miR-17 family members were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability and migration by cell count and wound healing assays. In addition, we using dual-luciferase assay and Western blot demonstrated the targeting relationship between the miRNA-17 family and RUNX3. These members of miR-17 family were highly expressed in HCC tissues, and the overexpression of the miR-17 family promoted the proliferation and migration of SMMC-7721 cells, whereas treatment with anti-miR17 inhibitors caused the opposite effects. Notably, we also found that inhibitors anti-each member of miR-17 can suppress the expression of the entire family member. In addition, they can bind to the 3' untranslated region of RUNX3 to regulate its expression at the translational level. Our results proved that miR-17 family has oncogenic characteristics, overexpression every member of the family contributed to HCC cell proliferation and migration by reducing the translation of RUNX3.

Emerging roles of tRNA-derived fragments in cancer.

tRNA-derived fragments (tRFs) are an emerging category of small non-coding RNAs that are generated from cleavage of mature tRNAs or tRNA precursors. The advance in high-throughput sequencing has contributed to the identification of increasing number of tRFs with critical functions in distinct physiological and pathophysiological processes. tRFs can regulate cell viability, differentiation, and homeostasis through multiple mechanisms and are thus considered as critical regulators of human diseases including cancer. In addition, increasing evidence suggest the extracellular tRFs may be utilized as promising diagnostic and prognostic biomarkers for cancer liquid biopsy. In this review, we focus on the biogenesis, classification and modification of tRFs, and summarize the multifaceted functions of tRFs with an emphasis on the current research status and perspectives of tRFs in cancer.

The cytoplasmic synthesis and coupled membrane translocation of eukaryotic polyphosphate by signal-activated VTC complex.

Inorganic polyphosphate (polyP) is an ancient energy metabolite and phosphate store that occurs ubiquitously in all organisms. The vacuolar transporter chaperone (VTC) complex integrates cytosolic polyP synthesis from ATP and polyP membrane translocation into the vacuolar lumen. In yeast and in other eukaryotes, polyP synthesis is regulated by inositol pyrophosphate (PP-InsP) nutrient messengers, directly sensed by the VTC complex. Here, we report the cryo-electron microscopy structure of signal-activated VTC complex at 3.0 Å resolution. Baker's yeast VTC subunits Vtc1, Vtc3, and Vtc4 assemble into a 3:1:1 complex. Fifteen trans-membrane helices form a novel membrane channel enabling the transport of newly synthesized polyP into the vacuolar lumen. PP-InsP binding orients the catalytic polymerase domain at the entrance of the trans-membrane channel, both activating the enzyme and coupling polyP synthesis and membrane translocation. Together with biochemical and cellular studies, our work provides mechanistic insights into the biogenesis of an ancient energy metabolite.

Insights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C.

Non-specific phospholipase C (NPC) hydrolyzes major membrane phospholipids to release diacylglycerol (DAG), a potent lipid-derived messenger regulating cell functions. Despite extensive studies on NPCs reveal their fundamental roles in plant growth and development, the mechanistic understanding of phospholipid-hydrolyzing by NPCs, remains largely unknown. Here we report the crystal structure of Arabidopsis NPC4 at a resolution of 2.1 Å. NPC4 is divided into a phosphoesterase domain (PD) and a C-terminal domain (CTD), and is structurally distinct from other characterized phospholipases. The previously uncharacterized CTD is indispensable for the full activity of NPC4. Mechanistically, CTD contributes NPC4 activity mainly via CTDα1-PD interaction, which ultimately stabilizes the catalytic pocket in PD. Together with a series of structure-guided biochemical studies, our work elucidates the structural basis and provides molecular mechanism of phospholipid hydrolysis by NPC4, and adds new insights into the members of phospholipase family.

RAB5C, a new mRNA binding target of HuR, regulates breast cancer cell proliferation.

The posttranscriptional control of gene expression mediated by RNA-binding proteins (RBPs) is essential to determine tumor cell fate. HuR is an RBP with increased expression in various cancer types. This study aimed to clarify the regulatory mechanism of HuR's contribution to breast cancer (BC) cell proliferation by inducing RAB5C expression. First, we analyzed the expression level of HuR and RAB5C in BC tissues and cell lines by immunohistochemistry, qRT-PCR, and western blot. Next, to further investigate the effect of HuR on RAB5C expression, we used short hairpin RNAs (shRNAs) to silence endogenous HuR expression in BC cell lines MCF7 and MDA-MB-231. The binding site of RAB5C mRNA and HuR was confirmed by RNA immunoprecipitation. Finally, the function of RAB5C was investigated using flow cytometry, colony formation, and MTT assays. We found that the expression of HuR and RAB5C was significantly upregulated in BC tissues and MCF-7 and MDA-MB231 cell lines. Importantly, RAB5C mRNA stability was increased through binding of HuR to its 3'UTR. Inhibition of HuR expression using shRNA decreased RAB5C mRNA, suggesting that HuR plays a role in regulating RAB5C expression level. In addition, suppression of RAB5C expression reduced BC cell growth. These results suggest RAB5C functions as an oncogene in BC cells, HuR promoted BC cell survival by facilitating RAB5C expression. Our findings suggest that HuR and RAB5C play important roles in BC cell survival.

Molecular Mechanisms of Mercury-Sensitive Aquaporins.

Aquaporins are transmembrane channels that allow for the passive permeation of water and other small molecules across biological membranes. Their channel activities are sensitive to mercury ions. Intriguingly, while most aquaporins are inhibited by mercury ions, several aquaporins are activated by mercury ions. The molecular basis of the opposing aquaporin regulation by mercury remains poorly understood. Herein, we investigated AqpZ inhibition and AQP6 activation upon binding of mercury ions using solid-state NMR (ssNMR) and molecular dynamics (MD) simulations. Based on the structure of the Hg-AqpZ complex constructed by MD simulations and ssNMR, we identified that the pore closure was caused by mercury-induced conformational changes of the key residue R189 in the selectivity filter region, while pore opening was caused by conformational changes of residues H181 and R196 in the selectivity filter region in AQP6. Both conformational changes were caused by the disruption of the H-bond network of R189/R196 by mercury. The molecular details provided a structural basis for mercury-mediated functional changes in aquaporins.

miR-3614-3p suppresses cell aggressiveness of human breast cancer by targeting AKT3 and HDAC1 expression.

Background: MicroRNAs (miRNAs) regulate various pathophysiological functions and pathobiological progression in various cancers. Our recent study reported that miR-3614-3p significantly suppressed the proliferation of breast cancer (BC) cells by downregulating its host gene TRIM25. However, other functional roles of miR-3614-3p migration and invasion in BC and its potential mechanisms are not clearly elucidated. Methods: In this study, we investigated miR-3614-3p regulation of AKT3 and HDAC1 expression in BC. miR-3614-3p and AKT3/HDAC1 mRNA expression levels were determined using quantitative real-time PCR (qRT-PCR) in MCF-7 and MDA-MB-231 BC cells. The effects of miR-3614-3p on migration and invasion were measured using wound healing and transwell migration assays. In BC cells, miR-3614-3p levels were remarkably low, and AKT3 and HDAC1 mRNA and protein levels were high as assessed by qRT-PCR and western blot. Finally, we investigated the role of AKT3/HDAC1 using silent interfering RNA (siRNA) and confirmed the targeting of AKT3 and HDAC1 3' UTR through miR-3614-3p using a luciferase reporter assay. Results: In the present study, we found that overexpression of miR-3614-3p markedly suppressed tumor cell invasion and migration independent of TRIM25, whereas other target genes, AKT3 and HDAC1, were involved. Moreover, we found that the resulting silencing of AKT3 and HDAC1 suppressed cell migration. Conclusions: miR-3614-3p is an anti-oncogene that can suppress BC cells by targeting AKT3 and HDAC1, revealing the potential role of miR-3614-3p in suppressing BC metastasis. Therefore, miR-3614 may act as a potential biomarker for BC prognosis.

Circular RNA EIF4G3 suppresses gastric cancer progression through inhibition of ß-catenin by promoting δ-catenin ubiquitin degradation and upregulating SIK1.

BACKGROUND: Increasing studies suggest that circular RNAs (circRNAs) are critical regulators of cancer development and progression. However, the biological roles and mechanisms of circRNAs in gastric cancer (GC) remain largely unknown. METHODS: We identified the differentially expressed circRNAs in GC by analyzing Gene Expression Omnibus (GEO) datasets. We explored the biological roles of circRNAs in GC by in vitro functional assays and in vivo animal studies. We performed tagged RNA affinity purification (TRAP), RNA immunoprecipitation (RIP), mass spectrometry (MS), RNA sequencing, luciferase reporter assays, and rescue experiments to investigate the mechanism of circRNAs in GC. RESULTS: Downregulated expression of circular RNA EIF4G3 (circEIF4G3; hsa_circ_0007991) was found in GC and was associated with poor clinical outcomes. Overexpression of circEIF4G3 suppressed GC growth and metastasis through the inhibition of ß-catenin signaling, whereas knockdown of circEIF4G3 showed the opposite effects. Mechanistic studies revealed that circEIF4G3 bound to δ-catenin protein to promote its TRIM25-mediated ubiquitin degradation and interacted with miR-4449 to upregulate SIK1 expression. CONCLUSION: Our findings uncovered a tumor suppressor function of circEIF4G3 in GC through the regulation of δ-catenin protein stability and miR-4449/SIK1 axis. CircEIF4G3 may act as a promising prognostic biomarker and therapeutic target for GC.

Solid-state NMR 13C and 15 N resonance assignments of Vibrio sp. SemiSWEET transporter in lipid bilayers.

The Sugar Will Eventually be Exported Transporter (SWEET) family is a new class of transporters that plays crucial roles in the cellular sugar transport process. Their bacterial homologs, known as SemiSWEETs, are among the smallest transporters and can be used as a prototype for studying the biological properties of sugar transporters. Here, a set of dipolar-based multidimensional solid-state NMR spectra were employed to investigate the structure of Vibrio sp. SemiSWEET (Vs-SemiSWEET) reconstituted in the native-like lipid bilayers. A nearly complete (88% of the amino acid residues) backbone and side-chain 13C and 15 N chemical shift assignments of Vs-SemiSWEET were obtained. The overall secondary structure of Vs-SemiSWEET predicted from the obtained 13C and 15 N chemical shifts is similar to that from X-ray crystallography, with some differences, reflecting the influence of the membrane environments to the structure of membrane proteins.

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy.

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.

Extensively sparse 13C labeling to simplify solid-state NMR 13C spectra of membrane proteins.

Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D 13C-13C spectra. Sparse labeling of 13C spins is an effective approach to simplify the 13C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate 13C-12C labeling, and demonstrate an enhanced dilution effect in 13C-13C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate 13C-12C labeling). The signal intensities of some other types of residues in 2D 13C-13C spectra are observed to be 20-50% weaker because of the unwanted reverse labeling. The extensively sparse 13C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D 13C-13C spectra of membrane proteins.