M6A RNA Methylation-Mediated Dysregulation of AGAP2-AS1 Promotes Trastuzumab Resistance of Breast Cancer.

INTRODUCTION: Trastuzumab is commonly used to treat human epidermal growth factor receptor-2-positive (HER2+) breast cancer, but its efficacy is often limited by chemotherapy resistance. Recent studies have indicated that long non-coding RNAs (lncRNAs) play important roles in tumor progression and response to therapy. However, the regulatory mechanisms associating lncRNAs and trastuzumab resistance remain unknown. METHODS: Quantitative polymerase chain reaction was performed to detect the expression of related genes. Western blot and immunofluorescence assays were used to evaluate protein expression levels. A series of gain- or loss-of-function assays confirmed the function of AGAP2-AS1 in trastuzumab resistance, both in vitro and in vivo. RNA immunoprecipitation and pull-down analyses were conducted to verify the interaction between METTL3/YTHDF2 and lncRNA AGAP2-AS1. RESULTS: AGAP2-AS1 was upregulated in trastuzumab-resistant cells and SKBR-3R-generated xenografts in nude mice. Silencing AGAP2-AS1 significantly decreased trastuzumab-induced cytotoxicity both in vitro and in vivo. Furthermore, m6A methylation of AGAP2-AS1 was reduced in trastuzumab-resistant cells compared to that in parental cells. In addition, METTL3 increased m6A methylation of AGAP2-AS1, which finally induced the suppressed AGAP2-AS1 expression. Moreover, YTHDF2 was essential for METTL3-mediated m6A methylation of AGAP2-AS1. Functionally, AGAP2-AS1 regulated trastuzumab resistance by inducing autophagy and increasing ATG5 expression. CONCLUSION: we demonstrated that METTL3/YTHDF2-mediated m6A methylation increased the expression of AGAP2-AS1, which could promote trastuzumab resistance in breast cancer. AGAP2-AS1 regulates trastuzumab resistance by inducing autophagy. Therefore, AGAP2-AS1 may be a promising predictive biomarker and therapeutic target in patients with breast cancer.

ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade.

Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance mechanisms. Using an ICB-insensitive tumor model, here we discover cisplatin enhances the anti-tumor effect of PD-L1 blockade and upregulates the expression of Ariadne RBR E3 ubiquitin-protein ligase 1 (ARIH1) in tumors. Arih1 overexpression promotes cytotoxic T cell infiltration, inhibits tumor growth, and potentiates PD-L1 blockade. ARIH1 mediates ubiquitination and degradation of DNA-PKcs to trigger activation of the STING pathway, which is blocked by the phospho-mimetic mutant T68E/S213D of cGAS protein. Using a high-throughput drug screen, we further identify that ACY738, less cytotoxic than cisplatin, effectively upregulates ARIH1 and activates STING signaling, sensitizing tumors to PD-L1 blockade. Our findings delineate a mechanism that tumors mediate ICB resistance through the loss of ARIH1 and ARIH1-DNA-PKcs-STING signaling and indicate that activating ARIH1 is an effective strategy to improve the efficacy of cancer immunotherapy.

The construction and analysis of a prognostic assessment model based on P53-related multi-genes in breast carcinoma.

BACKGROUND: Breast cancer ranks second in female tumor mortality, with an estimation of 2 million new cases diagnosed each year worldwide. METHODS: In our current study, we screened 13 genes highly distributed on the P53 phenotype which were significantly expressed and had a strong correlation with survival in the Cancer Genome Atlas breast cancer dataset. Least absolute shrinkage and selection operator Cox regression was conducted to construct the risk assessment model. Based on bioinformatics and statistical methods, we confirmed the credibility and validity of the model by training set and testing set. RESULTS: The result of comparing the other two previous hypoxia models was also satisfying. We also verified the model on one of the Gene Expression Omnibus datasets-GSE20685. Using clinical data from patients in the Cancer Genome Atlas, we acknowledged the risk score as an independent influence on breast cancer survival prognosis, and strong relevance was suggested between risk signature and age, lymphatic metastasis, tumor size and clinical stage by performing univariate and multivariate analysis. Immunology analysis demonstrated that the macrophages subset was positively associated with a risk score and other immune cell types had a negative effect with the risk score increases. The risk score was also emerging as a valuable prognostic factor for the prediction of chemotherapy drug curative effect because Gemcitabine, vinorelbine, paclitaxel and cisplatin known as a generic drug for breast cancer had more pleasing sensitivity in high-scored patients than low-scored patients. CONCLUSION: The P53-related risk assessment model is promising to be a potential predictor for the prognosis of patients with breast cancer and a powerful guide for the selection of therapeutic strategies.

Novel LncRNA AK023507 inhibits cell metastasis and proliferation in Papillary Thyroid Cancer through ß-catenin/Wnt Signaling Pathway.

INTRODUCTION: Papillary Thyroid Cancer (PTC) represents a commonly encountered type of thyroid malignancy whose occurrence and development is influenced by long non-coding RNA (LncRNA). A novel lncRNA (LncRNA AK023507), known to have tumor suppressive functions, was shown to prevent breast cancer cells from proliferating and metastasizing, but its mechanism in PTC is unclear. METHODS: Using PTC tissues and cell lines, the expression of LncRNA AK023507 was investigated by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). The effects of knockdown or overexpression of LncRNA AK023507 on cell growth and movement were investigated through various cell experiments in vitro. The presence of important functional proteins was determined by Western blotting, with the recovery experiment used for verification. RESULTS: LncRNA AK023507 was found to have low expression in both the PTC cell lines and tissue samples. Knockdown of LncRNA AK023507 in PTC cells significantly promoted cell proliferation, migration, and invasion, while overexpression of LncRNA AK023507 resulted in the opposite effects. Furthermore, LncRNA AK023507 could regulate the expression of ß-catenin/Wnt signaling pathway as confirmed by recovery experiment. CONCLUSION: By acting through the ß-catenin/Wnt signaling pathway, LncRNA AK023507 prevented PTC cells from proliferating and metastasizing. These novel findings indicate that LncRNA AK023507 could be of prognostic and diagnostic value as a potential biomarker of PTC.

The utility of sentinel Lymph node biopsy in the lateral neck in papillary thyroid carcinoma.

Background: Regional lymph node metastases (LNMs) are very common in papillary thyroid carcinoma (PTC) and associate with locoregional recurrence. The appropriate management of cervical lymph nodes is very important. Therefore, this study evaluated the application of sentinel lymph node biopsy (SLNB) in the lateral neck in PTC patients. Methods: This prospective study was conducted from 1 November 2015 to 31 December 2017 and recruited 78 PTC patients treated with SLNB in the lateral neck and prophylactic lateral neck dissection (compartments II-IV) followed by thyroidectomy or lobectomy and central neck dissection. Results: There were 78 PTC patients enrolled and sentinel lymph nodes (SLNs) were detected among 77 patients. A total of 30 patients were diagnosed with SLN metastases (SLNMs). The remaining 47 patients were pathologically negative of SLN, whereas 4 patients were found with metastases in the non-SLN samples. The detection rate, sensitivity, specificity, and accuracy rate of SLNB in the lateral neck were 98.7%, 87.1%, 98.7%, and 93.6%, respectively. However, the values varied greatly in each specific compartment of the lateral neck, and all of them were no more than 80%. These 34 PTC patients diagnosed with lateral compartment LNM (LLNM) were more likely to be younger (41.38 vs. 48.95 years old, p = 0.002) and exhibit extrathyroidal extension (56.8% vs. 31.7%, p = 0.026) and central compartment LNM (66.7% vs. 12.1%, p < 0.001). Tumors located in the upper third of the thyroid lobe also had a significantly higher probability of LLNM compared with those in middle or inferior location (66.7% vs. 35.3% vs. 34.8%, p = 0.044). At last, age (OR=0.912, p = 0.026), tumor location (upper vs inferior, OR=17.478, p = 0.011), and central compartment LNM (OR=25.364, p < 0.001) were independently predictive of LLNM. Conclusions: SLNB can help surgeons to identify some PTC patients who may benefit from therapeutic lateral neck dissection and protect some patients from prophylactic lateral neck dissection. However, it cannot accurately indicate specific lateral compartment-oriented neck dissection. Meanwhile, LLNM is more likely to occur in PTC patients with younger age or upper pole tumors or central compartment LNM.

Effects of Tamoxifen vs. Toremifene on fatty liver development and lipid profiles in breast Cancer.

BACKGROUND: Tamoxifen (TAM) and Toremifene (TOR), two kinds of selective estrogen receptor modulators (SERMs), have equal efficacy in breast cancer patients. However, TAM has been proved to affect serum lipid profiles and cause fatty liver disease. The study aimed to compare the effects of TAM and TOR on fatty liver development and lipid profiles. METHODS: This study performed a retrospective analysis of 308 SERMs-treated early breast cancer patients who were matched 1:1 based on propensity scores. The follow-up period was 3 years. The primary outcomes were fatty liver detected by ultrasonography or computed tomography (CT), variation in fibrosis indexes, and serum lipid profiles change. RESULTS: The cumulative incidence rate of new-onset fatty liver was higher in the TAM group than in the TOR group (113.2 vs. 67.2 per 1000 person-years, p < 0.001), and more severe fatty livers occurred in the TAM group (25.5 vs. 7.5 per 1000 person-years, p = 0.003). According to the Kaplan-Meier curves, TAM significantly increased the risk of new-onset fatty liver (25.97% vs. 17.53%, p = 0.0243) and the severe fatty liver (5.84% vs. 1.95%, p = 0.0429). TOR decreased the risk of new-onset fatty liver by 45% (hazard ratio = 0.55, p = 0.020) and showed lower fibrotic burden, independent of obesity, lipid, and liver enzyme levels. TOR increased triglycerides less than TAM, and TOR increased high-density lipoprotein cholesterol, while TAM did the opposite. No significant differences in total cholesterol and low-density lipoprotein cholesterol are observed between the two groups. CONCLUSIONS: TAM treatment is significantly associated with more severe fatty liver disease and liver fibrosis, while TOR is associated with an overall improvement in lipid profiles, which supports continuous monitoring of liver imaging and serum lipid levels during SERM treatment.

Long Non-Coding RNA NLIPMT as a Tumor Suppressor and Inhibitor of Cell Proliferation and Metastasis in Papillary Thyroid Carcinoma.

INTRODUCTION: Gene expression association studies of tumor samples have uncovered several long non-coding RNAs (lncRNAs) closely related to various types of cancer. Several lncRNAs have been reported to play essential roles in the progression of papillary thyroid carcinoma (PTC). Novel lncRNA inhibiting proliferation and metastasis (lnc-NLIPMT) is a known regulator of mammary cell proliferation and motility, but its involvement in PTC is unclear. MATERIALS AND METHODS: We investigated the role of lnc-NLIPMT in PTC by quantitative real-time polymerase chain reaction (qRT-PCR) on various PTC tissue samples and cell lines. We assessed the effects of overexpression or knockdown of lnc-NLIPMT on the proliferation, migration, and invasion of PTC cells using CCK-8, cell clone formation, and transwell assays. Changes in the expression of N-cadherin and vimentin were detected by immunoblotting. RESULTS: Our results revealed a downregulation of the expression of lnc-NLIPMT in PTC and a negative correlation between lnc-NLIPMT expression and tumor size (P=0.006). Overexpression of lnc-NLIPMT in TPC-1 and B-CPAP cells significantly suppressed cell proliferation, migration, and invasion, while lnc-NLIPMT knockdown had the opposite effect. In addition, lnc-NLIPMT played an important role in the regulation of the expression of N-cadherin and vimentin. CONCLUSION: lnc-NLIPMT inhibits cell proliferation and metastasis of PTC cells and is a potential diagnostic and prognostic biomarker in PTC.

Overexpression of novel lncRNA NLIPMT inhibits metastasis by reducing phosphorylated glycogen synthase kinase 3ß in breast cancer.

Long noncoding RNAs (lncRNAs) are considered as regulators of gene expression in cancers. However, cancer profiling has little focused on noncoding genes. Here, we reported that RP11-115N4.1 (here renamed novel lncRNA inhibiting proliferation and metastasis [NLIPMT]) was downregulated in breast cancer tissues. Ectopic expression of NLIPMT inhibited mammary cell proliferation, motility in vitro. Moreover, lnc-NLIPMT reduced the growth of implanted MDA-MB-231 cells in vivo. Mechanistically, glycogen synthase kinase 3ß (GSK3ß) was identified as an effector protein regulated by lnc-NLIPMT. Inhibition of GSK3ß activity restored NLIPMT-induced inhibition of proliferation and motility in breast cancer cells. These data reveal that lnc-NLIPMT functions as a driver of breast cancer progression and might serve as a potential target for antimetastatic therapies.

Oblique Colloidal Lithography for the Fabrication of Nonconcentric Features.

Herein, we describe the development of oblique colloidal lithography (OCL) and establish a systematic patterning strategy for creating libraries of nanosized nonconcentric plasmonic structures. This strategy combines OCL, capillary force lithography, and several wet and ion etching steps. Hexagonal arrays of nonconcentric gold features were created on glass substrates with highly controllable geometric parameters. The size, geometry, and eccentricity of the gold features could be independently tuned by controlling the experimental conditions. Gaps within surface elements could be shrunk to as small as 30 nm, while the total patterned area was about l cm2. The goal was to devise a method that offers a high degree of control over the resolution and morphology of asymmetric structures without the need to resort to electron beam lithography. This technique also enabled the development of numerous surface patterns through the stepwise fabrication of separate elements. Complex features, including dots-surrounded nonconcentric targets, nonconcentric hexagram-disks, and nonconcentric annular aperture arrays, were demonstrated, and their optical properties were characterized. Indeed, spectroscopic studies and FDTD simulations demonstrated that Fano resonances could readily be generated by the nonconcentric gold features. Consequently, our patterning strategy should enable the high-throughput investigation of plasmonic coupling and Fano resonances as a function of the physical parameters of the elements within the nanopattern array.

Stepwise molding, etching, and imprinting to form libraries of nanopatterned substrates.

Herein, we describe a novel colloidal lithographic strategy for the stepwise patterning of planar substrates with numerous complex and unique designs. In conjunction with colloidal self-assembly, imprint molding, and capillary force lithography, reactive ion etching was used to create complex libraries of nanoscale features. This combinatorial strategy affords the ability to develop an exponentially increasing number of two-dimensional nanoscale patterns with each sequential step in the process. Specifically, dots, triangles, circles, and lines could be assembled on the surface separately and in combination with each other. Numerous architectures are obtained for the first time with high uniformity and reproducibility. These hexagonal arrays were made from polystyrene and gold features, whereby each surface element could be tuned from the micrometer size scale down to line widths of ~35 nm. The patterned area could be 1 cm(2) or even larger. The techniques described herein can be combined with further steps to make even larger libraries. Moreover, these polymer and metal features may prove useful in optical, sensing, and electronic applications.

Fabrication of elliptical nanorings with highly tunable and multiple plasmonic resonances.

Herein, a new and facile patterning method is demonstrated for the scalable fabrication of gold elliptical rings (ERs) in a controlled manner over large areas. In this method, well-ordered hexagonally arrayed polystyrene (PS) rings, fabricated by colloidal lithography, were used as masters to generate poly(dimethylsiloxane) (PDMS) stamps with circular apertures. The stamps were then stretched and utilized as molds for creating elliptical PS rings by a capillary filling process. Through subsequent reactive ion etching and chemical wet-etching, the elliptical PS rings could be readily transferred into an underlying gold film, leading to the formation of gold ERs. Since the aspect ratio (AR) of the elliptical PS rings could be controlled by varying the applied strain during the capillary filling process, gold ERs with different ARs could be fabricated in a scalable manner. The optical properties of the gold ERs were characterized by UV-vis/NIR and IR extinction measurements. The ERs exhibited only odd modes of polarization-dependent plasmonic resonances at normal incidence. The experiments and corresponding theoretical studies illustrated that all resonant modes could be tuned across a broad spectral range from the visible to the mid infrared (550-4700 nm) by simply varying the AR of the ERs. Moreover, the experimental data were confirmed by COMSOL simulations.

Deflected capillary force lithography.

Herein we introduce a novel strategy based on capillary force lithography (CFL) to fabricate asymmetric polymeric ring structures by applying both shear and nomal forces to a poly(dimethylsiloxane) stamp. The mechanism for the formation of asymmetric rings is caused by the deflection of cylindrical PDMS pillars due to the shear load, which is therefore termed deflected CFL (dCFL). The asymmetric polymeric rings could be readily transferred to an underlying gold layer to generate split ring structures with tunable opening angles. Asymmetric structures based upon trigular and square-shaped pillars were also fabricated. These elements were formed into periodic arrays over surface areas as large as 1 cm(2) and may have optical and electromagnetic applications.

Generation of contact-printing based poly(ethylene glycol) gradient surfaces with micrometer-sized steps.

A surface with a density gradient of poly(ethylene glycol) (PEG) is an attractive substrate for combinatorial studies of biological phenomena. In this study, the generation of discrete step-wise density gradients of PEG utilizing a contact-printing approach is reported. The step-wise gradient template is achieved by contact-printing n-octadecyltrichlorosilane (OTS) to a glass from a hemispherical elastomeric stamp when the stamp is brought into contact with the substrate, and then step-wisely increasing the contact area as the corresponding contact-printing time for the step decreases. A PEG-silane is then used to backfill the unoccupied spaces of the contact printed OTS gradient to generate the OTS-PEG density gradient. Various characterizations, including water contact angle measurement, lateral force microscopy, and X-ray photoelectron spectroscopy, are conducted and confirmed that the surface coverage of OTS increases (or the coverage of PEG decreases) with the increase of contact-printing time of OTS. The step-wise gradient is illustrated by adsorption of a bovine serum albumin labeled with fluorescein isothiocyanate and subsequent attachment of fibroblasts. The amounts of protein adsorption and cellular attachment increase with the decrease of the surface coverage of PEG.

Porous polymer films templated by marangoni flow-induced water droplet arrays.

In this article, we report the development of a novel, simple, and cost-effective method for fabricating porous polymer films with controllable interpore distances, pore sizes, and arrangements using water droplets induced by Marangoni flow as templates. First, a spread-thin ethanol film on a partially water-wettable substrate is exposed to a humid airflow, facilitating the evaporation and recession of the ethanol film. Meanwhile, water in the airflow condenses on the ethanol film and accumulates near the receding contact line, which induces the formation of water fingers at the receding contact line and, finally, ordered arrays of water droplets after detachment. The formation of the hexagonal and square arrays of water droplets is due to the pinning and sliding of the water fingers on the silicone oxide (SiOx) and silicon (Si) substrates, respectively. By varying the thickness of the ethanol film spread on the Si substrate, the sliding velocity of water fingers can be tuned, subsequently leading to the fabrication of other arrangements. The interdroplet distance and droplet size can be dependently controlled by tuning the humidity of the airflow. The ordered arrays of water droplets on the substrate are then utilized to fabricate porous polymer films by dip-coating the substrate with a polystyrene solution. Films with hexagonal and square (and other arrangements) arrays of pores are fabricated on the silicon oxide (SiOx) and silicon (Si) substrates, respectively. The pore size can also be independently tuned by further condensation or evaporation of formed water droplets, leading to porous polymer films with both close- and non-close-packed arrays of pores. The ordered porous polymer films can be further used as templates for fabricating metal post patterns.

Dewetting of polystyrene thin films on poly(ethylene glycol)-modified surfaces as a simple approach for patterning proteins.

A simple technique for patterning proteins utilizing dewetted polystyrene (PS) droplets is demonstrated. A polystyrene thin film was spin coated on a poly(ethylene glycol) (PEG) silane-modified surface. As the PS film dewets from the surface, upon annealing, to form droplets, the PEG-silane-modified surface is exposed, which retains its capability to resist protein adsorption, and the PS droplets allow the selective adsorption of proteins. In contrast to the undewetted flat PS film, the droplet surface had a greater amount of adsorbed proteins. Atomic force microscopy scans reveal that the roughness of the droplet surface is higher, and a multilayer of proteins results on the droplet surface. Moreover, micro- and nanoscale droplet patterns can easily be achieved by tuning the thickness of PS thin films. Because dewetting approaches for generating ordered dewetting droplets have been successfully generated by others, those approaches could be easily combined with this technique to fabricate ordered protein patterns.

Marangoni flow-induced self-assembly of hexagonal and stripelike nanoparticle patterns.

We have developed a simple Marangoni flow-induced method for self-assembling nanoparticles (NPs) into both hexagonal and stripelike patterns. First, a NPs/ethanol suspension was spread on a slightly nonwettable and a wettable silicon oxide substrate. The Marangoni flow, induced by simultaneous evaporation of ethanol and condensation of water, leads to the formation of the corresponding hexagonal distributed circular NP rings and dotted stripes. The inter-ring spacing and ring size of the hexagonal patterns can be tuned by varying the relative humidity of the N2 stream blown over the slightly nonwettable substrate. Hexagonal patterns of circular NP patches can also be fabricated by lowering the evaporation of the condensed water droplets. On the wettable substrate, complex patterns result when the humidity of the N2 stream changes.

NaI-catalyzed highly regioselective ring-opening [1 + 2] cycloaddition reaction of cyclopropenes with imines: highly stereoselective synthesis of cis-vinylic aziridines.

cis-Vinylic aziridines were prepared highly stereoselectively via a NaI-catalyzed regioselective ring-opening [1 + 2] cycloaddition reaction of cyclopropenes with imines.

An X- (X= I, Br)-triggered ring-opening coupling reaction of cyclopropenes with organic halides.

Polyfunctionalized (E)-alk-1-enyl halides 3 were efficiently synthesized in high yields via a novel regio- and stereoselective X- (X = I or Br)-triggered ring-opening coupling reaction of cyclopropenes 1 with organic halides 2.