Laser capture microdissection-based spatiotemporal transcriptomes uncover regulatory networks during seed abortion in seedless Ponkan (Citrus reticulata).

Seed abortion is an important process in the formation of seedless characteristics in citrus fruits. However, the molecular regulatory mechanism underlying citrus seed abortion is poorly understood. Laser capture microdissection-based RNA-seq combined with Pacbio-seq was used to profile seed development in the Ponkan cultivars 'Huagan No. 4' (seedless Ponkan) (Citrus reticulata) and 'E'gan No. 1' (seeded Ponkan) (C. reticulata) in two types of seed tissue across three developmental stages. Through comparative transcriptome and dynamic phytohormone analyses, plant hormone signal, cell division and nutrient metabolism-related processes were revealed to play critical roles in the seed abortion of 'Huagan No. 4'. Moreover, several genes may play indispensable roles in seed abortion of 'Huagan No. 4', such as CrWRKY74, CrWRKY48 and CrMYB3R4. Overexpression of CrWRKY74 in Arabidopsis resulted in severe seed abortion. By analyzing the downstream regulatory network, we further determined that CrWRKY74 participated in seed abortion regulation by inducing abnormal programmed cell death. Of particular importance is that a preliminary model was proposed to depict the regulatory networks underlying seed abortion in citrus. The results of this study provide novel insights into the molecular mechanism across citrus seed development, and reveal the master role of CrWRKY74 in seed abortion of 'Huagan No. 4'.

Coexpression of TP53, BIM, and PTEN Enhances the Therapeutic Efficacy of Non-Small-Cell Lung Cancer.

For non-small-cell lung cancer (NSCLC), the ubiquitous occurrence of concurrent multiple genomic alterations poses challenges to single-gene therapy. To increase therapeutic efficacy, we used the branch-PCR method to develop a multigene nanovector, NP-TP53-BIM-PTEN, that carried three therapeutic gene expression cassettes for coexpression. NP-TP53-BIM-PTEN exhibited a uniform size of 104.8 ± 24.2 nm and high serum stability. In cell transfection tests, NP-TP53-BIM-PTEN could coexpress TP53, BIM, and PTEN in NCI-H1299 cells and induce cell apoptosis with a ratio of up to 94.9%. Furthermore, NP-TP53-BIM-PTEN also inhibited cell proliferation with a ratio of up to 42%. In a mouse model bearing an NCI-H1299 xenograft tumor, NP-TP53-BIM-PTEN exhibited a stronger inhibitory effect on the NCI-H1299 xenograft tumor than the other test vectors without any detectable side effects. These results exhibited the potential of NP-TP53-BIM-PTEN as an effective and safe multigene nanovector to enhance NSCLC therapy efficacy, which will provide a framework for genome therapy with multigene combinations.

Specific functions of single pistil S-RNases in S-gene homozygous Pyrus germplasm.

Gametophytic self-incompatibility (SI) is regulated by S-allele recognition; that is, pollen in a style with the same S-genotype will undergo programmed cell death and stop growing so that it is unable to complete double fertilization, ultimately resulting in the SI response. S-RNase is the female determinant of SI in pear (Pyrus). In the Pyrus genome, there are two different S-RNase alleles at the S-locus, which generate two different S-RNase products in the pistil. The extracted S-glycoprotein is actually a protein complex. In this study, artificial self-pollination was conducted at the bud stage to overcome SI in 'Huanghua' (S1S2) pear. Seven plants homozygous for S1-RNase and four homozygous for S2-RNase were selected from the selfed progeny of 'Huanghua' by S-gene molecular identification biotechnology. We investigated the function of single S-RNases isolated from the pistils of S-gene homozygous Pyrus germplasm. The pollen of 'Huanghua' could smoothly pass through the style of the S-gene homozygous germplasm and complete fertilization. S-RNases were extracted from flower styles of different genotypes and used to treat different types of pollen. The S-RNase from 'Huanghua' completely inhibited the growth of S1S2, S1S1, and S2S2 pollen, while the S-RNase from homozygous germplasm allowed some S1S2 pollen and different single genotypes of pollen to continue growing. These results further validate the core events of SI including cytoskeleton depolymerization and programmed cell death. By iTRAQ-based proteomic analysis of style proteins, a total of 13 S-RNase-related proteins were identified. In summary, we have created reliable S-RNase gene homozygous germplasm, which will play a crucial role in further research on SI in pear and in the development of the pear industry.

The effects of chemotherapy, primary tumor location and histological subtype on the survival of stage III colon cancer patients.

OBJECTIVE: Colon cancer (CC) is one of the most common cancers worldwide and has a poor prognosis. Surgery followed by adjuvant chemotherapy is the standard treatment strategy for stage III CC patients. Primary tumor location (PTL) is an important factor for the long-term survival of CC. However, the difference in the prognosis between the histological subtypes of mucinous adenocarcinoma (MAC) and nonspecific adenocarcinoma (AC) in stage III CC patients is unclear. The correlation of chemotherapy, PTL and histological subtype with the overall survival (OS) of stage III CC patients has not yet been explored. METHODS: Patients diagnosed with stage III CC from 2010 to 2016 in the Surveillance, Epidemiology, and End Results (SEER) database were retrieved. The clinicopathological features and OS were analyzed according to the chemotherapy, PTL and histological subtype. RESULTS: A total of 28,765 eligible stage III CC patients were enrolled in this study. The results showed that chemotherapy, left-sided CC (LCC) and AC were favorable prognostic factors for OS. Right-sided CC (RCC) had worse OS than LCC regardless of chemotherapy. MAC had worse OS than AC in the patients with chemotherapy, but the survival benefits disappeared in the patients without chemotherapy. Additionally, in LCC, MAC had worse OS than AC regardless of chemotherapy. However, in RCC, MAC had worse OS than AC in patients with chemotherapy but had similar OS to AC in patients without chemotherapy. In the AC group, RCC had worse OS than LCC regardless of chemotherapy. In the MAC group, RCC had comparable OS to LCC regardless of chemotherapy. Four subgroups, i.e., RCC/MAC, RCC/AC, LCC/MAC and LCC/AC, all showed benefits from chemotherapy. Among them, LCC/AC had the best OS, and RCC/MAC had the worst OS compared with the other three subgroups. CONCLUSION: The prognosis of MAC is worse than that of AC in stage III CC. LCC/AC has the best OS, while RCC/MAC has the worst OS but still benefits from chemotherapy. The impact of chemotherapy on survival is greater than that of histological subtype, but the impact of histological subtype on survival is similar to that of PTL.

Lipid droplet-specific near-infrared fluorescent probe for discriminating cancer and normal cells and diagnosing fatty liver.

Lipid droplets (LDs) is a newly essential organelle, which participates in carious physiological and pathological processes. LDs are considered as potential markers for disease diagnosis. Specific imaging of LDs is useful to understand their basic biological function and to diagnose diseases. Here we designed and synthesized two fluorescent probes based on the low polarity and high viscosity in LDs. The terminal probe ZH-2 exhibits lipophilicity, NIR emission, viscosity sensitivity, and LDs specificity. The probe has been successfully used for visualizing LDs metabolism, discriminating between normal and cancerous cells, and diagnosing fatty liver disease.

Identification of prognostic genes in the pancreatic adenocarcinoma immune microenvironment by integrated bioinformatics analysis.

PURPOSE: Pancreatic adenocarcinoma (PAAD) is one of the most common causes of death among solid tumors, and its pathogenesis remains to be clarified. This study aims to elucidate the value of immune/stromal-related genes in the prognosis of PAAD through comprehensive bioinformatics analysis based on the immune microenvironment and validated in Chinese pancreatic cancer patients. METHODS: Gene expression profiles of pancreatic cancer patients were obtained from TCGA database. Differentially expressed genes (DEGs) were identified based on the ESTIMATE algorithm. Gene co-expression networks were constructed using WGCNA. In the key module, survival analysis was used to reveal the prognostic value. Subsequently, we performed functional enrichment analysis to construct a protein-protein interaction (PPI) network. The relationship between tumor immune infiltration and hub genes was analyzed by TIMER and CIBERSORT. Finally, it was validated in the GEO database and in tissues of Chinese pancreatic cancer patients. RESULTS: In the TCGA pancreatic cancer cohort, a low immune/stromal score was associated with a good prognosis. After bioinformatic analysis, 57 genes were identified to be significantly associated with pancreatic cancer prognosis. Among them, up-regulation of four genes (COL6A3, PLAU, MMP11 and MMP14) indicated poor prognosis and was associated with multiple immune cell infiltration. IHC results showed that PLAU protein levels from Chinese pancreatic cancer tissues were significantly higher than those from adjacent non-tumor tissues and were also associated with tumor TNM stage and lymph node metastasis. CONCLUSION: In conclusion, this study demonstrates that PLAU may serve as a new diagnostic and therapeutic target, which is highly expressed in Chinese pancreatic cancer tissues and associated with lymph node metastasis.

Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing.

The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100-200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.

The Potential Application of Branch-PCR Assembled PTEN Gene Nanovector in Lung Cancer Gene Therapy.

Gene therapy offers an alternative and promising avenue to lung cancer treatment. Here, we used dibenzocyclooctyne (DBCO)-branched primers to construct a PTEN gene nanovector (NP-PTEN) through branch-PCR. NP-PTEN showed the nanoscale structure with biocompatible size (84.7±11.2 nm) and retained the improved serum stability compared to linear DNA. When transfected into NCI-H1299 cancer cells, NP-PTEN could overexpress PTEN protein to restore PTEN functions through the deactivation of PI3K-AKT-mTOR signaling pathway to inhibit cell proliferation and induce cell apoptosis. The apoptosis rate of NCI-H1299 cancer cells could reach up to 54.5 %±4.6 % when the transfection concentration of NP-PTEN was 4.0 µg/mL. In mice bearing NCI-H1299 tumor xenograft intratumorally administrated with NP-PTEN, the average tumor volume and tumor weight was separately reduced by 61.7 % and 63.9 %, respectively, compared with the PBS group on the 18th  day of administration. The anticancer efficacy of NP-PTEN in NCI-H1299 tumor xenograft suggests the promising therapeutic potential of branch-PCR assembled PTEN gene nanovectors in lung cancer gene therapy and also provided more opportunities to introduce two or more tumor suppressor genes as an all-in-one gene nanovector for multiple gene-based cancer gene therapy.

Identification of a pyroptosis-related lncRNA risk model for predicting prognosis and immune response in colon adenocarcinoma.

BACKGROUND: Colon adenocarcinoma (COAD) is one of the most common malignant tumors and is diagnosed at an advanced stage with a poor prognosis worldwide. Pyroptosis is involved in the initiation and progression of tumors. This research focused on constructing a pyroptosis-related ceRNA network to generate a reliable risk model for risk prediction and immune infiltration analysis of COAD. METHODS: Transcriptome data, miRNA-sequencing data, and clinical information were downloaded from the TCGA database. First, differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), and lncRNAs (DElncRNAs) were identified to construct a pyroptosis-related ceRNA network. Second, a pyroptosis-related lncRNA risk model was developed applying univariate Cox regression analysis and least absolute shrinkage and selection operator method (LASSO) regression analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were utilized to functionally annotate RNAs contained in the ceRNA network. In addition, Kaplan-Meier analysis, receiver operating characteristic (ROC) curves, univariate and multivariate Cox regression, and nomogram were applied to validate this risk model. Finally, the relationship of this risk model with immune cells and immune checkpoint blockade (ICB)-related genes was analyzed. RESULTS: A total of 5373 DEmRNAs, 1159 DElncRNAs, and 355 DEmiRNAs were identified. A pyroptosis-related ceRNA regulatory network containing 132 lncRNAs, 7 miRNAs, and 5 mRNAs was constructed, and a ceRNA-based pyroptosis-related risk model including 11 lncRNAs was built. The tumor tissues were classified into high- and low-risk groups according to the median risk score. Kaplan-Meier analysis showed that the high-risk group had a shorter survival time; ROC analysis, independent prognostic analysis, and nomogram further indicated the risk model was a significant independent prognostic factor what had an excellent ability to predict patients' risk. Moreover, immune infiltration analysis indicated that the risk model was related to immune infiltration cells (i.e., B cell naïve, T cell follicular helper, macrophage M1) and ICB-related genes (i.e., PD-1, CTLA4, HAVCR2). CONCLUSIONS: This pyroptosis-related lncRNA risk model possessed good prognostic value, and the ability to predict the outcome of ICB immunotherapy in COAD.

Multiple-Gene Regulation for Enhanced Antitumor Efficacy with Branch-PCR-Assembled TP53 and MYC Gene Nanovector.

Multiple proteins are involved in network regulation through the crosstalk of different signaling pathways in cancers. Here, we propose a novel strategy of genome therapy with branch-PCR-assembled gene nanovectors to perform network-based gene regulation at multiple levels for cancer therapy. To validate network-based multiplex-gene regulation for genome therapy, we chose to simultaneously target one tumor suppressor gene (TP53) and one oncogene (MYC) in two different signaling pathways. The results showed that, compared to gene nanovectors targeting single genes (NP-TP53 and NP-shMYC), branch-PCR-assembled gene nanovectors simultaneously expressing p53 proteins and MYC shRNA arrays (NP-TP53-shMYC) showed enhanced antitumor efficacy in both MDA-MB-231 cancer cells and an MDA-MB-231-tumor-bearing mouse model. These findings indicate the feasibility and effectiveness of genome therapy in cancer therapy.

Synthesis of KUE-siRNA Conjugates for Prostate Cancer Cell-Targeted Gene Silencing.

The delivery of siRNAs to selectively target cells poses a great challenge in RNAi-based cancer therapy. The lack of suitable cell-targeting methods seriously restricts the advance in delivering siRNAs to extrahepatic tissues. Based on prostate-specific membrane antigen (PSMA)-targeting ligands, we have synthesized a series of lysine-urea-glutamate (KUE)-siRNA conjugates and verified their effective cell uptake and gene silencing properties in prostate cancers. The results indicated that the KUE-siRNA conjugates could selectively enter PSMA+ LNCaP cells, eventually down-regulating STAT3 expression. Based on post-synthesis modification and receptor-mediated endocytosis, this strategy of constructing ligand-siRNA conjugates might provide a general method of siRNA delivery for cell-targeted gene silencing.

High-spatiotemporal-resolution transcriptomes provide insights into fruit development and ripening in Citrus sinensis.

Citrus fruit has a unique structure with soft leathery peel and pulp containing vascular bundles and several segments with many juice sacs. The function and morphology of each fruit tissue are different. Therefore, analysis at the organ-wide or mixed-tissue level inevitably obscures many tissue-specific phenomena. High-throughput RNA sequencing was used to profile Citrus sinensis fruit development based on four fruit tissue types and six development stages from young fruits to ripe fruits. Using a coexpression network analysis, modules of coexpressed genes and hub genes of tissue-specific networks were identified. Of particular, importance is the discovery of the regulatory network of phytohormones during citrus fruit development and ripening. A model was proposed to illustrate how ABF2 mediates the ABA signalling involved in sucrose transport, chlorophyll degradation, auxin homoeostasis, carotenoid and ABA biosynthesis, and cell wall metabolism during citrus fruit development. Moreover, we depicted the detailed spatiotemporal expression patterns of the genes involved in sucrose and citric acid metabolism in citrus fruit and identified several key genes that may play crucial roles in sucrose and citric acid accumulation in the juice sac, such as SWEET15 and CsPH8. The high spatial and temporal resolution of our data provides important insights into the molecular networks underlying citrus fruit development and ripening.

Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

BACKGROUND AND AIMS: Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. APPROACH AND RESULTS: We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. CONCLUSIONS: We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.

Potential risks of rare serious adverse effects related to long-term use of bisphosphonates: An overview of systematic reviews.

WHAT IS KNOWN AND OBJECTIVE: There have been concerns about a potential link between long-term use of bisphosphonates (BPs) for the treatment of osteoporosis and rare serious adverse effects, such as atypical femoral fractures. However, many reviews exist with conflicting conclusions about this issue. The aim of this overview of reviews was to systematically evaluate the risk of rare serious adverse effects of long-term use of BPs for the treatment of osteoporosis. METHODS: We identified systematic reviews with meta-analyses of randomized controlled trials (RCTs) and (or) observational studies published in English or Chinese that evaluated the safety of BPs through to December 2018. The Cochrane library, PubMed, Web of Science and hand-searching of reference lists and clinical practice guidelines were electronically searched for data sources. Carcinogenicity, atypical fracture, osteonecrosis of jaw (ONJ) and fracture union time were specified as the primary outcomes. The methodological quality of each systematic review was assessed by two reviewers using the Assessment of Multiple Systematic Reviews (AMSTAR) tool, and the quality of evidence for key outcomes was assessed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. RESULTS AND DISCUSSION: In total, 1376 potentially relevant citations were identified, of which only 8 systematic reviews with meta-analyses met the eligibility criteria. All the included reviews were published between 2012 and 2015 and documented the pooled estimates of effect size (relative risk [RR], odds ratio [OR] or hazard ratio [HR] and their 95% confidence intervals [95% CI]) for the incidence of adverse events. All included systematic reviews were of moderate or high quality. The median AMSTAR score was 7.5 (interquartile range, 5-10). However, evidence of the key outcomes was mainly of very low or moderate quality. BP treatment only increased the risk of atypical fracture and ONJ, and prolonged union time compared with placebo or other anti-osteoporosis drugs (P ï¼œ .05). WHAT IS NEW AND CONCLUSION: This study indicated that long-term use of BPs could increase the risk of rare serious adverse effects, but the relationship between long-term use of BPs and carcinogenicity was often uncertain. Therefore, high-quality research and more complete inclusion criteria are needed.

MafG/MYH9-LCN2 axis promotes liver fibrosis through inhibiting ferroptosis of hepatic stellate cells.

Hepatic stellate cells (HSCs) secrete extracellular matrix for collagen deposition, contributing to liver fibrosis. Ferroptosis is a novel type of programmed cell death induced by iron overload-dependent lipid peroxidation. Regulation of ferroptosis in hepatic stellate cells (HSCs) may have therapeutic potential for liver fibrosis. Here, we found that Maf bZIP transcription factor G (MafG) was upregulated in human and murine liver fibrosis. Interestingly, MafG knockdown increased HSCs ferroptosis, while MafG overexpression conferred resistance of HSCs to ferroptosis. Mechanistically, MafG physically interacted with non-muscle myosin heavy chain IIa (MYH9) to transcriptionally activate lipocalin 2 (LCN2) expression, a known suppressor for ferroptosis. Site-directed mutations of MARE motif blocked the binding of MafG to LCN2 promoter. Re-expression of LCN2 in MafG knockdown HSCs restored resistance to ferroptosis. In bile duct ligation (BDL)-induced mice model, we found that treatment with erastin alleviated murine liver fibrosis by inducing HSC ferroptosis. HSC-specific knowdown MafG based on adeno-associated virus 6 (AAV-6) improved erastin-induced HSC ferroptosis and alleviation of liver fibrosis. Taken together, MafG inhibited HSCs ferroptosis to promote liver fibrosis through transcriptionally activating LCN2 expression. These results suggest that MafG/MYH9-LCN2 signaling pathway could be a novel targets for the treatment of liver fibrosis.

Association of high LDL concentrations with erectile dysfunction from a Mendelian randomization study.

Lipid metabolism plays a key role in erectile dysfunction. Our purpose was to evaluate the influence of lipid-lowering drugs on erectile dysfunction employing a two-sample Mendelian randomization (MR) study. Genetic instruments were employed to represent the exposure of lipid-lowering drugs. Inverse variance-weighted MR (IVWMR) was employed to calculate the estimation of effects. IVW-MR analysis showed that the positive relationship between the expression of HMGCR and the risk of erectile dysfunction (odds ratio [OR] = 1.27, 95% confidence interval [CI] 1.03-1.57; p = 0.028). No significant relationship was detected between NPC1L1, PSK9 expression and erectile dysfunction. This MR study suggested that HMGCR inhibitors are a more desirable treatment modality for patients with ED.

The role and regulation of Maf proteins in cancer.

The Maf proteins (Mafs) belong to basic leucine zipper transcription factors and are members of the activator protein-1 (AP-1) superfamily. There are two subgroups of Mafs: large Mafs and small Mafs, which are involved in a wide range of biological processes, such as the cell cycle, proliferation, oxidative stress, and inflammation. Therefore, dysregulation of Mafs can affect cell fate and is closely associated with diverse diseases. Accumulating evidence has established both large and small Mafs as mediators of tumor development. In this review, we first briefly describe the structure and physiological functions of Mafs. Then we summarize the upstream regulatory mechanisms that control the expression and activity of Mafs. Furthermore, we discuss recent studies on the critical role of Mafs in cancer progression, including cancer proliferation, apoptosis, metastasis, tumor/stroma interaction and angiogenesis. We also review the clinical implications of Mafs, namely their potential possibilities and limitations as biomarkers and therapeutic targets in cancer.

Comprehensive analysis of necroptosis-related lncRNA signature with potential implications in tumor heterogeneity and prediction of prognosis in clear cell renal cell carcinoma.

BACKGROUND: Necroptosis has been reported to play a critical role in occurrence and progression of cancer. The dysregulation of long non-coding RNAs (lncRNAs) is associated with the progression and metastasis of clear cell renal cell carcinoma (CCRCC). However, research on necroptosis-related lncRNAs in the tumor heterogeneity and prognosis of CCRCC is not completely unclear. This study aimed to analysis the tumor heterogeneity among CCRCC subgroups and construct a CCRCC prognostic signature based on necroptosis-related lncRNAs. METHODS: Weighted gene co-expression network analysis (WGCNA) was performed to identify necroptosis-related lncRNAs. A preliminary classification of molecular subgroups was performed by non-negative matrix factorization (NMF) consensus clustering analysis. Comprehensive analyses, including fraction genome altered (FGA), tumor mutational burden (TMB), DNA methylation alterations, copy number variations (CNVs), and single nucleotide polymorphisms (SNPs), were performed to explore the potential factors for tumor heterogeneity among the three subgroups. Subsequently, we constructed a predictive signature by multivariate Cox regression. Nomogram, calibration curves, decision curve analysis (DCA), and time-dependent receiver-operating characteristics (ROC) were used to validate and evaluate the signature. Finally, immune correlation analyses, including immune-related signaling pathways, immune cell infiltration status and immune checkpoint gene expression level, were also performed. RESULTS: Seven necroptosis-related lncRNAs were screened out by WGCNA, and three subgroups were classified by NMF consensus clustering analysis. There were significant differences in survival prognosis, clinicopathological characteristics, enrichments of immune-related signaling pathway, degree of immune cell infiltration, and expression of immune checkpoint genes in the various subgroups. Most importantly, we found that 26 differentially expressed genes (DEGs) among the 3 subgroups were not affected by DNA methylation alterations, CNVs and SNPs. On the contrary, these DEGs were associated with the seven necroptosis-related lncRNAs. Subsequently, the identified RP11-133F8.2 and RP11-283G6.4 by multivariate Cox regression analysis were involved in the risk model, which could serve as an independent prognostic factor for CCRCC. Finally, qRT-PCR confirmed the differential expression of the two lncRNAs. CONCLUSIONS: These findings contributed to understanding the function of necroptosis-related lncRNAs in CCRCC and provided new insights of prognostic evaluation and optimal therapeutic strategy for CCRCC.

DNA methylation-mediated silencing of Neuronatin promotes hepatocellular carcinoma proliferation through the PI3K-Akt signaling pathway.

AIMS: To explore the methylation status, function, and underlying mechanism of the imprinted gene Neuronatin (NNAT) in hepatocellular carcinoma (HCC) progression. MAIN METHODS: Immunohistochemistry (IHC) was performed to evaluate the expression of NNAT in HCC samples. Bisulfite genomic sequencing PCR (BSP) was applied to examine the methylation status of the NNAT promoter. In addition, colony formation, 5-Ethynyl-20-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of NNAT in HCC cell proliferation. Furthermore, RNA-seq and phospho-specific protein microarray assays were conducted to illustrate the underlying mechanism by which NNAT regulates HCC progression. KEY FINDINGS: NNAT was obviously downregulated in HCC tissues, and its expression level was closely associated with tumor growth and patient prognosis. The downregulation of NNAT in HCC was induced by hypermethylation of CpG islands in the promoter region, and hypermethylation was correlated with overall survival of HCC. Moreover, the enforced expression of NNAT significantly inhibited HCC cell proliferation in vitro and in vivo. Transcriptome analysis showed that the alteration of NNAT expression was mainly related to dysregulation of the PI3K-Akt signaling pathway. Finally, phospho-specific antibody microarray detection further revealed that overexpressed NNAT can increase the phosphorylation levels of LKB1, Met, and elF4E and decrease the phosphorylation levels of PTEN, which are all involved in the PI3K-Akt signaling pathway. SIGNIFICANCE: Our research provides new insights into the epigenetic regulation of imprinted genes in tumorigenesis and implies that the imprinted gene NNAT may act as a prognostic biomarker and tumor suppressor in HCC.

Effectiveness and safety of pembrolizumab for patients with advanced non-small cell lung cancer in real-world studies and randomized controlled trials: A systematic review and meta-analysis.

Background: Several randomized controlled trials (RCTs) have confirmed the favorable clinical benefit of pembrolizumab in advanced non-small cell lung cancer (NSCLC). However, considering the strict inclusion and exclusion criteria in clinical research, there are certain differences between patients in the real-world, it is unclear whether the findings of clinical trials are fully representative of the treatment efficacy in patients who will eventually use it. Therefore, to further comprehensively assess the efficacy and safety of pembrolizumab in NSCLC, we conducted a systematic review and meta-analysis based on the latest RCTs and real-world studies (RWSs). Methods: We systematically searched PubMed, Embase, The Cochrane Library, The Web of Science, and clinical trials.gov as of December 2021. RCTs and RWSs of patients receiving pembrolizumab monotherapy or in combination with chemotherapy for advanced NSCLC were included. Results: The meta-analysis ultimately included 11 RCTs and 26 RWSs with a total of 10,695 patients. The primary outcomes of this study were overall survival (OS), progression-free survival (PFS), objective response rate (ORR), serious adverse events (SAEs), the incidence of severe pneumonia reactions, and drug-related mortality. Direct meta-analysis results showed that in RCTs, pembrolizumab in combination with chemotherapy was superior to chemotherapy in terms of OS (HR=0.60, 95%CI:0.50-0.73), PFS (HR=0.47, 95%CI:0.38-0.58) and ORR (OR=3.22, 95%CI:2.57-4.03); pembrolizumab monotherapy was superior to chemotherapy in terms of OS (HR=0.73, 95%CI:0.66-0.80) and ORR (OR=1.90, 95%CI:1.17-3.09), but comparable to chemotherapy in terms of PFS (HR=0.83, 95%CI:0.66-1.04). The ORR values in retrospective single-arm studies were 45% (40%-51%). Conclusion: In RCTs, pembrolizumab monotherapy or in combination with chemotherapy is more effective and safer than chemotherapy for advanced NSCLC. In RWSs, ECOG PS 0-1 was shown to correlate with PFS and OS for patients with NSCLC.