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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.

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.

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.

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.

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.

PurposeBone destruction and pain caused by cancer is one of the most devastating complications of cancer patients with bone metastases, and it seriously affects the quality of patients’ life. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule with increased expression in a variety of tumors. This study focused to clarify the specific function of EMMPRIN in bone metastasis of breast cancer.Materials and Methods Adenovirus with shRNA-EMMPRIN was transfected into MRMT-1 rat breast carcinoma cells, and the MRMT-1 cells with different expression levels of EMMPRIN were implanted into the bone marrow cavity of rat tibia. Next, the effect of down-regulation of EMMPRIN was evaluated as follows: bone damage was detected by X-ray radiological and tartrate-resistant acid phosphatase staining; the tumor burden was evaluated by hematoxylin and eosin staining; the test of pain-related behaviors was assessed used the bilateral paw withdrawal mechanical threshold; and the levels of secretory factors in tumor conditioned medium were determined by using enzyme-linked immunosorbent assay.ResultsWe found that down-regulation of EMMPRIN in tumor cells can simultaneously reduce tumor burden, relieve cancer-induced bone destruction and pain. ConclusionMaterials and Methods EMMPRIN is expected to be a therapeutic target for relieving bone metastasis of breast cancer and alleviating cancerinduced bone destruction and pain. The method of targeting EMMPRIN may be a promising strategy for the treatment of cancer in the future.

The bacterial mechanosensitive channel of large conductance (MscL) functions as a pressure-relief safety valve to prevent cells from lysing during sudden hypo-osmotic shock. The hydrophobic gate of MscL in the closed state forms a barrier to the permeation of ions and water molecules and can be switched to the open state for releasing solutions and ions. Currently, the gate-constituting residues and the functional role of these residues in the hydrophobic gate of MscL remain elusive and controversial. Here, we employ magic angle spinning solid-state nuclear magnetic resonance (ssNMR) techniques and functional assays to investigate the hydrophobic gate of MscL from Methanosarcina acetivorans (Ma-MscL) in lipid bilayers. We obtain chemical shift assignments of ∼70% residues of Ma-MscL and predict its 3D structure. Based on the structural characterization, we identify that the residues I21-T30 in the transmembrane helix 1 constitute the hydrophobic gate by detecting water distributions in the transmembrane pore using ssNMR H/D exchange and water-edited experiments. By using ssNMR structural characterization and functional assays, we reveal that the packing of aromatic rings of F23 in each subunit of Ma-MscL is critical to the hydrophobic gate, and hydrophilic substitutions of the other functionally important residues A22 and G26 modulate channel gating by attenuating hydrophobicity of constriction of F23.

Breast cancer brain metastasis (BCBM) is a devastating disease. Radiation therapy remains the mainstay for treatment of this disease. Unfortunately, its efficacy is limited by the dose that can be safely applied. One promising approach to overcoming this limitation is to sensitize BCBMs to radiation by inhibiting their ability to repair DNA damage. Here, we report a DNA repair suppressor, leucine-rich repeat-containing protein 31 (LRRC31), that was identified through a genome-wide CRISPR screen. We found that overexpression of LRRC31 suppresses DNA repair and sensitizes BCBMs to radiation. Mechanistically, LRRC31 interacts with Ku70/Ku80 and the ataxia telangiectasia mutated and RAD3-related (ATR) at the protein level, resulting in inhibition of DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) recruitment and activation, and disruption of the MutS homologue 2 (MSH2)-ATR module. We demonstrate that targeted delivery of the LRRC31 gene via nanoparticles improves the survival of tumour-bearing mice after irradiation. Collectively, our study suggests LRRC31 as a major DNA repair suppressor that can be targeted for cancer radiosensitizing therapy.

The adrenergic system contributes to the stress‐induced onset and progression of cancer. Adrenergic fibers are the primary source of norepinephrine (NE). The underlying mechanisms involved in NE‐induced colon cancer remain to be understood. In this study, we describe the function and regulatory network of NE in the progression of colon cancer. We demonstrate that NE‐induced phosphorylation of cAMP response element‐binding protein 1 (CREB1) promotes proliferation, migration, and invasion of human colon cancer cells. The downstream effector of NE, CREB1, bound to the promoter of miR‐373 and transcriptionally activated its expression. miR‐373 expression was shown to be necessary for NE‐induced cell proliferation, invasion, and tumor growth. We confirmed that proliferation and invasion of colon cancer cells are regulated in vitro and in vivo by miR‐373 through targeting of the tumor suppressors TIMP2 and APC. Our data suggest that NE promotes colon cancer cell proliferation and metastasis by activating the CREB1–miR‐373 axis. The study of this novel signaling axis may provide mechanistic insights into the neural regulation of colon cancer and help in the design of future clinical studies on stress biology in colorectal cancer., We propose a NE–CREB1–miR‐373 signaling axis in human colon cancer cells. NE promotes the progression of colon cancer via CREB1 activity‐dependent transcription, enhancing the expression of oncogenic miR‐373 and reducing the expression of the miR‐373 targets APC and TIMP2. This novel NE‐CREB1‐miR‐373 signaling axis may potentially serve as a new therapeutic target for colorectal cancer.

BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.

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

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 geneIn this study, we investigated miR-3614-3p regulation ofIn the present study, we found that overexpression of miR-3614-3p markedly suppressed tumor cell invasion and migration independent ofmiR-3614-3p is an anti-oncogene that can suppress BC cells by targeting

Background The cross-talk between RNA binding proteins (RBPs) and microRNAs (miRNAs) in the regulation of gene expression is a complex process. Here, we describe a new mode of regulation of TRIM25 expression mediated by an antagonistic interplay between IGF2BP3 and miR-3614-3p. Methods The expression level of TRIM25, IGF2BP3, pri-miR-3614 and miR-3614-3p in breast cancer (BC) tissues, non-tumor tissues and BC cell lines were detected by qRT-PCR, Western blot and Immunohistochemistry (IHC). Binding of miR-3614-3p and IGF2BP3 to TRIM25 RNA was verified using luciferase activation assays, RNA immunoprecipitation (RIP) and biotin pull-down assays. In vitro and in vivo loss- and gain-of-function studies were performed to reveal the effects and related mechanism of IGF2BP3-miR-3614-3p-TRIM25 axis in in breast cancer cells proliferation. Findings We found that an intragenic miRNA-3614-3p inhibits the expression of its host gene TRIM25 by binding to its 3′- untranslated region (UTR). Interestingly, IGF2BP3 can competitively occupy this binding site and inhibit miRNA-3614 maturation, thereby protecting TRIM25 mRNA from miR-3614-mediated degradation. The overexpression of miR-3614-3p dramatically inhibited breast cancer cell growth through the downregulation of TRIM25. Furthermore, the silencing of IGF2BP3 reduced TRIM25 expression, suppressed cell proliferation, and exhibited a synergistic effect with miR-3614-3p overexpression. Interpretation Collectively, these results demonstrate that control of TRIM25 RNA by an interplay between IGF2BP3 and miR-3614-3p represents a mechanism for breast cancer cell proliferation. Fund The scientific research and sharing platform construction project of Shaanxi Province, Opening Project of Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, China Postdoctoral Science Foundation and The National Natural Science Foundation of China.

Purpose: MicroRNAs (miRNA) have been reported in the regulation of various pathobiological progression in cancer. Our recent study has reported that miR-3614-3p significantly suppressed the proliferation of Breast Cancer (BC) cells through the downregulation its host gene TRIM25. However, the other functional role of miR3614-3p migration and invasion in BC and its mechanism have not been investigated thoroughly. Materials and methods: The MDA-MB-231 and MCF-7 BC cell lines were purchased. The cell line expression levels of miR-3614- 3p and AKT3/HDAC1 were determined by quantitative real-time PCR (qPCR). The wound healing assay and transwell migration assay were determined. We next measured protein levels of AKT3/HDAC1 by Western blot. Finally, we investigated the role of AKT3/HDAC1 using siRNA; and confirmed the targeting of 3’UTR of AKT3 and HDAC1 through miR-3614-3p using a luciferase reporter assay. Results: In the present research, we studied that overexpression of miR-3614-3p markedly suppressed tumor cell invasion and migration independent TRIM25, whereas through regulated another targets AKT3 and HDAC1 expression. Notably, TRIM25 is also a target gene of miR-3614 which bind to pri-miR-3614 caused TRIM25 silence. Conclusion: miR-3614-3p is an anti-oncogene that can suppress breast cancer cell aggressiveness by targeting AKT3 and HDAC1, which reveals the potential values of miR-3614-3p for suppression of metastasis of BC. Keywords: Breast cancer; miR-3614; AKT3; HDAC1.

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

We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion protein in KM12 colorectal carcinoma cells and inhibited downstream PLCγ1 signaling at sub-nanomolar concentrations. Both degraders also degraded human wild-type TRKA with similar potency. Interestingly, both degraders, especially 6, showed selectivity for the degradation of endogenous TPM3-TRKA over ectopically expressed ATP/GTP binding protein-like 4 (AGBL4)-TRKB or ETS variant transcription factor 6 (ETV6)-TRKC fusion proteins in KM12 cells. Global proteomic profiling assays demonstrated that 5 is highly selective for the intended target. TPM3-TRKA protein degradation induced by 5 and 6 was further confirmed to be mediated through cereblon and the ubiquitin-proteasome system. Compared with the parental TRK kinase inhibitor, both degraders exhibited higher potency for inhibiting growth of KM12 cells. Moreover, both 5 and 6 showed good plasma exposure levels in mice. Therefore, 5 and 6 are valuable chemical tool compounds for investigating the in vivo function of TRK fusion during tumorigenesis. Our study also paves the way for pharmacological degradation of TRK.

Background Increasing evidence indicates that circular RNAs (circRNAs) are dysregulated in human cancers. The biological roles of circRNAs in gastric cancer (GC) have not been well‐characterized. Methods The GEO database was used to analyze circRNA expression profile in GC. The expression level of target circRNA in tumor tissues and adjacent non‐tumor tissues was detected by reverse transcription‐quantitative PCR. Gene transfection was used to manipulate the expression of circRNAs. The biological roles of circRNAs in cell proliferation, migration, and invasion were determined by cell counting, colony formation, transwell migration, Matrigel invasion, and mouse xenograft tumor assays. The interactions between circRNAs and miRNAs were verified by RNA immunoprecipitation and luciferase reporter assays. Results We found that circHN1 was upregulated in GC tissues and cell lines compared to adjacent non‐tumor tissues and normal gastric epithelial cells. Additionally, circHN1 silencing significantly promoted GC cell growth, colony formation, migration, and invasion, whereas circHN1 overexpression had the opposite effects. CircHN1 overexpression also suppressed gastric cancer growth in the mouse xenograft tumor model. CircHN1 was mainly localized in the cytoplasm of GC cells and could bind to AGO2. MiR‐1248 and miR‐375 were predicted to interact with circHN1 by bioinformatic analyses. MiR‐1248 and miR‐375 overexpression inhibited the activity of the circHN1 luciferase reporter. Conclusion CircHN1 is aberrantly expressed in GC and affects the proliferation and migration of gastric cancer cells by acting as miRNA sponge.

Long non-coding RNAs (LncRNAs) have been suggested as important regulators of cancer development and progression in non-small cell lung cancer (NSCLC). Nevertheless, the biological roles and clinical significance of lncRNA UFC1 in NSCLC remain unclear. We detected the expression of UFC1 in tumor tissues, serum, and serum exosomes of NSCLC patients by qRT-PCR. Gene overexpression or silencing were used to examine the biological roles of UFC1 in NSCLC. RNA immunoprecipitation and ChIP assays were performed to evaluate the interaction between UFC1 and enhancer of zeste homolog 2 (EZH2) and the binding of EZH2 to PTEN gene promoter. Rescue study was used to access the importance of PTEN regulation by UFC1 in NSCLC progression. UFC1 expression was upregulated in tumor tissues, serum, and serum exosomes of NSCLC patients and high level of UFC1 was associated with tumor infiltration. UFC1 knockdown inhibited NSCLC cell proliferation, migration and invasion while promoted cell cycle arrest and apoptosis. UFC1 overexpression led to the opposite effects. Mechanistically, UFC1 bound to EZH2 and mediated its accumulation at the promoter region of PTEN gene, resulting in the trimethylation of H3K27 and the inhibition of PTEN expression. UFC1 knockdown inhibited NSCLC growth in mouse xenograft tumor models while the simultaneous depletion of PTEN reversed this effect. NSCLC cells derived exosomes could promote NSCLC cell proliferation, migration and invasion through the transfer of UFC1. Moreover, Exosome-transmitted UFC1 promotes NSCLC progression by inhibiting PTEN expression via EZH2-mediated epigenetic silencing. Exosome-mediated transmit of UFC1 may represent a new mechanism for NSCLC progression and provide a potential marker for NSCLC diagnosis.

Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. Here, we present a well-defined homo-trimeric structure of DgkA in phospholipid bilayers determined by magic angle spinning solid-state NMR (ssNMR) spectroscopy, using an approach combining intra-, inter-molecular paramagnetic relaxation enhancement (PRE)-derived distance restraints and CS-Rosetta calculations. The DgkA structure determined in lipid bilayers is different from the solution NMR structure. In addition, although ssNMR structure of DgkA shows a global folding similar to that determined by X-ray, these two structures differ in monomeric symmetry and dynamics. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins., Jianping Li et al. present the homo-trimeric structure of the small integral membrane protein diacylglycerol kinase (DgkA) in phospholipid bilayers determined by magic angle spinning solid-state NMR spectroscopy. They compare the structure with structures solved by solution NMR and X-ray crystallography and provide insights into the influence of membrane mimetic environments on membrane proteins.

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.

By integrating imaging and drug-delivery in a single system, fluorescent nano-multifunctional imaging platforms can offer simultaneous diagnosis and therapy to diseases like cancer. However, the synthesis of such system involves a tedious, time-consuming, and multi-step process. Herein we report a facile method based on simple ultrasonication to synthesize highly cross-linked, monodispersed fluorescent polyphosphazene nanoparticles from hexachlorocyclotriphosphazene (HCCP) and dichlorofluorescein (FD). Various functional groups (folic acid, PEG-NH2, and methylene blue) can be “fastened” in situ onto the poly(cyclotriphosphazene-co-dichlorofluorescein) (PCTPDF) nanoparticles to expand its application as nano-multifunctional platform. All the nanoparticles were characterized spectrophotometrically, and morphology was established by the images obtained from scanning electron microscope (SEM). The synthesized multifunctional nanoparticles exhibited low toxicity and penetrated through the cytomembranes of human colon cancer (HCT 116) cells. When applied to in vivo tumor imaging using biologically engineered mouse model, methylene blue functionalized (PCTPDF@MB) nanoparticles exhibited excellent photodynamic activity and imaging ability. Thus, PCTPDF nanoplatform based on multi-functional fluorescent nanoparticles might offer an efficient solution to new age theranostics. Apart from diagnostics application, PCTPDF, as a nanoplatform, could also be utilized to achieve more comprehensive application in modern analytic chemistry.

In this letter, we propose a robust heteronuclear dipolar recoupling method for proteins in magic-angle spinning (MAS) solid-state NMR. This method is as simple, robust and efficient as the well-known TEDOR in the aspect of magnetization transfer between 15N and 13C. Deriving from our recent band-selective dual back-to-back pulses (DBP) (Zhang et al., 2016), this method uses new phase-cycling schemes to realize broadband DBP (Bro-DBP). For broadband 15N-13C magnetization transfer (simultaneous 15N→13C' and 15N→13Cα), Bro-DBP has almost the same 15N→13Cα efficiency while offers 30-40% enhancement on 15N→13C' transfer, compared to TEDOR. Besides, Bro-DBP can also be used as a carbonyl (13C')-selected method, whose 15N→13C' efficiency is up to 1.7 times that of TEDOR and is also higher than that of band-selective DBP. The performance of Bro-DBP is demonstrated on the N-formyl-[U-13C,15N]-Met-Leu-Phe-OH (fMLF) peptide and the U-13C, 15N labeled β1 immunoglobulin binding domain of protein G (GB1) microcrystalline protein. Since Bro-DBP is as robust, simple and efficient as TEDOR, we believe it is very useful for protein studies in MAS solid-state NMR.

Numerous studies have demonstrated that microRNAs (miRNAs) play a key role in human carcinogenesis and metastasis. For example, miR‑299‑5p has previously been revealed to be dysregulated in several human cancers. However, the biological function of miR‑299‑5p in breast cancer remains unclear. The present study demonstrated that miR‑299‑5p was downregulated in breast cancer tissues and cell lines. The restoration of miR‑299‑5p expression suppressed cell migration and invasion, whereas inhibition of miR‑299‑5p promoted cell migration and invasion. In addition, in vivo studies demonstrated that miR‑299‑5p overexpression was able to inhibit tumour metastasis in nude mice. Mechanistically, through bioinformatics analysis and a dual‑luciferase assay, it was confirmed that miR‑299‑5p directly targets serine/threonine kinase 39 (STK39). Silencing STK39 inhibited cell metastasis and suppressed epithelial‑mesenchymal transition markers and matrix metalloproteinase expression, whereas restoration of STK39 expression was able to reverse miR‑299‑5p‑inhibited cell migration and invasion. Collectively, the results of the present study demonstrated that miR‑299‑5p supresses breast cancer cell migration and invasion by targeting STK39. These findings may provide novel insights into miR‑299‑5p and its potential diagnostic and therapeutic benefits in breast cancer.

Background: CircRNAs play important roles in cancer development and progression and have the potential to serve as cancer biomarkers. The aim of this study was to investigate the role of circular RNA CCDC66 (circCCDC66) in gastric cancer and to reveal the underlying mechanisms. Methods: The expression of circCCDC66 in GC tissues and cell lines was examined by qRT-PCR. The correlation between circCCDC66 expression level and clinicopathological characteristics was analyzed. The biological roles of circCCDC66 in GC cell apoptosis, proliferation, migration and invasion were determined by flow cytometry, cell counting, cell colony formation, wound healing, transwell migration and matrigel invasion assays. The role of circCCDC66 in GC growth was further confirmed by mouse xenograft tumor model. Western blot and qRT-PCR were used to explore the effects of circCCDC66 on epithelial-mesenchymal transition (EMT)-related gene and protein expression. Results: CircCCDC66 expression was elevated in both GC tissues and cell lines compared to adjacent normal tissues and normal gastric epithelial cell line. The upregulation of circCCDC66 in GC tissues was related to tumor stage and lymphatic metastasis. CircCCDC66 knockdown significantly inhibited GC cell proliferation, migration and invasion and induced cell apoptosis in GC cells. On the contrary, circCCDC66 overexpression had the opposite effects. In addition, circCCDC66 knockdown suppressed the tumorigenesis of GC cells in nude mice. Furthermore, circCCDC66 knockdown inhibited the activation of c-Myc and TGF-β signaling pathways and reversed EMT in GC cells. c-Myc and TGF-β interference blocked circCCDC66-mediated promotion of gastric cancer cell proliferation, migration and invasion. Conclusion: CircCCDC66 promotes GC growth and metastasis by activating c-Myc and TGF-β signaling pathways, suggesting that it may serve as a potential biomarker for GC.

New therapeutic strategies are needed for colorectal cancer (CRC) treatment. MicroRNAs are involved in cancer‑pertinent cellular processes, including chemoresistance. As miR‑302a is an embryonic stem cell‑specific microRNA, studies on miR‑302a have focused on its role in human stem cells. Studies analyzing miR‑302 function in cancer are limited. In this study, we used two human colon cancer cell lines, HCT116 and HT29, and evaluated the influence of miR‑302a on 5‑fluorouracil (5‑FU)‑induced cell death and viability inhibition. With bioinformatics tools, we hypothesized that insulin‑like growth factor‑1 receptor (IGF‑1R) is a novel target of miR‑302a, which we confirmed using a luciferase reporter assay and immunoblotting. Then, we designed siRNA against IGF‑1R and found that si‑IGF‑1R resembled the effect of miR‑302a on 5‑FU treatment. Both miR‑302a and si‑IGF‑1R inhibited Akt signaling. In conclusion, miR‑302a targeted IGF‑1R and enhanced 5‑FU‑induced cell death and viability inhibition in human colon cancer cells. Targeting miR‑302a may offer new therapeutic interventions in CRC.

We propose a robust band-selective heteronuclear 15N-13C recoupling method using dual back-to-back (BABA) pulses (DBP). It contains four 90° pulses in each rotor period and corresponding phase cycling on each channel (13C and 15N). DBP aims at rapid band-selective heteronuclear magnetization transfer between 15N and 13Cα/13C′, whose efficiency is close to that of the well-known SPECIFIC CP in membrane proteins with relatively short relaxation time in rotating frame (T1ρ). Compared to SPECIFIC CP, DBP is very simple to set up and highly robust to RF variations. Thus, it can reduce the efforts in experimental optimization, especially for low-sensitive samples, and is very suitable for long-time or quantitative experiments. The efficacy of DBP is demonstrated by the E. coli diacylglycerol kinase (DAGK) proteoliposome. We anticipate that DBP would be useful for (segments of) membrane proteins that undergo the μs-ms timescale motions in magic-angle spinning (MAS) solid-state NMR.