MLH1 Inhibits Metastatic Potential of Pancreatic Ductal Adenocarcinoma via Downregulation of GPRC5C.

DNA mismatch repair gene MutL homolog-1 (MLH1) has divergent effects in many cancers; however, its impact on the metastasis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, MLH1 stably overexpressed (OE) and knockdowned (KD) sublines were established. Wound healing and transwell assays were used to evaluate cell migration/invasion. In vivo metastasis was investigated in orthotopic implantation models (severe combined immunodeficiency mice). RT-qPCR and western blotting were adopted to show gene/protein expression. MLH1 downstream genes were screened by transcriptome sequencing. Tissue microarray-based immunohistochemistry was applied to determine protein expression in human specimens. In successfully generated sublines, OE cells presented weaker migration/invasion abilities, compared with controls, whereas in KD cells, these abilities were significantly stronger. The metastasis-inhibitory effect of MLH1 was also observed in mice. Mechanistically, G protein-coupled receptor, family C, group 5, member C (GPRC5C) was a key downstream gene of MLH1 in PDAC cells. Subsequently, transient GPRC5C silencing effectively inhibited cell migration/invasion and remarkably reversed the proinvasive effect of MLH1 knockdown in KD cells. In animal models and human PDAC tissues, tumoral GPRC5C expression, negatively associated with MLH1 expressions, was positively correlated with histologic grade, vessel invasion, and poor cancer-specific survival. In conclusion, MLH1 inhibits the metastatic potential of PDAC via downregulation of GPRC5C.

Construction of a Spatial-Confined Self-Stacking Catalytic Circuit for Rapid and Sensitive Imaging of Piwi-Interacting RNA in Living Cells.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that repress transposable elements to maintain genome integrity. The canonical catalytic hairpin assembly (CHA) circuit relies on random collisions of free-diffused reactant probes, which substantially slow down reaction efficiency and kinetics. Herein, we demonstrate the construction of a spatial-confined self-stacking catalytic circuit for rapid and sensitive imaging of piRNA in living cells based on intramolecular and intermolecular hybridization-accelerated CHA. We rationally design a 3WJ probe that not only accelerates the reaction kinetics by increasing the local concentration of reactant probes but also eliminates background signal leakage caused by cross-entanglement of preassembled probes. This strategy achieves high sensitivity and good specificity with shortened assay time. It can quantify intracellular piRNA expression at a single-cell level, discriminate piRNA expression in tissues of breast cancer patients and healthy persons, and in situ image piRNA in living cells, offering a new approach for early diagnosis and postoperative monitoring.

HK2 and LDHA upregulation mediate hexavalent chromium-induced carcinogenesis, cancer development and prognosis through miR-218 inhibition.

Hexavalent chromium [Cr(VI)] is one of the most common environmental contaminants due to its tremendous industrial applications, but its effects and mechanism remain to be investigated. Our previous studies showed that Cr(VI) exposure caused malignant transformation and tumorigenesis. This study showed that glycolytic proteins HK2 and LDHA levels were statistically significant changed in blood samples of Cr(VI)-exposed workers and in Cr-T cells compared to the control subjects and parental cells. HK2 and LDHA knockdown inhibited cell proliferation and angiogenesis, and higher HK2 and LDHA expression levels are associated with advanced stages and poor prognosis of lung cancer. We found that miR-218 levels were significantly decreased and miR-218 directly targeted HK2 and LDHA for inhibiting their expression. Overexpression of miR-218 inhibited glucose consumption and lactate production in Cr-T cells. Further study found that miR-218 inhibited tumor growth and angiogenesis by decreasing HK2 and LDHA expression in vivo. MiR-218 levels were negatively correlated with HK2 and LDHA expression levels and cancer development in human lung and other cancers. These results demonstrated that miR-218/HK2/LDHA pathway is vital for regulating Cr(VI)-induced carcinogenesis and human cancer development.

Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells.

Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3'-OH end. Subsequently, the TP is elongated by telomerase at the 3'-OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.

Construction of a Programmable Feedback Network with Continuously Activatable Molecular Beacon Fluorescence for One-Step Quantification of Long Noncoding RNAs in Clinical Breast Tissues.

Long noncoding RNAs (lncRNAs) are key regulators in numerous pathological and physiological processes, and their aberrant expression is implicated in many diseases. Herein, we develop a programmable feedback network with continuously activatable molecular beacon (MB) fluorescence for one-step quantification of mammalian-metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) in clinical breast tissues. We introduce a functional MB with three domains, including a substrate for lncRNA MALAT1 recognition, a template for strand displacement amplification (SDA), and a reporter for signal output with FAM fluorescence being quenched by BHQ1. When MALAT1 is present, it recognizes and unfolds the MB, leading to the recovery of FAM fluorescence. Once the MB is opened, multiple rounds of SDA reaction are automatically initiated by recruiting primer, KF DNA polymerase, and Nt.BbvCI nicking enzyme, inducing the opening of more MBs and the dissociation of more FAM/BHQ1 pairs. Consequently, a feedback network is constructed through multicycle cascade SDA, achieving the exponential accumulation of fluorescence signals for accurate quantification of MALAT1. In this assay, only two oligonucleotides (i.e., MB and primer) are involved for the establishment of a feedback amplification network, greatly simplifying the design of the reaction system. Moreover, this assay requires only one step to realize the isothermal exponential amplification for real-time monitoring of MALAT1 with attomolar sensitivity. This assay displays single-base mismatch selectivity with high anti-interference capability, and it can further quantify endogenous MALAT1 at the single-cell level and differentiate MALAT1 expression between breast cancer patient tissues and healthy person tissues.

RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues.

N6-methyladenosine (m6A) is an ubiquitous post-transcriptional modification catalyzed by METTL3/14 complex in eukaryotic mRNAs. The abnormal METTL3/14 complex activity affects multiple steps of RNA metabolism and may induce various diseases. Herein, we demonstrate the RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues. METTL3/14 complex can catalyze the methylation of RNA probe to prevent it from being cleaved by MazF. The intact RNA probe is recognized by the magnetic bead (MB)-capture probe conjugates to induce duplex-specific nuclease (DSN)-assisted cyclic digestion, exposing numerous shorter ssDNAs with 3'-OH end. The shorter ssDNAs on the MB surface can act as the primers to initiate terminal deoxynucleotidyl transferase (TdT)-enhanced tyramide signal amplification (TSA), forming the Cy5 fluorescence-encoded nanostructures. After magnetic separation, the Cy5 fluorescence-encoded nanostructures are digested by DNase I to release abundant Cy5 fluorophores that can be simply quantified by fluorescence measurement. This assay achieves good specificity and high sensitivity with a detection limit of 58.8 aM, and it can screen METTL3/14 complex inhibitors and quantify METTL3/14 complex activity at the single-cell level. Furthermore, this assay can differentiate the METTL3/14 complex level in breast cancer patient tissues and healthy volunteer tissues.

Elevated NOX4 promotes tumorigenesis and acquired EGFR-TKIs resistance via enhancing IL-8/PD-L1 signaling in NSCLC.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used for human non-small-cell lung cancer (NSCLC) treatment. However, acquired resistance to EGFR-TKIs is the major barrier of treatment success, and new resistance mechanism remains to be elucidated. In this study, we found that elevated NADPH oxidase 4 (NOX4) expression was associated with acquired EGFR-TKIs resistance. Gefitinib is the first-generation FDA-approved EGFR-TKI, and osimertinib is the third-generation FDA-approved EGFR-TKI. We demonstrated that NOX4 knockdown in the EGFR-TKI resistant cells enabled the cells to become sensitive to gefitinib and osimertinib treatment, while forced expression of NOX4 in the sensitive parental cells was sufficient to induce resistance to gefitinib and osimertinib in the cells. To elucidate the mechanism of NOX4 upregulation in increasing TKIs resistance, we found that knockdown of NOX4 significantly down-regulated the expression of transcription factor YY1. YY1 bound directly to the promoter region of IL-8 to transcriptionally activate IL-8 expression. Interestingly, knockdown of NOX4 and IL-8 decreased programmed death ligand 1 (PD-L1) expression, which provide new insight on TKIs resistance and immune escape. We found that patients with higher NOX4 and IL-8 expression levels showed a shorter survival time compared to those with lower NOX4 and IL-8 expression levels in response to the anti-PD-L1 therapy. Knockdown of NOX4, YY1 or IL-8 alone inhibited angiogenesis and tumor growth. Furthermore, the combination of NOX4 inhibitor GKT137831 and gefitinib had synergistic effect to inhibit cell proliferation and tumor growth and to increase cellular apoptosis. These findings demonstrated that NOX4 and YY1 were essential for mediating the acquired EGFR-TKIs resistance. IL-8 and PD-L1 are two downstream targets of NOX4 to regulate TKIs resistance and immunotherapy. These molecules may be used as potential new biomarkers and therapeutic targets for overcoming TKIs resistance in the future.

Dysregulation of MiR-199a/IL8 pathway in chronic Cr (VI)-induced tumor growth and angiogenesis.

Hexavalent chromium [Cr(VI)] is a well-known environmental carcinogen. Recent studies revealed that chronic exposure of human bronchial epithelial cells (BEAS-2B, B2B) to Cr(VI) activated several signaling pathways and induced cell malignant transformation and tumor growth. However, new mechanisms of Cr(VI) in inducing carcinogenesis remains to be elucidated. This study showed that miR-199a expression levels were significantly lower in Cr(VI)-transformed Cr-T cells. By using the mouse model, the expression levels of miR-199a were significantly decreased in blood samples and lung tissues of mice intranasally exposed to Cr(VI) for 12 weeks compared to the solvent exposure control. Overexpression of miR-199a inhibited tube formation and angiogenesis. C-X-C motif chemokine ligand 8 (CXCL8, IL8) levels were significantly higher in blood samples of Cr (VI)-exposed workers compared to normal workers, and forced expression of miR-199a in the cells suppressed IL8 levels. miR-199a suppression induced expression of hypoxia-inducible factor 1α (HIF-1α) and nuclear factor kappa B (NF-κB) p65 to increase IL8 expression. With animal experiment, the results showed that miR-199a overexpression inhibited tumor growth and angiogenesis through inhibiting IL8, HIF-1α and NF-κB p65 expression in vivo. These results show that miR-199a/IL8 pathway is important in Cr(VI)-induced carcinogenesis and angiogenesis.

microRNA-497 slows esophageal cancer development and reverses chemotherapy resistance through its target QKI.

Esophageal cancer (EC) is considered one of the most lethal cancers in human beings, and multiple miRNAs have been investigated to be involved in EC development by targeting their target genes. However, the function and related mechanism of miRNA-497 on EC tumorigenesis remain uncertain. This study first demonstrated that the expression levels of miR-497 in esophageal cancer specimens and cells were down-regulated. Forced expression of miR-497 inhibited cell proliferation, tube formation and migration in EC cells. To further investigate the potential molecular mechanism of miR-497 suppression in regulating EC, we found that miR-497 directly binds to the 3'-untranslational region of QKI, miR-497 overexpression suppressed QKI expression. We further found that overexpression of miR-497 enhanced the effect of chemotherapy in EC cell lines, and prevented the tumor growth of EC in vivo. Our findings indicated that miR-497 suppression increased QKI expression and therapeutic resistance of esophageal cancer, which is likely to be a biomarker of EC progression and potential therapeutic target.

RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues.

Fat mass and obesity-associated enzyme (FTO) can dynamically regulate N6-methyladenosine modification, and it is engaged in various cellular functions. Herein, we demonstrate the RNA demethylation-driven functional supramolecular structure for label-free detection of m6A modification eraser FTO in human breast tissues. The presence of FTO catalyzes the removal of methyl group in m6A, causing the cleavage of demethylated DNA by DpnII and the release of DNA primer. The resultant DNA primer hybridizes with circular template to initiate isothermal rolling circle amplification (RCA), producing abundant long ssDNA polymers with repeating sequences of G-quadruplex. Subsequently, N-methylmesoporphyrin IX (NMM) is selectively embedded into G-quadruplex DNAzyme to form a supramolecular NMM-G-quadruplex structure for the generation of an amplified fluorescence signal. Benefiting from high selectivity of DpnII toward demethylated DNA, high amplification efficiency of RCA, and high signal-to-noise ratio of G-quadruplex-NMM system, this assay can sensitively detect FTO with a limit of detection (LOD) of 3.10 × 10-16 M, screen RNA demethylase inhibitors, quantify FTO activity in cancer cells, and discriminate FTO activity between breast cancer patient tissues and healthy person tissues. Importantly, this assay can be homogeneously conducted in a label-free manner, with great potential in RNA demethylases-related pathogenesis research and clinical diagnostics.

Target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 activity.

We demonstrate that target-activated cascade transcription amplification lights up RNA aptamers for label-free detection of metalloproteinase-2 (MMP-2) activity with zero background. This assay exhibits good specificity and high sensitivity with a limit of detection (LOD) of 0.6 fM. Moreover, it can analyze enzyme kinetic parameters, screen inhibitors, and accurately quantify MMP-2 in cancer cells and clinical serums.

Five glycosylated phenolic derivatives from the bark of Ilex rotunda Thunb. and their anti-inflammatory activities.

Five new glycosylated phenolic derivatives, rotundosides A-E (1-5), and three known glycosides (6-8) were isolated from the 95% alcohol extract of the bark of Ilex rotunda. Their structures were elucidated by extensive spectroscopic analysis and comparison with the literature data. All new compounds possessed a [5-O-(E)-caffeoyl]-ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranosyl group. The anti-inflammatory properties of all isolated compounds were evaluated using a modified nitric oxide (NO) production in lipopolysaccharide (LPS)-induced leukemia cells in mouse macrophage (RAW264.7) method. Compound 8, dracunculifoside H, showed significant anti-inflammatory activity in vitro.

METTL3-mediated N6-methyladenosine modification and HDAC5/YY1 promote IFFO1 downregulation in tumor development and chemo-resistance.

Ovarian cancer (OC) is a malignant tumor that seriously threatens women's health. Due to the difficulty of early diagnosis, most patients exhibit advanced disease or peritoneal metastasis at diagnosis. We discovered that IFFO1 is a novel tumor suppressor, but its role in tumorigenesis, development and chemoresistance is unknown. In this study, IFFO1 levels were downregulated across cancers, leading to the acceleration of tumor development, metastasis and/or cisplatin resistance. Overexpression of IFFO1 inhibited the translocation of ß-catenin to the nucleus and decreased tumor metastasis and cisplatin resistance. Furthermore, we demonstrated that IFFO1 was regulated at both the transcriptional and posttranscriptional levels. At the transcriptional level, the recruitment of HDAC5 inhibited IFFO1 expression, which is mediated by the transcription factor YY1, and the METTL3/YTHDF2 axis regulated the mRNA stability of IFFO1 in an m6A-dependent manner. Mice injected with IFFO1-overexpressing cells had lower ascites volumes and tumor weights throughout the peritoneal cavity than those injected with parental cells expressing the vector control. In conclusion, we demonstrated that IFFO1 is a novel tumor suppressor that inhibits tumor metastasis and reverses drug resistance in ovarian cancer. IFFO1 was downregulated at both the transcriptional level and posttranscriptional level by histone deacetylase and RNA methylation, respectively, and the IFFO1 signaling pathway was identified as a potential therapeutic target for cancer.

[Study on the molecular mechanism of circ_0000326 regulating the proliferation, invasion and migration of oral squamous cell carcinoma HSC3 cells by targeting miR-567].

PURPOSE: To explore the molecular mechanism of circ_0000326 regulating proliferation, invasion and migration of oral squamous cell carcinoma HSC3 cells. METHODS: Cancerous tissue and adjacent tissue specimens of 45 patients with oral squamous cell carcinoma (OSCC) admitted to the Second People's Hospital of Hefei from March 2020 to June 2021 were collected. qRT-PCR was used to detect the expression levels of circ_0000326 and miR-567. CCK-8, plate clone formation test, scratch test and Transwell test were used to detect cell proliferation, clone formation, migration and invasion. Dual luciferase reporter experiment was used to detect the targeting relationship between circ_0000326 and miR-567. Western blot was used to quantify E-cadherin and N-cadherin protein. SPSS 21.0 software package was used for statistical analysis. RESULTS: circ_0000326 expression was 4.01±0.29 in OSCC and 1.00±0.13 in paracancerous tissues, while miR-567 expression was 0.28±0.03 and 1.00±0.10, respectively, with significant differences. Compared with the si-NC group, the cell viability and the number of cell clones in the si-circ_0000326 group were significantly decreased(P＜0.05). Compared with the si-NC group, the number of invasive cells and scratch healing rate in the si-circ_0000326 group were significantly decreased (P＜0.05), the expression level of E-cadherin protein was significantly increased (P＜0.05), and the expression level of N-cadherin protein was significantly decreased(P＜0.05). Additionally, circ_0000326 targeted miR-567. miR-567 expression was 1.00±0.00 in pcDNA group, 0.44±0.04 in pcDNA-circ_0000326 group, 0.99±0.06 in si-NC group, and 2.92±0.25 in si-circ_0000326 group with significant differences. Compared with miR-NC group, the cell viability, scratch healing rate, the number of cell clones and the number of invasive cells of miR-567 group were decreased, while E-cadherin protein level was increased(P＜0.05). Compared with si-circ_0000326+anti-miR-NC group, the cell viability, scratch healing rate, N-cadherin protein level, the number of cell clones and the number of invasive cells of si-circ_0000326+anti-miR-567 group were increased(P＜0.05), while E-cadherin protein level was decreased(P＜0.05). CONCLUSIONS: Interference with the expression of circ_0000326 could reduce the ability of OSCC cell proliferation, colony formation, migration and invasion by promoting the expression of miR-567.

Enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of cytosine deaminase activity in cancer cells.

APOBEC3A (A3A) is a cytidine deaminase with critical roles in molecular diagnostics. Herein, we demonstrate the enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of A3A activity in cancer cells. Target A3A can convert cytosine (C) in substrate probe to uracil (U), and then the template binds with substrate probe to form a dsDNA containing U/A base pairs. Uracil DNA glycosylase (UDG) excises the U base to produce an apurinic/apyrimidinic (AP) site that can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1) to obtain the substrate fragment with 3'-OH end. Subsequently, the substrate fragment initiates cyclic enzymatic repairing amplification (ERA), releasing trigger-1 and trigger-2. The resultant trigger-1 can act as the primer to induce multiple cycles of cyclic ERA, producing numerous trigger-1 and trigger-2. The hybridization of trigger-2 with signal probe forms the dsDNA duplexes with an AP site, inducing the cyclic cleavage of signal probes by APE1 to release abundant Cy5 molecules from the AuNPs. Released Cy5 molecules can be easily quantified by single-molecule imaging. This nanosensor allows for specific and sensitive detection of A3A activity with a detection limit of 0.855 aM, and it can further measure kinetic parameters, screen inhibitors, and quantify endogenous A3A activity at the single-cell level, with prospect application in disease diagnostics and therapy.

Construction of a phos-tag-directed self-assembled fluorescent magnetobiosensor for the simultaneous detection of multiple protein kinases.

Protein kinases play important roles in regulating various cellular processes and may function as potential diagnostic and therapeutic targets for various diseases including cancers. Herein, we construct a phos-tag-directed self-assembled fluorescent magnetobiosensor to simultaneously detect multiple protein kinases with good selectivity and high sensitivity. In the presence of protein kinases (i.e., PKA and Akt1), their substrate peptides (i.e., a FITC-labeled substrate peptide and a Cy5-labeled substrate peptide) are phosphorylated, and are then specifically recognized and captured by a biotinylated phos-tag to generate biotinylated substrate peptides for the assembly of magnetic bead (MB)-peptides-FITC/Cy5 nanostructures. After magnetic separation, the phosphorylated substrate peptides are disassembled from the MB-peptides-FITC/Cy5 nanostructures using deionized water at 80 °C, releasing FITC and Cy5 molecules. The released FITC and Cy5 molecules are detected by steady-state fluorescence measurements, with FITC indicating PKA and Cy5 indicating Akt1. This magnetobiosensor only involves one phos-tag without the requirement of radiolabeling, antibody screening, carboxypeptidase Y (CPY) cleavage, and cumbersome chemical/enzyme reactions. The introduction of magnetic separation can effectively eliminate the interference from complex real samples, generating an extremely low background signal. Moreover, this magnetobiosensor can accurately measure cellular protein kinase activities and screen inhibitors, with great potential for kinase-related biomedical research and therapeutic applications.

Corrigendum: Regulation of microrna-497-targeting AKT2 influences tumor growth and chemoresistance to cisplatin in lung cancer.

[This corrects the article DOI: 10.3389/fcell.2020.00840.].

18ß-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells.

BACKGROUND: Gastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. AIM: To explore the mechanism by which 18ß-glycyrrhetinic acid (18ß-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells. METHODS: Cell counting kit-8 assay was used to detect the effects of 18ß-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18ß-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18ß-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells. RESULTS: The results indicated that 18ß-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18ß-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18ß-GRA intervention compared with control. We found that the intervention of 18ß-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18ß-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18ß-GRA concentration. CONCLUSION: 18ß-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

Programmable Ligation-Transcription Circuit-Driven Cascade Amplification Machinery for Multiple Long Noncoding RNAs Detection in Lung Tissues.

The measurement of long noncoding RNAs (lncRNAs) is essential to diagnosis and treatment of various diseases such as cancers. Herein, we develop a simple method to simultaneously detect multiple lncRNAs using programmable ligation-transcription circuit-driven cascade amplification and single-molecule counting. The presence of targets lncRNA HOTAIR and lncRNA MALAT1 activates the ligation-transcription circuits to produce two corresponding functional RNAs. The functional RNAs then cyclically initiate the digestion of signal probes by duplex-specific nuclease to liberate Cy5 and Cy3 molecules. After magnetic separation, the liberated Cy5 and Cy3 molecules are measured by single-molecule counting. In this assay, a single lncRNA can activate ligation-transcription circuit to generate abundant functional RNAs, endowing this assay with high sensitivity. Integration of single-molecule counting ensures the high sensitivity. This method shows extremely high sensitivity with a limit of detection (LOD) of 0.043 aM for HOX gene antisense intergenic RNA (lncRNA HOTAIR) and 0.126 aM for mammalian metastasis-related lung adenocarcinoma transcript 1 (lncRNA MALAT1). Importantly, this method enables simultaneous measurement of multiple endogenous lncRNAs at the single-cell level, and it may discriminate the expressions of various lncRNA in lung tumor tissues of nonsmall cell lung cancer (NSCLC) patients and their corresponding healthy adjacent tissues, offering a promising platform for clinical diagnosis and biomedical research.

A label-free and self-circulated fluorescent biosensor for sensitive detection of ten-eleven translocation 1 in cancer cells.

We develop for the first time a label-free and self-circulated fluorescent biosensor for sensitive detection of ten-eleven translocation 1 (TET1) activity in cancer cells. This biosensor displays good specificity and high sensitivity without the need for any antibodies/exogenous primers and it can accurately quantify TET1 activity at the single-cell level. Moreover, it can be employed for the measurement of kinetic parameters and screening of inhibitors.