Association of triglyceride glucose index with stroke: from two large cohort studies and mendelian randomization analysis.

INTRODUCTION: The triglyceride glucose index (TyG) is associated with cardiovascular diseases; however, its association with stroke remains unclear. This study aimed to elucidate this relationship by examining two extensive cohort studies using two-sample Mendelian randomization (MR). METHODS: Using data from the 1999-2018 National Health and Nutrition Examination Survey (NHANES) and the Medical Information Mart for Intensive Care (MIMIC)-IV, the correlation between TyG (continuous and quartile) and stroke was examined using multivariate Cox regression models and sensitivity analyses. Two-sample MR was employed to establish causality between TyG and stroke using the inverse variance weighting method. Genome-wide association study catalog queries were performed for single nucleotide polymorphism-mapped genes, and the STRING platform used to assess protein interactions. Functional annotation and enrichment analyses were also conducted. RESULTS: From the NHANES and MIMIC-IV cohorts, we included 740 and 589 participants with stroke, respectively. After adjusting for covariates, TyG was linearly associated with the risk of stroke death (NHANES: hazard ratio [HR] 0.64, 95% confidence interval [CI]: 0.41-0.99, P=0.047; Q3 vs. Q1, HR 0.62, 95%CI: 0.40-0.96, P=0.033; MIMIC-IV: HR 0.46, 95%CI: 0.27-0.80, P=0.006; Q3 vs. Q1, HR 0.32, 95%CI: 0.12-0.86; Q4 vs. Q1, HR 0.30, 95%CI: 0.10-0.89, P=0.030, P for trend=0.017). Two-sample MR analysis showed genetic prediction supported a causal association between a higher TyG and a reduced risk of stroke (odds ratio 0.711, 95%CI: 0.641-0.788, P=7.64e-11). CONCLUSIONS: TyG was causally associated with a reduced risk of stroke. TyG is a critical factor for stroke risk management.

Development of a prognostic model for long-term survival of young patients with bladder cancer: a retrospective analysis of the SEER Database.

OBJECTIVES: This study aims to present the clinical characteristics of young patients with bladder cancer (YBCa), evaluate related risk factors and construct a nomogram based on data acquired from the Surveillance, Epidemiology, and End Results (SEER) Database. DESIGN: Retrospective analysis of the SEER Database (2004-2015) for primary YBCa. SETTING AND PARTICIPANTS: Data for YBCa (defined as those aged 40 years or younger) were extracted from the SEER Database, which covers approximately 28% of the US population, using the SEER*Stat software (V.8.4.0.1). A total of 1233 YBCa were identified. Patients were randomly assigned to the training and validation sets. The database included clinicopathological features, demographic information and survival outcomes, such as age, gender, race, year of diagnosis, marital status at diagnosis, primary tumour site, histological type, tumour grade, tumour, node, metastases (TNM) staging, treatment regimen for the primary tumour, cause of death and survival time. A nomogram model was developed using univariate and multivariate analyses. The prediction model was validated using the consistency index (C-index), calibration curve and receiver operating characteristic curve. PRIMARY OUTCOME MEASURES: 3-year, 5-year and 10-year overall survival (OS). RESULTS: 1233 YBCa from 2004 to 2015 were randomly assigned to the training set (n=865) and validation set (n=368). Age, marital status, tumour grade, histological type and TNM staging were included in the nomogram. The C-index of the model was 0.876. The 3-year, 5-year and 10-year OS area under the curve values for the training and validation sets were 0.949, 0.923 and 0.856, and 0.919, 0.890 and 0.904, respectively. Calibration plots showed that the nomogram had a robust predictive accuracy. CONCLUSIONS: To our knowledge, this is the first study to establish a precise nomogram predicting the 3-year, 5-year and 10-year OS in YBCa based on multivariate analyses. Our nomogram may serve as a valuable reference for future diagnostics and individualised treatments for YBCa. However, external validation is warranted to assess the accuracy and generalisability of our prognostic model.

TBC1D1 represses glioma progression by altering the integrity of the cytoskeleton.

BACKGROUND: Glioma is one of the most aggressive malignant brain tumors and is characterized by invasive growth and poor prognosis. TBC1D1, a member of the TBC family, is associated with the development of various malignancies. However, the role of TBC1D1 in glioma-genesis remains unclear. METHODS: The effect of TBC1D1 on the prognosis of glioma patients and related influencing factors were analyzed in the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases. Expression of TBC1D1 in glioma cell lines was detected by western blotting. Cell viability and proliferation were measured by EdU and Colony formation assays, respectively. Transwell and wound healing assays were performed to determine the cell migration and invasion capacities. Immunofluorescence was used to observe actin morphology in the cytoskeleton. RESULTS: We discovered that high TBC1D1 expression in gliomas led to poor prognosis. Downregulation of TBC1D1 in glioma cells significantly inhibited multiple important functions, such as proliferation, migration, and invasion. We further demonstrated that the tumor-inhibitory effect of TBC1D1 might occur through the P-LIMK/cofilin pathway, destroying the cytoskeletal structure and affecting the depolymerization of F-actin, thereby inhibiting glioma migration. CONCLUSION: TBC1D1 affects the balance and integrity of the actin cytoskeleton via cofilin, thereby altering the morphology and aggressiveness of glioma cells. This study provides a new perspective on its role in tumorigenesis, thereby identifying a potential therapeutic target for the treatment of gliomas.

CAV1 and KRT5 are potential targets for prostate cancer.

Prostate cancer is the most common malignant tumor of male urogenital system that occurs in prostate epithelium. However, relationship between CAV1 and KRT5 and prostate cancer remains unclear. The prostate cancer datasets GSE114740 and GSE200879 were downloaded from Gene Expression Omnibus generated by GPL11154 and GPL32170. De-batch processing was performed, differentially expressed genes (DEGs) were screened, and weighted gene co-expression network analysis. The construction and analysis of protein-protein interaction network, functional enrichment analysis, gene set enrichment analysis. Gene expression heat map was drawn and immune infiltration analysis was performed. Comparative toxicogenomics database analysis were performed to find the disease most related to core gene. In addition, the cell experiment was performed to verify the role of CAV1 and KRT5 by western blot. Divided into 4 groups: control, prostate cancer, prostate cancer-over expression, and prostate cancer- knock out. TargetScan screened miRNAs that regulated central DEGs; 770 DEGs were identified. According to Gene Ontology analysis, they were mainly concentrated in actin binding and G protein coupled receptor binding. In Kyoto Encyclopedia of Gene and Genome analysis, they were mainly concentrated in PI3K-Akt signal pathway, MAPK signal pathway, and ErbB signal pathway. The intersection of enrichment terms of differentially expressed genes and GOKEGG enrichment terms was mainly concentrated in ErbB signaling pathway and MAPK signaling pathway. Three important modules were generated. The protein-protein interaction network obtained 8 core genes (CAV1, BDNF, TGFB3, FGFR1, PRKCA, DLG4, SNAI2, KRT5). Heat map of gene expression showed that core genes (CAV1, TGFB3, FGFR1, SNAI2, KRT5) are highly expressed in prostate cancer tissues and low in normal tissues. Comparative toxicogenomics database analysis showed that core genes (CAV1, TGFB3, FGFR1, SNAI2, KRT5) were associated with prostate tumor, cancer, tumor metastasis, necrosis, and inflammation. CAV1 and KRT5 are up-regulated in prostate cancer. CAV1 and KRT5 are highly expressed in prostate cancer. The higher expression of CAV1 and KRT5, the worse prognosis.

Rapid generation of CD19 CAR-T cells by minicircle DNA enables anti-tumor activity and prevents fatal CAR-B leukemia.

Manufacturing chimeric antigen receptor (CAR)-T cells using viral vectors is expensive and time-consuming. In addition, during viral transduction, genes encoding CARs are randomly integrated into the genome, which can cause oncogenesis or produce devastating CAR-tumor cells. Here, using a virus-free and non-transgenic minicircle DNA (mcDNA) vector, we enabled the rapid generation of CD19 CAR-T cells within two days. Furthermore, we demonstrated in vitro and in xenograft models that the antitumor effects of CD19 CAR-T cells produced by mcDNA are as effective as those produced by viral vectors. Finally, we showed that our manufacturing process avoids the production of fatal CAR-tumor cells. Taken together, we have provided a fast, effective, and therapeutically safe method for generating CD19 CAR-T cells for the treatment of leukemia.

Potential roles of microRNAs and long noncoding RNAs as diagnostic, prognostic and therapeutic biomarkers in coronary artery disease.

Coronary artery disease (CAD), which is mainly caused by atherosclerotic processes in coronary arteries, became a significant health issue. MicroRNAs (miRNAs), and long noncoding RNAs (lncRNAs), have been shown to be stable in plasma and could thereby be adopted as biomarkers for CAD diagnosis and treatment. MiRNAs can regulate CAD development through different pathways and mechanisms, including modulation of vascular smooth muscle cell (VSMC) activity, inflammatory responses, myocardial injury, angiogenesis, and leukocyte adhesion. Similarly, previous studies have indicated that the causal effects of lncRNAs in CAD pathogenesis and their utility in CAD diagnosis and treatment, has been found to lead to cell cycle transition, proliferation dysregulation, and migration in favour of CAD development. Differential expression of miRNAs and lncRNAs in CAD patients has been identified and served as diagnostic, prognostic and therapeutic biomarkers for the assessment of CAD patients. Thus, in the current review, we summarize the functions of miRNAs and lncRNAs, which aimed to identify novel targets for the CAD diagnosis, prognosis, and treatment.

RPS14 promotes the development and progression of glioma via p53 signaling pathway.

Glioma is a common primary intracranial brain disease that exhibits an increasing incidence and mortality rate. Accumulating evidences have suggested that Ribosomal protein S14 (RPS14) was involved in cell proliferation and tumor progression. Nevertheless, the biological function and underlying mechanism of RPS14 in glioma are still largely unclear. Herein, we found that RPS14 was overexpressed in glioma. In the loss-of-function experiments, RPS14 depletion markedly suppressed glioma cell proliferation, migration and prompted cell apoptosis in vitro. Further study suggested that RPS14 depletion inhibited tumor growth of glioma in vivo. Additionally, human phospho-kinase array profiling and Western blot analysis revealed that the effects of RPS14 knockdown on glioma may be closely associated with p53 signaling pathway. Further study indicated that addition of p53 inhibitor pifithrin-α (PFT-α) could attenuate the influences of RPS14 knockdown on cell proliferation and apoptosis. Taken together, our findings suggested that RPS14 exhibits a pro-oncogenic role in glioma progression and may be act as a novel potential therapeutic target for gliomas.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like "off-target" toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment. METHODS: The peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups. RESULTS: The mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal "off-target" combination existed on CoG133-CAR T cells and major organs. CONCLUSION: Our study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

Anti-HER2 scFv-CCL19-IL7 recombinant protein inhibited gastric tumor growth in vivo.

HER-2 targeted therapies, such as monoclonal antibodies (mAbs) and CAR-T cell therapy have been applied in the treatment of various of cancers. However, the anti-HER2 CAR-T cell therapy are limited by its expensive production procedure and fatal side effects such as cytokine storm or "On target, off tumor". The application of anti-HER2 mAbs to the soild tumor are also plagued by the patients resistant with different mechanisms. Thus, the recombinant protein technology can be presented as an attractive methods in advantage its less toxic and lower cost. In this study, we produced a HER-2-targeting recombinant protein, which is the fusion of the anti-HER-2 single chain fragment variable domain, CCL19 and IL7 (HCI fusion protein). Our results showed that the recombinant protein can induce the specific lysis effects of immune cells on HER-2-positive gastric tumor cells and can suppress gastric tumor growth in a xenograft model by chemotactic autoimmune cell infiltration into tumor tissues and activated T cells. Taken together, our results revealed that the HCI fusion protein can be applied as a subsequent clinical drug in treating HER-2 positive gastric tumors.

Folate Receptor-Alpha Targeted 7x19 CAR-γδT Suppressed Triple-Negative Breast Cancer Xenograft Model in Mice.

Background: Triple-negative breast cancer (TNBC) is the worst prognosis subtype of breast cancer due to lack of specific targets. Recent studies have shown that immunotherapy may solve that problem by targeting folate receptor-alpha (FRα). Methods: Gene modified γδ T cells were manufactured to express FRa specific chimeric antigen receptor (FRa CAR) and secrete interleukin-7 (IL-7) and chemokine C-C motif ligand 19 (CCL19). CAR-γδT cells that secrete IL-7 and CCL19 (7 × 19 CAR-γδT) were evaluated for their antitumor activity both in vitro and in vivo. Results: 7 × 19 CAR-γδT showed remarkable antitumor activity in vitro. Combined with PBMC, 7 × 19 CAR-γδT inhibited TNBC xenograft model growth superiorly compared with single-application or conventional CAR-γδT cells. Histopathological analyses showed increased DC or T cells infiltration to tumor tissues. Conclusion: Taken together, our results showed that 7 × 19 CAR-γδT have remarkable anti-TNBC tumor activity and showed a broad application prospect in the treatment of incurable TNBC patients.

m(6)A demethylase ALKBH5 inhibits cell proliferation and the metastasis of colorectal cancer by regulating the FOXO3/miR-21/SPRY2 axis.

OBJECTIVE: Colorectal cancer is a common malignancy worldwide. This research aimed to investigate the role of α-ketoglutarate-dependent dioxygenase alkB homologue 5 (ALKBH5), a N6-methyladenosine (m(6)A) demethylase, on the cell proliferation and metastasis of colorectal cancer. METHODS: The interaction relationship between FOXO3, miR-21, and SPRY2 were predicted by starBase 2.0 and determined using RIP, CHIP, and dual-luciferase reporter assays. Quantitative reverse transcription PCR (RT-qPCR) and western blot were used to measure the gene and miRNA expressions of ALKBH5, FOXO3, miR-21, and SPRY2. The cell proliferation was determined using CCK8 and colony formation assays. The metastatic abilities were measured using wound healing and transwell assays. RESULTS: In colorectal cancer, downregulated ALKBH5 is related to poor prognosis. Rescued ALKBH5 suppresses the proliferation and metastasis of colorectal cancer cells. The role of ALKBH5 is achieved by reducing the m(6)A modification of forkhead box O3 (FOXO3), which enhances its stability. FOXO3 targets miR-21 and increases the SPRY2 expressions. The antitumor effects of ALKBH5 can be blocked by FOXO3 knockdown, which is reversed by the miR-21 inhibitor. CONCLUSION: ALKBH5 plays an antitumor role in colorectal cancer by regulating the FOXO3/miR-21/SPRY2 axis, providing a new direction for colorectal cancer therapy.

Therapeutic effects of CXCL9-overexpressing human umbilical cord mesenchymal stem cells on liver fibrosis in rats.

Umbilical cord mesenchymal stem cells (UC-MSCs) transplantation has become a promising treatment for liver fibrosis. However, UC-MSCs have limited anti-fibrosis ability, and their homing ability of UC-MSCs to the injured liver seems to be poor. In our study, we aimed to determine if the CXCL9-overexpressing UC-MSCs could have synergistic anti-fibrosis effects and whether it can promote the homing ability of UC-MSCs. Overexpression of CXCL9 in UC-MSCs (CXCL9-UC-MSCs) was attained by transfecting the lenti-CXCL9-mCherry to naive UC-MSCs. The therapeutic effect of transducted CXCL9-UC-MSCs on both repairing of hepatic fibrosis and target homing were evaluated by comparing with the control of UC-MSCs transfected with empty lenti-mCherry vector. The results revealed that the liver function of CXCL9-UC-MSCs treated group was significantly improved when compared with that of control UC-MSCs (P < 0.05), and the histopathology indicated an obvious decrease of the collagen fiber content and significant disappearing of pseudo-lobules with basically normal morphology of hepatic lobules. Furthermore, liver frozen sections confirmed that CXCL9-UC-MSCs have significantly stronger chemotaxis and stable persistence in the injured liver tissues. In summary, overexpression of CXCL9 could improve the efficacy of UC-MSCs therapy for liver fibrosis repairing on account of an enhanced ability of UC-MSCs in homing to and staying in the injured sites of liver fibrosis in rat models.

Severe bleeding following off-pump coronary artery bypass grafting: predictive factors and risk model.

BACKGROUND: Severe bleeding following cardiac surgery remains a troublesome complication, but to date, there is a lack of comprehensive predictive models for the risk of severe bleeding following off-pump coronary artery bypass grafting (OPCABG). This study aims to analyze relevant indicators of severe bleeding after isolated OPCABG and establish a corresponding risk assessment model. METHODS: The clinical data of 584 patients who underwent OPCABG from January 2018 to April 2020 were retrospectively analyzed. We gathered the preoperative baseline data and postoperative data immediately after intensive care unit admission and used multifactor logistic regression to screen the potential predictors of severe bleeding, upon which we established a predictive model. Using the consistency index and calibration curve, decision curve, and clinical impact curve analysis, we evaluated the performance of the model. RESULTS: This study is the first to establish a risk assessment and prediction model for severe bleeding following isolated OPCABG. Eight independent risk factors were identified: male sex, aspirin/clopidogrel withdrawal time, platelet count, fibrinogen level, C-reactive protein, serum creatinine, and total bilirubin. Among the 483 patients in the training group, 138 patients (28.6%) had severe bleeding; among the 101 patients in the verification group, 25 patients (24.8%) had severe bleeding. Receiver operating characteristic (ROC) curve analysis for the internal training group revealed a convincing performance with a concordance index (C-index) of 0.859, while the area under the ROC curve for the external validation data was 0.807. Decision curve analysis showed that the model was useful for both groups. CONCLUSIONS: Although there are some limitations, the model can effectively predict the probability of severe bleeding following isolated OPCABG and is therefore worthy of further exploration and verification.

"Felodipine-indomethacin" co-amorphous supersaturating drug delivery systems: "Spring-parachute" process, stability, in vivo bioavailability, and underlying molecular mechanisms.

Amorphous solid dispersions (ASD) are one of most commonly used supersaturating drug delivery systems (SDDS) to formulate insoluble active pharmaceutical ingredients. However, the development of polymer-guided stabilization of ASD systems faces many obstacles. To overcome these shortcomings, co-amorphous supersaturable formulations have emerged as an alternative formulation strategy for poorly soluble compounds. Noteworthily, current researches around co-amorphous system (CAS) are mostly focused on preparation and characterization of these systems, but more detailed investigations of their supersaturation ("spring-parachute" process), stability, in vivo bioavailability and molecular mechanisms are inadequate and need to be clarified. In present study, we chose pharmacological relevant BCS II drugs to fabricate and characterize "felodipine-indomethacin" CAS. To enrich the current inadequate but key knowledge on CAS studies, we carried out following highlighted investigations including dissolution/solubility, semi-continuous "spring-parachute" process, long-term stability profile of amorphous state, in vivo bioavailability and underlying molecular mechanisms (molecular interaction, molecular miscibility and crystallization inhibition). Generally, the research provides some key information in the field of current "drug-drug" CAS supersaturable formulations.

Gut microbiota mediates the effects of curcumin on enhancing Ucp1-dependent thermogenesis and improving high-fat diet-induced obesity.

Due to extremely poor systemic bioavailability, the mechanism by which curcumin increases energy expenditure remains unelucidated. Accumulating evidence suggests a strong association between the gut microbiota (GM) and energy metabolism. We investigated whether the GM mediates the effects of curcumin on improving energy homeostasis. High-fat diet (HFD)-fed wild type, uncoupling protein 1 (Ucp1) knockout and G protein-coupled membrane receptor 5 (TGR5) knockout mice were treated with curcumin (100 mg kg-1 d-1, p.o.). Curcumin-treated HFD-fed mice displayed decreased body weight gain and augmented cold tolerance due to enhanced adaptive thermogenesis as compared with that in control mice. The anti-obesity effects of curcumin were abolished by Ucp1 knockout. 16S ribosomal DNA sequencing analysis revealed that curcumin restructured the GM in HFD-fed mice. Fecal microbiota transplantation (FMT) and endogenous GM depletion indicated that the GM mediated the enhanced effect of curcumin on Ucp1-dependent thermogenesis. Curcumin altered bile acid (BA) metabolism with increased fractions of circulating deoxycholic acid (DCA) and lithocholic acid (LCA), which are the two most potent ligands for TGR5. Consistently, the enhanced effect of curcumin on Ucp1-dependent thermogenesis was eliminated by TGR5 knockout. Curcumin requires the GM and TGR5 to activate the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in thermogenic adipose tissue. Here, we demonstrated that the GM mediates the effects of curcumin on enhancing Ucp1-dependent thermogenesis and ameliorating HFD-induced obesity by influencing BA metabolism. We disclosed the potential of nutritional and pharmacologic manipulations of the GM to enhance Ucp1-dependent thermogenesis in the prevention and treatment of obesity.

Downregulated CMTM8 Correlates with Poor Prognosis in Gastric Cancer Patients.

This study aimed to examine the expression and clinical significance of chemokine-like factor-like MARVEL transmembrane domain-containing family member 8 (CMTM8) in gastric cancer (GC). The mRNA and protein expression of CMTM8 were detected by bioinformatics analysis and immunohistochemistry (IHC), respectively. Bioinformatics analysis found that there was a high mRNA expression of CMTM8 in GC tissues, but failed to identify a significant relationship with the clinicopathological features or prognosis of GC patients. However, IHC results showed that the positive expression of CMTM8 protein in GC tissues was significantly lower than that of adjacent nontumor tissues and correlated with differentiation, tumor node metastasis stage, and distal metastasis of GC patients (p < 0.05). Moreover, the survival time of GC patients with negative CMTM8 protein expression group was shorter than that of positive CMTM8 protein expression group by Kaplan-Meier survival analysis (p < 0.05). Cox proportional hazards model (COX) regression analysis indicated that CMTM8 protein was an independent protective factor for the overall survival of GC patients. Further Gene Set Enrichment Analysis suggested that CMTM8 may be involved in regulating the calcium signaling pathway, cell adhesion molecules, and cytokine-cytokine receptor interaction in GC. Our study shows that CMTM8 protein is downregulated in GC tissues, and CMTM8 protein expression is related to GC metastasis and the prognosis of GC patients.

Inhibition of galectin-3 augments the antitumor efficacy of PD-L1 blockade in non-small-cell lung cancer.

Multiple clinical trials have shown that monoclonal antibodies (mAbs) against programmed death-ligand 1 (PD-1/PD-L1) can benefit patients with lung cancer by increasing their progression-free survival and overall survival. However, a significant proportion of patients do not respond to anti-PD-1/PD-L1 mAbs. In the present study, we investigated whether galectin (Gal)-3 inhibitors can enhance the antitumor effect of PD-L1 blockade. Using the NSCLC-derived cell line A549, we examined the expression of Gal-3 in lung cancer cells under hypoxic conditions and investigated the regulatory effect of Gal-3 on PD-L1 expression, which is mediated by the STAT3 pathway. We also explored whether Gal-3 inhibition can facilitate the cytotoxic effect of T cells induced by PD-L1 blockade. The effects of combined use of a Gal-3 inhibitor and PD-L1 blockade on tumor growth and T-cell function were also investigated, and we found that hypoxia increased the expression and secretion of Gal-3 by lung cancer cells. Gal-3 increased PD-L1 expression via the upregulation of STAT3 phosphorylation, and administration of a Gal-3 inhibitor enhanced the effect of PD-L1 blockade on the cytotoxic activity of T cells against cancer cells in vitro. In a mouse xenograft model, the combination of a Gal-3 inhibitor and PD-L1 blockade synergistically suppressed tumor growth. Furthermore, the administration of a Gal-3 inhibitor enhanced T-cell infiltration and granzyme B release in tumors. Collectively, our results show that Gal-3 increases PD-L1 expression in lung cancer cells and that the administration of a Gal-3 inhibitor as an adjuvant enhanced the antitumor activity of PD-L1 blockade.

Construction and application of a human scFv phage display library based on Cre­LoxP recombination for anti­PCSK9 antibody selection.

A large human natural single­chain fragment variable (scFv) phage library was constructed based on Cre­LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription­quantitative PCR. Two­step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through Cre­LoxP enzyme­mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10­10 M towards PCSK9. In vitro, the PCSK9/low­density lipoprotein receptor (LDLR) pathway of Hep­G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3­fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre­LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.

lncRNA LIFR­AS1 inhibits gastric carcinoma cell proliferation, migration and invasion by sponging miR­4698.

The vital functions of long non­coding (lnc)RNAs have been verified in gastric carcinoma (GC). However, as a novel cancer­related lncRNA, the influence of leukemia inhibitory factor receptor antisense RNA 1 (LIFR­AS1) in GC cell biological behaviors remains unreported. The present study explored the biological effects of lncRNA LIFR­AS1 on GC progression. Reverse transcription­quantitative PCR was performed to examine lncRNA LIFR­AS1 expression in GC tissues and cells. Cell Counting Kit­8, 5­ethynyl­2'­deoxyuridine incorporation, cell wound healing and Transwell invasion assays were used to assess the functions of lncRNA LIFR­AS1 in GC cell proliferation, migration and invasion. Additionally, associations among lncRNA LIFR­AS1, microRNA (miR)­4698 and microtubule­associated tumor suppressor 1 (MTUS1) were investigated via bioinformatics software and a luciferase reporter system. In addition, western blotting was used to examine the expression of MEK and ERK. Decreased lncRNA LIFR­AS1 expression was observed in GC tissues and cells. Upregulated lncRNA LIFR­AS1 inhibited GC cell proliferation, migration and invasion. Upregulated miR­4698 and downregulated MTUS1 were identified in GC tissues and cells. The inhibitory interaction between lncRNA LIFR­AS1 and miR­4698 was confirmed. Additionally, MTUS1 was predicted as a target gene of miR­4698 positively regulated by lncRNA LIFR­AS1. The MEK/ERK pathway was inhibited by lncRNA LIFR­AS1 via regulating MTUS1. These findings revealed the inhibitory functions of lncRNA LIFR­AS1 in GC cell proliferation, migration and invasion. The process was mediated via miR­4698, MTUS1 and the MEK/ERK pathway.