Engrailed 2 serves as a master regulator of the super-enhancer in the TNC gene locus in non-small cell lung cancer.

Engrailed 2 (EN2) is a homeodomain-containing protein that is dysregulated in many types of cancer. However, the role of EN2 in non-small cell lung cancer (NSCLC) and the mechanism underlying its biological function are largely unclear. Here, we showed that EN2 played an oncogenic function in NSCLC and greatly enhanced the malignant phenotype of NSCLC cells. Meanwhile, EN2 was able to boost the expression of a well-studied oncogenic Tenascin-C (TNC) gene, which in turn activated the AKT signaling pathway. Interestingly, we found that EN2 directly bound to the super-enhancer (SE) region in the TNC locus. The histone marker H3K27ac was also enriched in the region, indicating the activation of the SE. Treatment of the cells with JQ1, an inhibitor of SE activity, abrogated the effect of EN2 on the expression of TNC and phosphorylation of AKT-Ser473. Collectively, our work unveils a novel mode of EN2 function, in which EN2 governs the SE in the TNC locus, consequently activating the oncogenic TNC-AKT axis in NSCLC.

A novel nomogram for predicting the prolonged length of stay in post-anesthesia care unit after elective operation.

BACKGROUND: Prolonged length of stay in post-anesthesia care unit (PLOS in PACU) is a combination of risk factors and complications that can compromise quality of care and operating room efficiency. Our study aimed to develop a nomogram to predict PLOS in PACU of patients undergoing elective surgery. METHODS: Data from 24017 patients were collected. Least absolute shrinkage and selection operator (LASSO) was used to screen variables. A logistic regression model was built on variables determined by a combined method of forward selection and backward elimination. Nomogram was designed with the model. The nomogram performance was evaluated with the area under the receiver operating characteristic curve (AUC) for discrimination, calibration plot for consistency between predictions and actuality, and decision curve analysis (DCA) for clinical application value. RESULTS: A nomogram was established based on the selected ten variables, including age, BMI < 21 kg/m2, American society of Anesthesiologists Physical Status (ASA), surgery type, chill, delirium, pain, naloxone, operation duration and blood transfusion. The C-index value was 0.773 [95% confidence interval (CI) = 0.765 - 0.781] in the development set and 0.757 (95% CI = 0.744-0.770) in the validation set. The AUC was > 0.75 for the prediction of PLOS in PACU. The calibration curves revealed high consistencies between the predicted and actual probability. The DCA showed that if the threshold probability is over 10% , using the models to predict PLOS in PACU and implement intervention adds more benefit. CONCLUSIONS: This study presented a nomogram to facilitate individualized prediction of PLOS in PACU for patients undergoing elective surgery.

Efficacy and safety of anlotinib plus XELOX regimen as first-line therapy for mCRC: a single-arm, multicenter, phase II study (ALTER-C-001).

Background: Anlotinib showed encouraging anti-tumor activity in metastatic colorectal cancer (mCRC). This study was designed to assess the efficacy and safety of anlotinib plus XELOX as first-line therapy in mCRC patients. Materials and Methods: Eligible patients aged ≥18 with mCRC were enrolled in this multicenter, single-arm, phase II, exploratory study. Patients received at least 6 cycles of anlotinib, oxaliplatin, and capecitabine as initial therapy. Subsequently, patients received anlotinib monotherapy as maintenance therapy until tumor progression or intolerable toxicity. The primary endpoint was progression-free survival (PFS). Results: Thirty-one patients were included between December 2019 and March 2022. The median follow-up was 17.5 (95% CI, 3.0-17.5) months. The median PFS was 8.3 (95% CI, 6.3-10.0) months, with 6- and 12-month PFS rates of 82.3% (95% CI, 59.2%-93.0%) and 18.9% (95% CI, 4.8%-40.1%), respectively. Fifteen (48.4%) achieved partial response for an ORR of 48.4% (95% CI, 30.2%-66.9%). The disease control rate was 71.0% (95% CI, 52.0%-85.8%) due to 7 (22.6%) stable diseases. The median duration of response was 6.0 (95% CI, 3.6-8.0) months and 1 patient had the longest ongoing response of 17.3 months. Of 24 patients with evaluable imaging, 23 (74.2%) obtained tumor shrinkage. The median PFS (11.0 vs. 6.9 months) and ORR (66.7% vs. 60.0%) for patients with RAS/BRAF wild-type were numerically better than those with mutation. Three patients are still ongoing treatment. The grade 3 or more treatment-emergent adverse events (TEAEs) were mainly hypertension (12.9%) and decreased neutrophil count (12.9%). Four (12.9%) had serious TEAEs, primarily including abdominal pain and incomplete intestinal obstruction. Conclusion: Anlotinib plus XELOX as first-line therapy in patients with mCRC showed anti-tumor activity and safety profile, which is worth further investigation. Clinical Trial Registration: chictr.org.cn, identifier ChiCTR1900028417.

Amplified Drug Delivery System with a Pair of Master Keys Triggering Precise Drug Release for Chemo-Photothermal Therapy.

A versatile drug delivery system (DDS) enabling highly effective and targeting oncotherapy has always been of great significance in medical research. In the development of a stimuli-responsive DDS, compared with a single-factor stimulation DDS, a multifactor activation DDS has higher therapeutic specificity between diseased and normal tissue, but there are challenges in drug-release efficiency and united targeting cancer therapy. Herein, a novel dual-microRNA (dual-miRNA)-mediated 1:N-amplified DDS is fabricated. The gold nanocage (AuNC) was synthesized and used as a carrier. A DNA bridge motif as a nanolock (DNA bridge nanolock) was designed and modified on the surface of AuNCs, which could seal the holes of AuNCs. Using the dual-miRNAs as a pair of master keys, through DNA strand migration and DNAzyme self-assembly, a cell endogenous substance Mg2+-dependent DNAzyme cyclic shear reaction could perform the function of the master keys to open multiple locks for the enhanced release of doxorubicin from the AuNCs. In addition, under near-infrared irradiation, via absorption of light and heat release, the AuNC is activated to perform the function of photothermal therapy. Thereby, the system achieves precise chemo-photothermal therapy. Using the in vitro and in vivo anti-tumor analysis, the DDS could be proved to present a novel design of enhanced and targeted drug-release system for highly effective cancer therapy.

KDM2A plays a dual role in regulating the expression of malignancy-related genes in esophageal squamous cell carcinoma.

KDM2A is a histone demethylase, which primarily catalyzes the demethylation of H3K36me2. Abnormal expression of KDM2A is observed in many types of cancers; however, the molecular events connected to KDM2A expression remain unclear. We report that KDM2A performs an oncogenic function in esophageal squamous cell carcinoma (ESCC) and is robustly expressed in ESCC cells. ShRNA-mediated knockdown of KDM2A resulted in a significant inhibition of the malignant phenotype of ESCC cell lines, whereas ectopic expression of KDM2A showed the opposite effect. We also analyzed the function of KDM2A using a CRISPR-CAS9 depletion system and subsequent rescue experiment, which also indicated a cancerous role of KDM2A. Interestingly, analysis of the gene expression network controlled by KDM2A using RNA-seq revealed an unexpected feature: KDM2A could induce expression of a set of well-documented oncogenic genes, including IL6 and LAT2, while simultaneously suppressing another set of oncogenes, including MAT2A and HMGCS1. Targeted inhibition of the upregulated oncogene in the KDM2A-depleted cells led to a synergistic suppressive effect on the malignant phenotype of ESCC cells. Our results revealed the dual role of KDM2A in ESCC cells, which may have therapeutic implications.

Multiple-mRNA-controlled and heat-driven drug release from gold nanocages in targeted chemo-photothermal therapy for tumors.

Multifunctional drug delivery systems enabling effective drug delivery and comprehensive treatment are critical to successful cancer treatment. Overcoming nonspecific release and off-target effects remains challenging in precise drug delivery. Here, we design triple-interlocked drug delivery systems to perform specific cancer cell recognition, controlled drug release and effective comprehensive therapy. Gold nanocages (AuNCs) comprise a novel class of nanostructures possessing hollow interiors and porous walls. AuNCs are employed as a drug carrier and photothermal transducer due to their unique structure and photothermal properties. A smart triple-interlocked I-type DNA nanostructure is modified on the surface of the AuNCs, and molecules of the anticancer drug doxorubicin (DOX) are loaded as molecular cargo and blocked. The triple-interlocked nanostructure can be unlocked by binding with three types of tumor-related mRNAs, which act as "keys" to the triple locks, sequentially, which leads to precise drug release. Additionally, fluorescence-imaging-oriented chemical-photothermal synergistic treatment is achieved under illumination with infrared light. This drug delivery system, which combines the advantages of AuNCs and interlocked I-type DNA, successfully demonstrates effective and precise imaging, drug release and photothermal therapy. This multifunctional triple-interlocked drug delivery system could be used as a potential carrier for effective cancer-targeting comprehensive chemotherapy and photothermal therapy treatments.

A One-Two-Three Multifunctional System for Enhanced Imaging and Detection of Intracellular MicroRNA and Chemogene Therapy.

Simultaneous imaging, diagnosis, and therapy can offer an effective strategy for cancer treatment. However, the complex probe design, poor drug release efficiency, and multidrug resistance remain tremendous challenges to cancer treatment. Here, a novel one-two-three system is built for enhanced imaging and detection of miRNA-21 (miR-21) overexpressed in cancer cell and chemogene therapy. The system consists of dual-mode DNA robot nanoprobes assembled by two types of hairpin DNAs and three-way branch DNAs modified on gold nanoparticles, with intercalating anticancer drugs (doxorubicin), into DNA duplex GC base pairs. In the system, via intracellular ATP-accelerated cyclic reaction triggered by miR-21, fluorescence and SERS signals were alternated with DNA structure switch, and the precise SERS detection of miRNA and fluorescence imaging oriented "on-demand" release of two types of anticancer drugs (anti-miR-21 and Dox) are achieved. Thus, "one-two-three" means one kind of miR-21-triggered endogenous substance accelerated cyclic reaction, two modes of signal switch, and three functions including enhanced imaging, detection, and comprehensive treatment. The one-two-three system has some notable merits. First, ATP as an endogenous substance promotes DNA structure switching and accelerates the cyclic reaction. Second, the treatment with a dual-mode signal switch is more reliable and accurate and can provide more abundant information than a single-mode treatment platform. Thus, the imaging and detection of intracellular miRNA and effective comprehensive therapy are realized. In vivo results indicate that the system can provide new insights and strategies for diagnosis and therapy.

SERS-Microfluidic Approach for the Quantitative Detection of miRNA Using DNAzyme-Mediated Reciprocal Signal Amplification.

MicroRNAs (miRNAs) play important roles in biological processes. Designing a sensitive, selective, and rapid method of miRNA detection is crucial for biological research. Here, with a reciprocal signal amplification (RSA) probe, this work established a novel surface-enhanced Raman scattering (SERS)-microfluidic approach for the quantitative analysis of miRNA. First, via a DNAzyme self-assemble cycle reaction, two types of SERS signals produce amplified reciprocal changes. The sum of the absolute signal value is first adopted for the quantitative analysis of miRNA, which results in an enhanced response and a reduced blank value. Furthermore, the assay is integrated in an electric drive microfluidic mixing reactor that enables physical mixing and enriching of the reactants for more rapid and enhanced detection sensitivity. The protocol owns the merits of the SERS technology, amplified reciprocal signals, and a microfluidic chip, with a detection limit of 2.92 fM for miR-141 in 40 min. In addition, successful determination of miRNA in a variety of cells proved the practicability of the assay. Compared with the reported strategies for miRNA analysis, this work avoids a complex and time-consuming procedure and enhances the sensitivity and specificity. The method opens a promising way for biomolecular chip detection and research.

Fluorescent-Raman Binary Star Ratio Probe for MicroRNA Detection and Imaging in Living Cells.

The expression of microRNAs (miRNAs) is critical in gene regulation and has been counted into disease diagnosis marks. Precise imaging and quantification of miRNAs could afford the important information for clinical diagnosis. Here, two smart binary star ratio (BSR) probes were designed and constructed, and miRNA triggered the connection of the binary star probes and the reciprocal changes of dual signals in living cells. This multifunctional probe integrates fluorescence and surface enhanced Raman scattering (SERS) imaging, with enzyme-free numerator signal amplification for dual-mode imaging and dual-signal quantitative analysis of miRNA. First, compared with the single-mode ratio imaging method, using fluorescence-SERS complementary ratio imaging, this probe enables more accurate imaging contrast for direct visualization signal changes in living cells. Multiscale information about the dynamic behavior of miRNA and the probe is acquired. Next, via SERS reverse signal ratio response and a novel enzyme-free numerator signal amplification, the amplified signal and reduced black value were achieved in the quantification of miRNA. More importantly, BSR probes showed good stability in cells and were successfully used for accurate tracing and quantification of miR-203 from MCF-7 cells. Therefore, the reported BSR probe is a potential tool for the reliable monitoring of biomolecule dynamics in living cells.

An accurate and ultrasensitive SERS sensor with Au-Se interface for bioimaging and in situ quantitation.

We propose a stable and highly sensitive Au-Se SERS nanoprobe for bioimaging and in situ quantitation, which aims to break through the limitations of traditional Au-S SERS nanoprobes, such as interference from biothiols and unsatisfactory SERS efficiency.

A seesaw ratiometric probe for dual-spectrum imaging and detection of telomerase activity in single living cells.

Herein, a seesaw ratiometric (SR) probe is designed which integrates fluorescence and surface enhanced Raman scattering (SERS) technology. Fluorescence imaging enables tracking of the spatiotemporal dynamic behaviour of telomerase. Meanwhile, SERS reverse ratiometric measurement can enable sensitive detection of telomerase activity in single living cells.

Triggerable Mutually Amplified Signal Probe Based SERS-Microfluidics Platform for the Efficient Enrichment and Quantitative Detection of miRNA.

Sensitive detection of microRNAs (miRNAs) that serve as a disease marker could advance the diagnosis and treatment of diseases. Many methods used for quantitative detection of miRNAs, such as PCR-based approaches or the hybridization chain reaction, have presented challenges due to the complicated and time-consuming-procedures that are required. In this manuscript, a simple triggerable mutually amplified signal (TMAS) probe was designed and enriched within the center of a microfluidic chip and then used for one-step quantitative detection of microRNAs via surface enhanced Raman scattering (SERS) technology. First, many mutually amplified double strands are produced via an enzyme-free target-strand displacement recycling reaction initiated by the target miRNA, that result in the generation of an enhanced SERS signal. Second, microfluidic chips that utilize alternating current (AC) electrokinetic flow technology produce efficient mixing and rapid concentration to improve the DNA hybridization rate and further enhance the SERS signal intensity. This method enables the sensitive and rapid detection of miR-21 in human breast cancer cells within 30 min with a detection limit of 2.33 fM. Compared with traditional methods, this novel method overcomes the shortcomings resulting from complex operations, and has the advantages of high sensitivity, short assay time, and reduced sample usage.

A Dual-Signal Twinkling Probe for Fluorescence-SERS Dual Spectrum Imaging and Detection of miRNA in Single Living Cell via Absolute Value Coupling of Reciprocal Signals.

Imaging and detecting microRNAs (miRNAs) is of central importance in tumor cell analysis. It stays challenging to establish simple, accurate, and sensitive analytical assays for imaging and detection of miRNA in a single living cell, because of intracellular complex environment and miRNA sequence similarity. Herein, we designed a dual-signal twinkling probe (DSTP) with triplex-stem structure which employed a fluorescence-SERS signal reciprocal switch. The spatiotemporal dynamics of the miRNA molecular and intracellular uptake of the probe are monitored by fluorescence-SERS signal switch of the DSTP. Meanwhile, using the surface-enhanced Raman scattering (SERS) signals of DSTP, the measure of absolute value coupling of reciprocal signals is first used to real-time detection of miRNA. Through simultaneous enhancing the target response signal value and reducing blank value, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, the probe shows excellent reversibility and specificity in real quantitative detection of intracellular miRNA. miR-203 was successfully monitored in MCF-7, in accord with the results in vitro as well as in cell lysates. We anticipate that this new dual-signal twinkling and dual-spectrum switch method will be generally useful to image and detect various types of biomolecules in single living cell.

Correction to: The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer.

AbstractAfter the publication of this article [1] it came to our attention that there were some errors in two of the figures.

Wnt inhibitory factor-1-mediated autophagy inhibits Wnt/ß-catenin signaling by downregulating dishevelled-2 expression in non-small cell lung cancer cells.

Wnt inhibitory factor­1 (WIF­1) is an important antagonist of Wnt/ß­catenin signaling by binding to Wnt ligands. The downregulation of WIF­1 leads to the development of non­small cell lung cancer (NSCLC). The upregulation of WIF­1 significantly inhibits proliferation and induces apoptosis by inhibiting Wnt/ß­catenin signaling in NSCLC. However, the mechanisms underlying the inhibition of Wnt/ß­catenin signaling by WIF­1­mediated autophagy are poorly understood. Thus, in this study, we aimed to shed some light into these mechanisms. The upregulation of WIF­1­induced autophagy in NSCLC cells was detected by transmission electron microscopy, acridine orange staining, punctate GFP­LC3 and immunoblotting­based LC3 flux assay. Subsequently, WIF­1­mediated autophagy was blocked in NSCLC cells and the effects of WIF­1­mediated autophagy blocking were examined on the proliferation and apoptosis of NSCLC cells in vitro. Western blot analysis was used to investigate the molecular mechanisms effected by WIF­1­mediated autophagy in NSCLC cells. Finally, combination treatment with WIF­1 and an autophagy agonist was used to examine the tumor growth inhibitory effects of WIF­1 in vivo. The results revealed that the upregulation of WIF­1 induced autophagy in NSCLC cells. WIF­1­mediated autophagy was demonstrated to inhibit Wnt/ß­catenin signaling by downregulating dishevelled­2 (Dvl2), which contributed to the inhibition of the proliferation and the promotion of the apoptosis of NSCLC cells. Moreover, the induction of autophagy mediated by WIF­1 was associated with to suppression of the activation of the phosphoinositide 3­kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Finally, we found that transfection with a WIF­1 gene overexpression vector in combination with treatment with the autophagy agonist, everolimus (RAD001) exerted synergistic antitumor effects on A549 subcutaneous tumor xenografts and pulmonary metastasis in mice. On the whole, the findings of this study demonstrated that WIF­1­mediated autophagy inhibits Wnt/ß­catenin signaling by downregulating Dvl2 expression in NSCLC cells. This may a novel molecular mechanism through which WIF­1 inhibits Wnt/ß­catenin signaling. This study may provide a theoretical basis for joint therapy of NSCLC with WIF­1 and autophagic agonists in clinical practice.

A DNA-linker-DNA bifunctional probe for simultaneous SERS detection of miRNAs via symmetric signal amplification.

In this work, using a dual functional DNA-linker-DNA (DLD) probe, a new concept of a symmetric signal amplification (SSA) reaction is introduced to simultaneously analyze miRNAs. By coupling the surface-enhanced Raman scattering (SERS) technology with symmetric amplification modes, this flexible biosensing system exhibits high sensitivity and specificity.

Prospective investigation of intravenous patient-controlled analgesia with hydromorphone or sufentanil: impact on mood, opioid adverse effects, and recovery.

BACKGROUND: Radical surgery for colorectal cancer, associated with moderate to severe postoperative pain, needs multimodal analgesia with opioid for analgesia. Despite considerable advancements, the psychological implications and other side effects with opioid remain substantially unresolved. This study aimed to investigate the impact on mood, side effects relative to opioid, and recovery of the patients with hydromorphone or sufentanil intravenous patient-controlled analgesia (IV-PCA) in a multimodal perioperative analgesia regimen undergoing radical surgery for colorectal cancer. METHODS: Eighty patients undergoing elective laparoscopic or open radical surgery for colorectal cancer under general anesthesia were randomized to receive postoperative IV-PCA with either sufentanil (group S) or hydromorphone (group H). All patients received additionally flurbiprofen axetil 50 mg 30 min before the end of surgery and wound infiltration with 10 ml of 0.75% ropivacaine at the end of surgery. The primary endpoint was mood changes at 48 and 96 h after surgery. The secondary endpoints were the incidence of opioid-related adverse effects, recovery results and patient satisfaction after surgery. RESULTS: Seventy-two patients completed the study finally. There were no significant differences between the two groups with respect to preoperative parameters, surgical and anesthetic characteristics (P > 0.05). No obvious significant differences were observed in VAS score (at rest and during mobilization) and rescue analgesics use (P > 0.05). Compared with group S, the anger scores in the group H at 48 h and 96 h after surgery were significantly lower (P = 0.012 and 0.005; respectively), but the incidences of pruritus and nausea were higher (P = 0.028 and 0.008; respectively). There were no significant differences in the incidences of vomiting, respiratory depression, dizziness, Ramsay score, and hemodynamic changes between the two groups (P > 0.05). Moreover, there were no significant differences in the time to gastrointestinal recovery, time to drainage tube removal, time to walk, hospital stay after surgery and patient satisfaction between the two groups (P > 0.05). CONCLUSIONS: Under the similar analgesia effect with different opoiods postoperatively, hydromorphone IV-PCA resulted in an improved mood, however, a higher occurrence of pruritus and nausea while compared to sufentanil IV-PCA in a multimodal perioperative analgesia regimen. Both regimens of opioid with IV-PCA may serve as promising candidates for good postoperative pain management, and provide with similar postoperative recovery for the patients undergoing radical surgery for colorectal cancer. TRIAL REGISTRATION: This study was registered with the Chinese Clinical Trial Registry on September 20, 2015 (URL: http://www.chictr.org.cn . Registry number: ChiCTR-IPR-15007112).

Fluorescence and SERS Imaging for the Simultaneous Absolute Quantification of Multiple miRNAs in Living Cells.

The simultaneous imaging and quantification of multiple intracellular microRNAs (miRNAs) are particularly desirable for the early diagnosis of cancers. However, simultaneous direct imaging with absolute quantification of multiple intracellular RNAs remains a great challenge, particularly for miRNAs, which have significantly different expression levels in living cells. We designed dual-signal switchable (DSS) nanoprobes using the fluorescence-Raman signal switch. The intracellular uptake and dynamic behaviors of the probe are monitored by its fluorescence signal. Meanwhile, real-time quantitative detection of multiple miRNAs is made possible by measurements of the surface-enhanced Raman spectroscopy (SERS) ratios. Moreover, the signal 1:n ratio amplification mode only responds to low-abundance miRNA (asymmetric signal amplification mode) for simultaneous visualization and quantitative detection of significantly different levels of miRNAs in living cells. miR-21 and miR-203 were successfully detected in living MCF-7 cells, in agreement with in vitro results from the same batch of cell lysates. The reported dual-spectrum imaging method promises to offer a new strategy for the intracellular imaging and detection of various types of biomolecules.

Microarray-based analysis and clinical validation identify ubiquitin-conjugating enzyme E2E1 (UBE2E1) as a prognostic factor in acute myeloid leukemia.

BACKGROUND: Previous research suggested that single gene expression might be correlated with acute myeloid leukemia (AML) survival. Therefore, we conducted a systematical analysis for AML prognostic gene expressions. METHODS: We performed a microarray-based analysis for correlations between gene expression and adult AML overall survival (OS) using datasets GSE12417 and GSE8970. Positive findings were validated in an independent cohort of 50 newly diagnosed, non-acute promyelocytic leukemia (APL) AML patients by quantitative RT-PCR and survival analysis. RESULTS: Microarray-based analysis suggested that expression of eight genes was each associated with 1-year and 3-year AML OS in both GSE12417 and GSE8970 datasets (p < 0.05). Next, we validated our findings in an independent cohort of AML samples collected in our hospital. We found that ubiquitin-conjugating enzyme E2E1 (UBE2E1) expression was adversely correlated with AML survival (p = 0.04). Multivariable analysis showed that UBE2E1 high patients had a significant shorter OS and shorter progression-free survival after adjusting other known prognostic factors (p = 0.03). At last, we found that UBE2E1 expression was negatively correlated with patients' response to induction chemotherapy (p < 0.05). CONCLUSIONS: In summary, we demonstrated that UBE2E1 expression was a novel prognostic factor in adult, non-APL AML patients.

Sensitive SERS detection of miRNA via enzyme-free DNA machine signal amplification.

In this work, an enzyme-free signal amplified detection platform is described for miRNA detection with a DNA fueled molecular machine. Coupling SERS technology with multiple amplification modes, this flexible biosensing system exhibits high sensitivity and specificity.