A prognostic exosome-related long non-coding RNAs risk model related to the immune microenvironment and therapeutic responses for patients with liver hepatocellular carcinoma.

Background: Liver hepatocellular carcinoma (LIHC) is the third largest cause of cancer mortality. Exosomes are vital regulators in the development of cancer. However, the mechanisms regarding the association of exosome-related long non-coding RNAs (lncRNAs) in LIHC are not clear. Methods: LIHC RNA sequences and exosome-associated genes were collected according to The Cancer Genome Atlas (TCGA), Hepatocellular Carcinoma Cell DataBase (HCCDB) and ExoBCD databases, and exosome-related lncRNAs with prognostic differential expression were screened as candidate lncRNAs using Spearman's method and univariate Cox regression analysis. Candidate lncRNAs were then used to construct a prognostic model and mRNA-lncRNA co-expression network. Differentially expressed genes (DEGs) in low- and high-risk groups were identified and enrichment analysis was performed for up- and down-regulated DEGs, respectively. The expression of immune checkpoint-related genes, immune escape potential and microsatellite instability among different risk groups were further analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) and transwell assay were applied for detecting gene expression levels and invasion and migration ability. Results: Based on 17 prognostical exosome-associated lncRNAs, four hub lncRNAs (BACE1_AS, DSTNP2, PLGLA, and SNHG3) were selected for constructing a prognostic model, which was demonstrated to be an independent prognostic variable for LIHC. High risk score was indicative of poorer overall survival, lower anti-tumor immune cells, higher genomic instability, higher immune escape potential, and less benefit for immunotherapy. The qRT-PCR test verified the expression level of the lncRNAs in LIHC cells, and the inhibitory effect of BACE1_AS on immune checkpoint genes levels. BACE1_AS silence also depressed the ability of migration and invasion of LIHC cells. Conclusion: The Risk model constructed by exosome-associated lncRNAs could well predict immunotherapy response and prognostic outcomes for LIHC patients. We comprehensively reveal the clinical features of prognostical exosome-related lncRNAs and their potential ability to predict immunotherapeutic response of patients with LIHC and their prognosis.

Three molecular subtypes and a five-gene signature for hepatocellular carcinoma based on m7G-related classification.

BACKGROUND: The current research investigated the heterogeneity of hepatocellular carcinoma (HCC) based on the expression of N7-methylguanosine (m7G)-related genes as a classification model and developed a risk model predictive of HCC prognosis, key pathological behaviors and molecular events of HCC. METHODS: The RNA sequencing data of HCC were extracted from The Cancer Genome Atlas (TCGA)-live cancer (LIHC) database, hepatocellular carcinoman database (HCCDB) and Gene Expression Omnibus database, respectively. According to the expression level of 29 m7G-related genes, a consensus clustering analysis was conducted. The least absolute shrinkage and selection operator (LASSO) regression analysis and COX regression algorithm were applied to create a risk prediction model based on normalized expression of five characteristic genes weighted by coefficients. Tumor microenvironment (TME) analysis was performed using the MCP-Counter, TIMER, CIBERSORT and ESTIMATE algorithms. The Tumor Immune Dysfunction and Exclusion algorithm was applied to assess the responses to immunotherapy in different clusters and risk groups. In addition, patient sensitivity to common chemotherapeutic drugs was determined by the biochemical half-maximal inhibitory concentration using the R package pRRophetic. RESULTS: Three molecular subtypes of HCC were defined based on the expression level of m7G-associated genes, each of which had its specific survival rate, genomic variation status, TME status and immunotherapy response. In addition, drug sensitivity analysis showed that the C1 subtype was more sensitive to a number of conventional oncolytic drugs (including paclitaxel, imatinib, CGP-082996, pyrimethamine, salubrinal and vinorelbine). The current five-gene risk prediction model accurately predicted HCC prognosis and revealed the degree of somatic mutations, immune microenvironment status and specific biological events. CONCLUSION: In this study, three heterogeneous molecular subtypes of HCC were defined based on m7G-related genes as a classification model, and a five-gene risk prediction model was created for predicting HCC prognosis, providing a potential assessment tool for understanding the genomic variation, immune microenvironment status and key pathological mechanisms during HCC development.

Discovery of Potent and Oral Bioavailable MAT2A Inhibitors for the Treatment of MTAP-Deleted Tumors.

Inhibition of methionine adenosyltransferase 2A (MAT2A) has received significant interest because of its implication as a synthetic lethal target in methylthioadenosine phosphorylase (MTAP)-deleted cancers. Here, we report the discovery of a series of 3H-pyrido[1,2-c]pyrimidin-3-one derivatives as novel MAT2A inhibitors. The selected compound 30 exhibited high potency for MAT2A inhibition and a favorable pharmacokinetic profile. Furthermore, in an HCT-116 MTAP-deleted xenograft model, compound 30 showed better in vivo potency than current clinical compound AG-270.

Long non-coding RNA SLC7A11 antisense RNA1 promotes oral squamous cell carcinoma progression by regulating ubiquitination of K-homology type splicing regulatory protein.

OBJECTIVES: This article aims to elucidate the role of Long non-coding RNA SLC7A11 antisense RNA1 (SLC7A11-AS1) in oral squamous cell carcinoma, which are expected to be useful for the oral squamous cell carcinoma diagnosis and treatment. DESIGN: SLC7A11-AS1 expression was detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in oral squamous cell carcinoma cell lines. Cellular localization of SLC7A11-AS1C was detected by fluorescence in situ hybridization (FISH) assays and subcellular fractionation assay. Biological functions of SLC7A11-AS1 were explored by 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), wounding healing, and transwell invasion assays in vitro, as well as mice xenograft experiments and metastasis assays in vivo. RNA pull-down, RNA immunoprecipitation, co-immunoprecipitation, ubiquitination assays, and rescue experiments were performed to determine the molecular mechanism of SLC7A11-AS1 in oral squamous cell carcinoma. RESULTS: SLC7A11-AS1 is overexpressed in oral cancer tissues and cell lines. Functionally, knockdown of SLC7A11-AS1 reduced the proliferation, migration, and invasion of oral squamous cell carcinoma cells in vitro and inhibited tumor growth as well as metastasis in vivo. Mechanistically, SLC7A11-AS1 impeded the interaction between K-homology type splicing regulatory protein (KHSRP) and kelch-like 12 (KLHL12), maintaining the stability of KHSRP by restraining KHSRP degradation through the ubiquitination-proteasome pathway. Furthermore, KHSRP overexpression recovered the malignant behaviors inhibited by SLC7A11-AS1 knockdown in oral cancer cells. CONCLUSION: SLC7A11-AS1 promoted oral squamous cell carcinoma development by interacting with KHSRP and maintaining KHSRP stability by preventing its degradation via the ubiquitination-proteasome pathway. Thus, SLC7A11-AS1 is a potential therapeutic target for oral cancer.

Exploring the role of tumor stemness and the potential of stemness-related risk model in the prognosis of intrahepatic cholangiocarcinoma.

Background: Tumor stem cells (TSCs) have been widely reported to play a critical role in tumor progression and metastasis. We explored the role of tumor stemness in intrahepatic cholangiocarcinoma (iCCA) and established a prognostic risk model related to tumor stemness for prognosis prediction and clinical treatment guidance in iCCA patients. Materials and Methods: The expression profiles of iCCA samples (E-MTAB-6389 and GSE107943 cohorts) were used in the study. One-class logistic regression algorithm calculated the mRNA stemness index (mRNAsi). The mRNAsi-related genes were used as a basis for the identification of mRNAsi-related molecular subtypes through consensus clustering. The immune characteristics and biological pathways of different subtypes were assessed. The mRNAsi-related risk model was constructed with differentially expressed genes (DEGs) between subtypes. Results: The patients with high mRNAsi had longer overall survival than that with low mRNAsi. Two subtypes were identified with that C2 had higher mRNAsi and better prognosis than C1. Tumor-related pathways such as TGF-ß and epithelial-mesenchymal transition (EMT) were activated in C1. C1 had higher enrichment of cancer-associated fibroblasts and tumor-associated macrophages, as well as higher immune response and angiogenesis score than C2. We screened a total 98 prognostic DEGs between C1 and C2. Based on the prognostic DEGs, we constructed a risk model containing three genes (ANO1, CD109, and CTNND2) that could divide iCCA samples into high- and low-risk groups. The two groups had distinct prognosis and immune characteristics. Notably, the risk score was negatively associated with mRNAsi (R = -0.53). High-risk group had higher enrichment score of T cell inflamed GEP, INF-γ, and cytolytic activity, and lower score of estimated IC50 of 5-fluorouracil and cisplatin than low-risk group. Conclusions: This study clarified the important role of tumor stemness in iCCA and developed an mRNAsi-related risk model for predicting the prognosis and supporting the clinical treatment in iCCA patients. The three genes (ANO1, CD109, and CTNND2) may serve as potential targets for iCCA treatment.

Wide-range stiffness gradient PVA/HA hydrogel to investigate stem cell differentiation behavior.

Although stiffness-controllable substrates have been developed to investigate the effect of stiffness on cell behavior and function, the use of separate substrates with different degrees of stiffness, substrates with a narrow range stiffness gradient, toxicity of residues, different surface composition, complex fabrication procedures/devices, and low cell adhesion are still considered as hurdles of conventional techniques. In this study, a cylindrical polyvinyl alcohol (PVA)/hyaluronic acid (HA) hydrogel with a wide-range stiffness gradient (between ∼20kPa and ∼200kPa) and cell adhesiveness was prepared by a liquid nitrogen (LN2)-contacting gradual freezing-thawing method that does not use any additives or specific devices to produce the stiffness gradient hydrogel. From an in vitro cell culture using the stiffness gradient PVA/HA hydrogel, it was observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve cell, ∼40kPa for muscle cell, ∼80kPa for chondrocyte, and ∼190kPa for osteoblast). The PVA/HA hydrogel with a wide range of stiffness spectrum can be a useful tool for basic studies related with the stem cell differentiation, cell reprogramming, cell migration, and tissue regeneration in terms of substrate stiffness. STATEMENT OF SIGNIFICANCE: It is postulated that the stiffness of the extracellular matrix influences cell behavior. To prove this concept, various techniques to prepare substrates with a stiffness gradient have been developed. However, the narrow ranges of stiffness gradient and complex fabrication procedures/devices are still remained as limitations. Herein, we develop a substrate (hydrogel) with a wide-range stiffness gradient using a gradual freezing-thawing method which does not need specific devices to produce a stiffness gradient hydrogel. From cell culture experiments using the hydrogel, it is observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve, ∼40kPa for muscle, ∼80kPa for cartilage, and ∼190kPa for bone in our hydrogel system).

Novel variants in MLL confer to bladder cancer recurrence identified by whole-exome sequencing.

Bladder cancer (BC) is distinguished by high rate of recurrence after surgery, but the underlying mechanisms remain poorly understood. Here we performed the whole-exome sequencing of 37 BC individuals including 20 primary and 17 recurrent samples in which the primary and recurrent samples were not from the same patient. We uncovered that MLL, EP400, PRDM2, ANK3 and CHD5 exclusively altered in recurrent BCs. Specifically, the recurrent BCs and bladder cancer cells with MLL mutation displayed increased histone H3 tri-methyl K4 (H3K4me3) modification in tissue and cell levels and showed enhanced expression of GATA4 and ETS1 downstream. What's more, MLL mutated bladder cancer cells obtained with CRISPR/Cas9 showed increased ability of drug-resistance to epirubicin (a chemotherapy drug for bladder cancer) than wild type cells. Additionally, the BC patients with high expression of GATA4 and ETS1 significantly displayed shorter lifespan than patients with low expression. Our study provided an overview of the genetic basis of recrudescent bladder cancer and discovered that genetic alterations of MLL were involved in BC relapse. The increased modification of H3K4me3 and expression of GATA4 and ETS1 would be the promising targets for the diagnosis and therapy of relapsed bladder cancer.

An epigenetic biomarker combination of PCDH17 and POU4F2 detects bladder cancer accurately by methylation analyses of urine sediment DNA in Han Chinese.

To develop a routine and effectual procedure of detecting bladder cancer (BlCa), an optimized combination of epigenetic biomarkers that work synergistically with high sensitivity and specificity is necessary. In this study, methylation levels of seven biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 148 individuals-which including 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients,20 prostate cancer (PC) patients, and 30 healthy volunteers (HV)-were quantified by qMSP using the urine sediment DNA. Receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of possible combinations were calculated through logistic regression model. Subsequently, ROC curves of the three best performing combinations were constructed. Then, we validated the three best performing combinations and POU4F2 in another 72 UCC, 21 IUC, 26 KC and 22 PC, and 23 HV urine samples. The combination of POU4F2/PCDH17 has yielded the highest sensitivity and specificity of 90.00% and 93.96% in all the 312 individuals, showing the capability of detecting BlCa effectively among pathologically varied sample groups.

Creating stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing-thawing method to investigate stem cell differentiation behaviors.

Polyvinyl alcohol (PVA) cylindrical hydrogel with a stiffness gradient was prepared using a simple liquid nitrogen (LN2)-contacting gradual freezing and thawing method in order to investigate the effects of substrate stiffness on stem cell differentiation into specific cell types. The prepared cylindrical PVA hydrogel showed a gradually increasing stiffness along the longitudinal direction from the top at approximately 1 kPa to the bottom (LN2 contacted side) at approximately 24 kPa. From the in vitro culture of bone marrow stem cells, it was observed that each soft (∼1 kPa) and stiff (∼24 kPa) hydrogel section promotes effective neurogenesis and osteogenesis of the cells, respectively, with the tendency to gradually decrease toward the opposing characteristic's side. The stiffness gradient cylindrical PVA hydrogel fabricated using this simple gradual freezing and thawing method can be a useful tool for basic studies, including the determination of optimum stiffness ranges for a variety of stem cell differentiations, as well as the investigation of cell migration in terms of substrate stiffness.

Radiofrequency ablation versus hepatic resection for small hepatocellular carcinoma: a meta-analysis of randomized controlled trials.

BACKGROUND AND GOALS: Whether radiofrequency ablation or hepatic resection is superior for improving the survival in patients with small hepatocellular carcinoma (HCC) remains controversial. A meta-analysis of randomized controlled trials was performed to examine this issue. METHODS: PubMed, EMBASE, and Cochrane Library databases were used to identify all randomized controlled trials comparing the survival between small HCC patients receiving radiofrequency ablation and hepatic resection. The hazard ratio (HR) was pooled to compare the overall survival and recurrence-free survival rates. The odds ratio was pooled to compare the incidence of treatment-related complications. The mean difference was pooled to compare the hospitalization duration. Heterogeneity among studies was assessed. RESULTS: Three randomized controlled trials were included in this meta-analysis. All patients met the Milan criteria. Hepatic resection was superior to radiofrequency ablation for the improvement of overall survival [HR=1.41; 95% confidence interval (CI), 1.06-1.89; P=0.02] and recurrence-free survival (HR=1.41; 95% CI, 1.14-1.74; P=0.001). Heterogeneity among studies was not significant (overall survival: P=0.14; recurrence-free survival: P=0.28). Patients treated with hepatic resection had a significantly higher incidence of treatment-related complications (odds ratio=0.12; 95% CI, 0.03-0.47; P=0.002) and a significantly longer hospitalization duration (mean difference: -8.77; 95% CI, -10.36 to -7.18; P<0.00001) than those treated with radiofrequency ablation. Heterogeneity among studies was significant (treatment-related complications: P=0.006; hospitalization duration: P=0.003). No hospital death occurred in the 2 groups. CONCLUSIONS: Evidence from the meta-analysis of randomized controlled trials suggested that hepatic resection might improve the overall survival and recurrence-free survival in small HCC patients, whereas increase the complications and hospitalization duration. However, this conclusion should be explained with caution, due to the absence of further subgroup analysis with respect to the outcome in patients with different tumor size (<3 and 3 to 5 cm).