Extracellular ATP contributes to the reactive oxygen species burst and exaggerated mitochondrial damage in D-galactosamine and lipopolysaccharide-induced fulminant hepatitis.

Fulminant hepatitis (FH) is a severe clinical syndrome leading to hepatic failure and even mortality. D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) challenge is commonly used to establish an FH mouse model, but the mechanism underlying D-GalN/LPS-induced liver injury is incompletely understood. Previously, it has been reported that extracellular ATP that can be released under cytotoxic and inflammatory stresses serves as a damage signal to induce potassium ion efflux and trigger the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome activation through binding to P2X7 receptor. In this study, we tried to investigate whether it contributed to the fulminant hepatitis (FH) induced by D-GalN plus LPS. In an in vitro cellular model, D-GalN plus extracellular ATP, instead of D-GalN alone, induced pyroptosis and apoptosis, accompanied by mitochondrial reactive oxygen species (ROS) burst, and the oligomerization of Drp1, Bcl-2, and Bak, as well as the loss of mitochondrial membrane potential in LPS-primed macrophages, well reproducing the events induced by D-GalN and LPS in vivo. Moreover, these events in the cellular model were markedly suppressed by both A-804598 (an ATP receptor P2X7R inhibitor) and glibenclamide (an ATP-sensitive potassium ion channel inhibitor); in the FH mouse model, administration of A-804598 significantly mitigated D-GalN/LPS-induced hepatic injury, mitochondrial damage, and the activation of apoptosis and pyroptosis signaling, corroborating the contribution of extracellular ATP to the cell death. Collectively, our data suggest that extracellular ATP acts as an autologous damage-associated molecular pattern to augment mitochondrial damage, hepatic cell death, and liver injury in D-GalN/LPS-induced FH mouse model.

Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION: P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

ZNF471 Interacts with BANP to Reduce Tumour Malignancy by Inactivating PI3K/AKT/mTOR Signalling but is Frequently Silenced by Aberrant Promoter Methylation in Renal Cell Carcinoma.

Background: Renal cell carcinoma (RCC) is one of the most common malignant tumours of the urinary system. However, the aetiology and pathogenesis of RCC remain unclear. The C2H2 zinc finger protein (ZNF) family is the largest transcriptional regulatory factor family found in mammals, and Krüppel-associated box domain-containing zinc finger proteins (KRAB-ZFPs) constitute the largest subfamily of the C2H2 zinc finger protein family and play an important role in the occurrence and development of tumours. The aim of this study was to explore the role of abnormal methylation of ZNF471 in the development of renal carcinoma. Methods: In this study, we first used the TCGA and EWAS Data Hub databases to analyse the expression and methylation levels of ZNF471 in renal carcinoma tissues and adjacent normal tissues. Second, we collected samples of renal cancer and adjacent normal tissues at Peking University First Hospital to investigate the expression and methylation level of ZNF471 in renal cancer tissues and the relationships between these levels and the clinicopathological features and prognosis of patients with renal cancer. Next, we investigated the effects of ZNF471 on the proliferation, metastasis, cell cycle progression, and apoptosis of renal cell carcinoma cells by cell biology experiments. Finally, we elucidated the underlying molecular mechanisms of ZNF471 in renal cell carcinoma by transcriptome sequencing, bioinformatics analysis and molecular biology experiments. Results: The expression of ZNF471 in renal carcinoma tissues and cell lines was significantly lower than that in adjacent normal tissues and cell lines due to abnormal promoter CpG methylation. Furthermore, the expression of ZNF471 in renal carcinoma tissues was negatively correlated with tumour stage and grade in patients with renal carcinoma. The results of the cell biology experiments showed that ZNF471 could significantly inhibit the proliferation, migration and cell cycle progression of renal cell carcinoma cells and promote apoptosis in these cells. In addition, ZNF471 could interact with BANP and suppress the malignant phenotype of RCC by inactivating the PI3K/AKT/mTOR signalling pathway. Conclusions: As an important tumour suppressor, ZNF471 can interact with BANP in renal cancer cells and inhibit the activation of the PI3K/AKT/mTOR signalling pathway, thereby inhibiting the occurrence and development of renal cancer.

First Report of Lecanicillium aphanocladii Causing Rot of Sparassis crispa in China.

Sparassis crispa, also known as cauliflower mushroom, is a new popularly edible mushroom in China, also a medicinal mushroom, which possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. (Han et al., 2018). In recent years, the artificial cultivation of S. crispa has gained considerable public attention in China. In 2023, approximately 20% of S. crispa (about 0.05 ha of the planting area) showed obvious rot with white molds symptoms in mushroom hothouse, located in Shuangliu county, Sichuan province, China (GPS, 104°7'51"N, 30°25'2"E). Infected fruiting bodies were covered by white mycelia that later turned red or fuchsia. In the final stages of infection, the S. crispa fruiting bodies turned dark red or brown before rotting. The pathogen was isolated from the margin of the lesions by plating onto potato dextrose agar (PDA), and incubated at 25℃ in the dark for a week. Five pure culture fungal isolates were obtained. Collected isolates with similar morphology were described as Lecanicillium spp. (Zare et al., 2001). The colonies were raised, covered with white, the reverse side were violet brown, produced diffusing reddish-purple pigment. Conidiogenous cells produced singly, in pairs, verticillate or in dense irregular clusters on prostrate hyphae, at first flask-shaped, tapering into threadlike neck, with a size of 3.0-6.2×0.8-2.2 µm. Conidia were solitary, oval to subglobose, and 2.3-4.0×1.1-2.1 µm in size, similar to L. aphanocladii (Higo et al., 2021). For pathogenicity testing, ten fruiting bodies of S. crispa (planted in the bottles) were selected. Fungal cake of the isolate Bx-Ljb of L. aphanocladii were applied to the fruiting body of S. crispa, whereas pieces of sterile PDA medium were used as controls. All the bottles were incubated at 19±1℃, 85-100% relative humidity, and 18 h of light in the mushroom hothouse. A week later, the inoculated fruiting bodies developed brown spots and gradually expanding, with symptoms similar to the original diseased fruiting bodies. The controls remained healthy. The same fungus was reisolated from the infected fruiting bodies and subsequently identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. For molecular identification, the DNA of the isolates was extracted using a Fungi Genomic DNA Extraction kit (Solarbio, Beijing). The SSU, LSU, and TEF1-α genes were amplified with the primer as previously described (Zhou et al., 2018). The generated sequences were deposited in GenBank with accession numbers OR206377, OR206378, and OR204702, respectively. BLASTn analyses showed >99.2% identity with previously deposited sequences of L. aphanocladii. Based on the maximum likelihood method, phylogenetic analysis revealed 99% bootstrap support values with L. aphanocladii. The fungus was identified as L. aphanocladii based on morphological and multilocus phylogenetic analyses. To our knowledge, there are two reports of L. aphanocladii on fruiting bodies of Tremella fuciformis and Morchella sextelata in China, and this is the first report of this fungus causing rot of S. crispa in China. It may be a reminder that the risk of L. aphanocladii in mushroom production in China is gradually increasing. These results will contribute to developing managemental strategies for this disease in S. crispa.

Combining pathological risk factors and T, N staging to optimize the assessment for risk stratification and prognostication in low-risk stage III colon cancer.

BACKGROUND: This study aimed to investigate the combined pathological risk factors (PRFs) to stratify low-risk (pT1-3N1) stage III colon cancer (CC), providing a basis for individualized treatment in the future. PATIENTS AND METHODS: PRFs for low-risk stage III CC were identified using COX model. Low-risk stage III CC was risk-grouped combining with PRFs, and survival analysis were performed using Kaplan-Meier. The Surveillance, Epidemiology, and End Results (SEER) databases was used for external validation. RESULTS: Nine hundred sixty-two stage III CC patients were included with 634 (65.9%) as low risk and 328 (34.1%) as high risk. Poor differentiation (OS: P = 0.048; DFS: P = 0.011), perineural invasion (OS: P = 0.003; DFS: P < 0.001) and tumor deposits (OS: P = 0.012; DFS: P = 0.003) were identified as PRFs. The prognosis of low-risk CC combined with 2 PRFs (OS: HR = 3.871, 95%CI, 2.004-7.479, P < 0.001; DFS: HR = 3.479, 95%CI, 2.158-5.610, P < 0.001) or 3 PRFs (OS: HR = 5.915, 95%CI, 1.953-17.420, P = 0.002; DFS: HR = 5.915, 95%CI, 2.623-13.335, P < 0.001) was similar to that of high-risk CC (OS: HR = 3.927, 95%CI, 2.317-6.656, P < 0.001; DFS: HR = 4.132, 95%CI, 2.858-5.974, P < 0.001). In the SEER database, 18,547 CC patients were enrolled with 10,023 (54.0%) as low risk and 8524 (46.0%) as high risk. Low-risk CC combined with 2 PRFs (OS: HR = 1.857, 95%CI, 1.613-2.139, P < 0.001) was similar to that of high-risk CC without PRFs (HR = 1.876, 95%CI, 1.731-2.033, P < 0.001). CONCLUSION: Combined PRFs improved the risk stratification of low-risk stage III CC, which could reduce the incidence of undertreatment and guide adjuvant chemotherapy.

Potassium ion efflux induces exaggerated mitochondrial damage and non-pyroptotic necrosis when energy metabolism is blocked.

Damage-associated molecular patterns (DAMPs) such as extracellular ATP and nigericin (a bacterial toxin) not only act as potassium ion (K+) efflux inducers to activate NLRP3 inflammasome, leading to pyroptosis, but also induce cell death independently of NLRP3 expression. However, the roles of energy metabolism in determining NLRP3-dependent pyroptosis and -independent necrosis upon K+ efflux are incompletely understood. Here we established cellular models by pharmacological blockade of energy metabolism, followed by stimulation with a K+ efflux inducer (ATP or nigericin). Two energy metabolic inhibitors, namely CPI-613 that targets α-ketoglutarate dehydrogenase and pyruvate dehydrogenase (a rate-limiting enzyme) and 2-deoxy-d-glucose (2-DG) that targets hexokinase, are recruited in this study, and Nlrp3 gene knockout macrophages were used. Our data showed that CPI-613 and 2-DG dose-dependently inhibited NLRP3 inflammasome activation, but profoundly increased cell death in the presence of ATP or nigericin. The cell death was K+ efflux-induced but NLRP3-independent, which was associated with abrupt reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, and oligomerization of mitochondrial proteins, all indicating mitochondrial damage. Notably, the cell death induced by K+ efflux and blockade of energy metabolism was distinct from pyroptosis, apoptosis, necroptosis or ferroptosis. Furthermore, fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, significantly suppressed CPI-613+nigericin-induced mitochondrial damage and cell death. Collectively, our data show that energy deficiency diverts NLRP3 inflammasome activation-dependent pyroptosis to Nlrp3-independent necrosis upon K+ efflux inducers, which can be dampened by high-energy intermediate, highlighting a critical role of energy metabolism in cell survival and death under inflammatory conditions.

Tax1 binding protein 3 regulates osteogenic and adipogenic differentiation through inactivating Wnt/ß-catenin signalling.

Tax1 binding protein 3 (Tax1bp3) is a PDZ domain-containing protein that is overexpressed in cancer. Previous studies recognized Tax1bp3 as an inhibitor of ß-catenin. Till now it is not known whether Tax1bp3 regulates osteogenic and adipogenic differentiation of mesenchymal progenitor cells. In the current study, the data showed that Tax1bp3 was expressed in bone and was increased in the progenitor cells when induced toward osteoblast and adipocyte differentiation. The overexpression of Tax1bp3 in the progenitor cells inhibited osteogenic differentiation and conversely stimulated adipogenic differentiation, and the knockdown of Tax1bp3 affected the differentiation of the progenitor cells oppositely. Ex vivo experiments using the primary calvarial osteoblasts from osteoblast-specific Tax1bp3 knock-in mice also demonstrated the anti-osteogenic and pro-adipogenic function of Tax1bp3. Mechanistic investigations revealed that Tax1bp3 inhibited the activation of canonical Wnt/ß-catenin and bone morphogenetic proteins (BMPs)/Smads signalling pathways. Taken together, the current study has provided evidences demonstrating that Tax1bp3 inactivates Wnt/ß-catenin and BMPs/Smads signalling pathways and reciprocally regulates osteogenic and adipogenic differentiation from mesenchymal progenitor cells. The inactivation of Wnt/ß-catenin signalling may be involved in the reciprocal role of Tax1bp3.

Pyroptosis-Related Gene Signature Predicts the Prognosis of ccRCC Using TCGA and Single-Cell RNA Seq Database.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent type of renal carcinoma, which is not sensitive to both radiotherapy and chemotherapy. The objective response rate of metastatic renal cancer to targeted drugs and immunotherapy is unsatisfactory. Pyroptosis, proven as an inflammatory form of programmed cell death, could be activated by some inflammasomes, while could create a tumor-suppressing environment by releasing inflammatory factors in the tumor. To explore indicators predicting the prognosis of ccRCC and the effect of antitumor therapy, we constructed a pyroptosis risk model containing 4 genes after 11 pyroptosis-related genes of 516 ccRCC cases in the TCGA database were scanned. Based on the risk score, 516 ccRCC cases were divided into two groups for functional enrichment analysis and immune profile to seek functional pathways and potential therapeutic targets. Besides, those results were verified in GSE29609 and single-cell transcriptomic data. The study suggests that the conducted pyroptosis model could predict the prognosis of ccRCC and reflect the immune microenvironment, which may help in immune checkpoint inhibitor treatment.

Silver nano-reporter enables simple and ultrasensitive profiling of microRNAs on a nanoflower-like microelectrode array on glass.

MicroRNAs (miRNAs) are small non-coding RNAs with ~ 22 nucleotides, playing important roles in the post-transcriptional regulation of gene expression. The expression profiles of many miRNAs are closely related to the occurrence and progression of cancer and can be used as biomarkers for cancer diagnosis and prognosis. However, their intrinsic properties, such as short length, low abundance and high sequence homology, represent great challenges in miRNA detection of clinical samples. To overcome these challenges, we developed a simple, ultrasensitive detection platform of electrochemical miRNAs chip (e-miRchip) with a novel signal amplification strategy using silver nanoparticle reporters (AgNRs) for multiplexed, direct, electronic profiling of miRNAs. A two-step hybridization strategy was used to detect miRNAs, where the target miRNA hybridizes with a stem-loop probe to unlock the probe first, and the opened stem-loop can further hybridize with AgNRs for signaling amplification. To enhance the detection sensitivity, the gold nanoflower electrodes (GNEs) were constructed in the microaperture arrays of the e-miRchips by electroplating. With the optimal size of the GNEs, the e-miRchip showed excellent performance for miR-21 detection with a detection limit of 0.56 fM and a linear range extended from 1 fM to 10 pM. The e-miRchip also exhibited good specificity in differentiating the 3-base mismatched sequences of the target miRNA. In addition, the e-miRchip was able to directly detect miR-21 expression in the total RNA extracts or cell lysates collected from lung cancer cells and normal cells. This work demonstrated the developed e-miRchip as an efficient and promising miniaturized point-of-care diagnostic device for the early diagnosis and prognosis of cancers.

ZNF677 suppresses renal cell carcinoma progression through N6-methyladenosine and transcriptional repression of CDKN3.

BACKGROUND: Studies on biological functions of N6-methyladenosine (m6 A) modification in mRNA have sprung up in recent years. Previous studies have reported m6 A can determine mRNA fate and play a pivotal role in tumour development and progression. The zinc finger protein 677 (ZNF677) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence-specific DNA. METHODS: The expression of ZNF677 and its clinicopathological impact were evaluated in renal cell carcinoma (RCC) patients. The m6 A level of ZNF677 was determined by m6 A methylated RNA immunoprecipitation-sequencing (MeRIP-seq) and MeRIP-qPCR in RCC tissues and adjacent normal tissues. RNA immunoprecipitation-qPCR (RIP-qPCR) and luciferase assays were performed to identify the targeted effect of IGF2BP2 and YTHDF1 on ZNF677. RCC cells and subcutaneous models uncovered the role of ZNF677 methylated by CRISPR/dCas13b-METTL3 in tumour growth. ZNF677-binding sites in the CDKN3 promoter were investigated by chromatin immunoprecipitation (ChIP) and luciferase assays. RESULTS: ZNF677 is frequently downregulated in RCC tissues and its low expression is associated with unfavourable prognosis and decreased m6 A modification level. Further, we find the m6 A-modified coding sequence (CDS) of ZNF677 positively regulates its translation and mRNA stability via binding with YTHDF1 and IGF2BP2, respectively. Targeted specific methylation of ZNF677 m6 A by CRISPR/dCas13b-METLL3 system can significantly increase the m6 A and expression level of ZNF677, and dramatically inhibit cell proliferation and induce cell apoptosis of RCC cells. In addition, ZNF677 exerted its tumour suppressor functions in RCC cells through transcriptional repression of CDKN3 via binding to its promoter. In vitro and clinical data confirm the negative roles of ZNF677/CDKN3 in tumour growth and progression of RCC. CONCLUSION: ZNF677 functions as a tumour suppressor and is frequently silenced via m6 A modification in RCC, which may highlight m6 A methylation-based approach for RCC diagnosis and therapy.

Precisely NIR-II-activated and pH-responsive cascade catalytic nanoreactor for controlled drug release and self-enhanced synergetic therapy.

Mesoporous polydopamine (MPDA) and MPDA-based nanosystems have been widely used in the field of photothermal therapy (PTT) and drug delivery. However, synthesis of the corresponding nanoplatforms for efficient PTT and controlled drug release simultaneously in the second near infrared (NIR-II) region remains a great challenge. Herein, a NIR-II and pH dual-responsive HMPDA@Cu2-xSe cascade catalytic nanoplatform was constructed by assembling hollow mesoporous polydopamine (HMPDA) with ultra-small Cu2-xSe, which could compensate the inadequate NIR-II-induced PTT effect of HMPDA and enhance the efficacy of chemodynamic therapy (CDT) simultaneously under NIR-II laser irradiation. Meanwhile, doxorubicin (DOX) and glucose oxidase (GOx) were encapsulated into the synthesized HMPDA@Cu2-xSe using the photothermal-induced phase change material (PCM) tetradecyl (1-TD) as a gatekeeper to achieve the controlled release of the cargo. Under 1064 nm laser, the generated heat could cause 1-TD melting, resulting in the release of large amounts of DOX and GOx. The released GOx could further catalyze glucose to H2O2 and gluconic acid, which in turn promoted the effects of PTT/CDT and the release of drugs. In vitro and in vivo experiments showed that the synthesized HMPDA@Cu2-xSe-DOX-GOx@PCM (HMPC-D/G@PCM) nanosystem exhibited a significant tumor cell inhibition effect by combining different treatment modes. Thus, this smart nanoplatform with multiple stimuli-activated cascade reactions provided a new idea for designing effective multi-modal combination therapy for tumors.

Metastasis suppressor 1 controls osteoblast differentiation and bone homeostasis through regulating Src-Wnt/ß-catenin signaling.

Metastasis suppressor 1 (MTSS1) plays an inhibitory role in tumorigenesis and metastasis of a variety of cancers. To date, the function of MTSS1 in the differentiation of marrow stromal progenitor cells remains to be explored. In the current study, we investigated whether and how MTSS1 has a role in osteoblast differentiation and bone homeostasis. Our data showed that MTSS1 mRNA was upregulated during osteoblast differentiation and downregulated in the osteoblastic lineage cells of ovariectomized and aged mice. Functional studies revealed that MTSS1 promoted the osteogenic differentiation from marrow stromal progenitor cells. Mechanistic explorations uncovered that the inactivation of Src and afterward activation of canonical Wnt signaling were involved in osteoblast differentiation induced by MTSS1. The enhanced osteogenic differentiation induced by MTSS1 overexpression was attenuated when Src was simultaneously overexpressed, and conversely, the inhibition of osteogenic differentiation by MTSS1 siRNA was rescued when the Src inhibitor was supplemented to the culture. Finally, the in vivo transfection of MTSS1 siRNA to the marrow of mice significantly reduced the trabecular bone mass, along with the reduction of trabecular osteoblasts, the accumulation of marrow adipocytes, and the increase of phospho-Src-positive cells on the trabeculae. No change in the number of osteoclasts was observed. This study has unraveled that MTSS1 contributes to osteoblast differentiation and bone homeostasis through regulating Src-Wnt/ß-catenin signaling. It also suggests the potential of MTSS1 as a new target for the treatment of osteoporosis.

A multifunctional nanoamplifier with self-enhanced acidity and hypoxia relief for combined photothermal/photodynamic/starvation therapy.

Phototherapies, including photothermal therapy (PTT) and photodynamic therapy (PDT) have been potential noninvasive therapeutic modality with high efficiency, however, there still exist some intrinsic limitations that impede their clinical applications. Herein, taking the advantages of the synergistic effect and high reactivity of manganese dioxide (MnO2) nanosheets and glucose oxidase (GOx), multifunctional MPDA@MnO2-MB-GOx nanoamplifier was constructed for enhanced PTT, PDT, and starvation therapy. In tumor microenvironment (TME), MnO2 nanosheets on the surface of mesoporous polydopamine (MPDA) could react with endogenous hydrogen peroxide (H2O2) and generate oxygen (O2) to relieve tumor hypoxia, thus enhancing the efficacy of PDT and GOx catalysis. Glucose consumption under the catalysis of GOx will enhance the acidity of TME and increase intracellular H2O2 concentration, which in turn promotes the production of O2 by MnO2 nanosheets, thus forming efficient cascade reaction and maximizing the efficacy of the functional agents. Furthermore, the heat generated by MPDA under the irradiation of 808 nm laser can accelerate chemical reactions, thus further enhancing synergistic therapeutic efficacy. In vitro/vivo results emphasize that enhanced cancer cell death and tumor inhibition are gained by modulating unfavorable TME with the functional nanosystem, which highlights the promise of the synthesized MPDA@MnO2-MB-GOx nanomaterial to overcome the limitations of phototherapy.

Transcriptome-Wide Map of N6-Methyladenosine Methylome Profiling in Human Bladder Cancer.

N6-Methyladenosine (m6A) is the most widespread internal RNA modification in several species. In spite of latest advances in researching the biological roles of m6A, its function in the development and progression of bladder cancer remains unclear. In this study, we used MeRIPty -55-seq and RNA-seq methods to obtain a comprehensive transcriptome-wide m6A profiling and gene expression pattern in bladder cancer and paired normal adjacent tissues. Our findings showed that there were 2,331 hypomethylated and 3,819 hypermethylated mRNAs, 32 hypomethylated and 105 hypermethylated lncRNAs, and 15 hypomethylated and 238 hypermethylated circRNAs in bladder cancer tissues compared to adjacent normal tissues. Furthermore, m6A is most often harbored in the coding sequence (CDS), with some near the start and stop codons between two groups. Functional enrichment analysis revealed that differentially methylated mRNAs, lncRNAs, and circRNAs were mostly enriched in transcriptional misregulation in cancer and TNF signaling pathway. We also found that different m6A methylation levels of gene might regulate its expression. In summary, our results for the first time provide an m6A landscape of human bladder cancer, which expand the understanding of m6A modifications and uncover the regulation of mRNAs, lncRNAs, and circRNAs through m6A modification in bladder cancer.

Silencing of the TRIM58 Gene by Aberrant Promoter Methylation is Associated with a Poor Patient Outcome and Promotes Cell Proliferation and Migration in Clear Cell Renal Cell Carcinoma.

To investigate the underlying molecular mechanism of tripartite motif-containing 58 (TRIM58) in the development of clear cell renal cell carcinoma (ccRCC), we explored TRIM58 expression and methylation in tumor tissues and the association with clinicopathological features and prognosis of tissue samples; Moreover, we examined the direct gene transcription of TRIM58-specific DNA demethyltransferase (TRIM58-TET1) by the CRISPR-dCas9 fused with the catalytic domain of TET1 and the biological functions in RCC cells. In this study, we demonstrate that TRIM58 is frequently downregulated by promoter methylation in ccRCC tissues, associated significantly with tumor nuclear grade and poor patient survival. TRIM58-TET1 directly induces demethylation of TRIM58 CpG islands, and activates TRIM58 transcription in RCC cell lines. Besides, DNA demethylation of TRIM58 by TRIM58-TET1 significantly inhibits cell proliferation and migration Overall, our results demonstrate that TRIM58 is inactivated by promoter methylation, associates with tumor nuclear grade and poor survival, and TRIM58 DNA demethylation could directly activate TRIM58 transcription and inhibit cell proliferation and migration in RCC cell lines.

m6A-mRNA Methylation Regulates Gene Expression and Programmable m6A Modification of Cellular RNAs With CRISPR-Cas13b in Renal Cell Carcinoma.

Background: N6-methyladenosine (m6A) is the most extensive messenger RNA modification. Despite recent advances in the biological roles of m6A, its role in the development and progression of renal cell carcinoma (RCC) remains unclear. Methods: In this study, we gained the transcriptome-wide m6A profile and gene expression pattern in RCC and paired adjacent peritumoral tissues by meRIP-seq and RNA-seq. m6A modifications of mRNAs were validated by meRIP-qPCR in tissues, and targeted methylation or demethylation was validated by using a CRISPR-Cas13b-based tool in RCC cell lines. Results: Our findings showed that there were 13,805 m6A peaks among 5,568 coding gene transcripts (mRNAs) in adjacent tissues and 24,730 m6A peaks among 6,866 mRNAs in tumor tissues. Furthermore, m6A modification sites were usually located in the coding sequences (CDS), and some near the start and stop codons. Gene Ontology analysis revealed that coding genes had differential N6-methyladenosine sites and were enriched in kidney development and cancer-related signaling pathways. We also found that different levels of m6A modifications could regulate gene expression. Conclusion: In summary, our results provided evidence for studying the potential function of RNA m6A modification and m6A-mediated gene expression regulation in human RCC.

NIR-/pH-Responsive Nanocarriers Based on Mesoporous Hollow Polydopamine for Codelivery of Hydrophilic/Hydrophobic Drugs and Photothermal Synergetic Therapy.

Combined therapy system has become an efficient strategy to overcome drug resistance and strengthen therapeutic effects. Herein, an efficient NIR-/pH-triggered dual-drug-loaded nanoplatform was designed for combined chemo-photothermal therapy. The hydrophobic anticancer drug bortezomib (BTZ) was first loaded in mesoporous polydopamine nanospheres (MPDAs) through the acid-sensitive borate ester bond. Afterward, pH-responsive carboxymethyl chitosan (CMCS) conjugated on the surface of MPDA could capture another anticancer drug doxorubicin (DOX) and exhibited controlled release behavior in an acidic tumor microenvironment. Meanwhile, under NIR laser irradiation, hyperthermia produced by the photothermal conversion agent MPDA could efficiently ablate cancer cells and further promote drug release. In vitro and in vivo experiments emphasized that the synthesized MPDA-BTZ@CMCS-DOX nanostructure exhibited efficient accumulation in the tumor site, resulting in sustained release of BTZ and DOX and realizing NIR-/pH-triggered chemotherapy and photothermal synergistic ablation of cancer.

Synthetic Artificial Long Non-coding RNA Shows Higher Efficiency in Specific Malignant Phenotype Inhibition Compared to the CRISPR/Cas Systems.

Objective: Both oncogenic transcription factors (TFs) and microRNAs (miRNAs) play an important regulator in human cancer by transcriptional and post-transcriptional regulation, respectively. These phenomena raise questions about the ability of artificial device to regulate miRNAs and TFs simultaneously. In this study, we aimed to construct an artificial long non-coding RNA, "alncRNA," which imitated CRISPR/Cas systems and to illuminate its therapeutic effects in bladder cancer cell lines. At the same time, we also compared the efficiency of alncRNA and CRISPR/Cas systems in regulating gene expression. Study Design and Methods: Based on engineering principles of synthetic biology, we combined tandem arrayed cDNA sequences of aptamer for TFs with tandem arrayed cDNA copies of binding sites for the miRNAs to construct alncRNA. In order to prove the utility of this platform, we chose ß -catenin, NF-κB, miR-940, and miR-495 as the functional targets and used the bladder cancer cell lines 5637 and T24 as the test models. Real-time Quantitative PCR (qPCR), dual-luciferase assay and relative phenotypic experiments were applied to severally test the expression of relative gene and therapeutic effects of our devices. Result: Dual-luciferase assay indicated alncRNA could inhibit transcriptional activity of TFs. What's more, the result of qPCR showed that expression levels of the relative TFs target genes and miRNAs were reduced by corresponding alncRNA and the inhibitory effect was better than CRIPSR dCas9-KRAB. By functional experiments, decreased cell proliferation, increased apoptosis, and motility inhibition were observed in alncRNA-infected bladder cells. Conclusion: In summary, our synthetic devices indeed function as anti-tumor regulator, which synchronously accomplish transcriptional and post-transcriptional regulation in bladder cancer cell and show higher efficiency in specific malignant phenotype inhibition compared to the CRISPR/Cas systems. Most importantly, Anti-cancer effects were induced by the synthetic alncRNA in the bladder cancer lines. Our devices, therefore, provides a novel strategy for cancer therapy and could be a useful "weapon" for cancer cell.

[Perpendicular and transverse needling of "Sanyinjiao" (SP6) relieves abdominal pain by regulating arginine vasopressin and its receptor expression levels in uterus and hypothalamus in cold-stasis type dysmenorrhea rats].

OBJECTIVE: To observe the effect of perpendicular and subcutaneous transverse needling at "Sanyinjiao" (SP6) on visceral pain behavior, arginine vasopressin (AVP) content in the serum, uterine tissues, spinal cord and hypothalamus and expression of AVP receptors AVPR1A and AVPR1B in the uterine tissues, spinal cord and hypothalamus in cold-stasis (stasis due to pathogenic cold) type dysmenorrhea rats, so as to explore their mechanisms underlying pain relief. METHODS: Forty female SD rats were randomly divided into blank control, model, perpendicular needling and transverse needling groups, with 10 rats in each group. The cold-stasis dysmenorrhea rat model was established by exposure in a freezer (-25 ℃) for 4 h, once daily for 5 days, and subcutaneous injection of estradiol benzoate (once daily for 10 days) and intra-abdominal injection of oxytocin injection (once). For rats of the two acupuncture groups, acupuncture needles were inserted into the bilateral SP6 perpendicularly or transversely to a depth of about 4-5 mm, and retained for 20 min. The abdominal pain behavior was assessed by recording the writhing latency and scaling the rats' writhing reactions after modeling. The contents of AVP in the serum, uterus, spinal cord and hypothalamus tissues were assayed using ELISA and the expression of AVPR1A and AVPR1B in the uterus, spinal cord and hypothalamus was measured by using Western blot and quantitative real time-PCR, respectively. RESULTS: After mode-ling and compared with the blank control group, the writhing latency was significantly shortened (P<0.05), and the writhing score in the first 20 min was significantly increased (P<0.01) in the model group. After the intervention, the writhing latency was significantly prolonged (P<0.01), and the writhing scores in 20 min were significantly decreased (P<0.01) in the two needling groups. The AVP contents were obviously increased in the serum and uterine tissue (P<0.05, P<0.01) but decreased appa-rently in the spinal cord and hypothalamus tissues (P<0.01, P<0.05), and the expression levels of AVPR1A or AVPR1B protein and mRNA were markedly increased in the uterine tissues (P<0.01, P<0.05), and significantly decreased in the spinal cord and hypotha-lamus in the model group relevant to the control group (P<0.05, P<0.01). Following the intervention, The AVP content in the serum of the perpendicular needling group (P<0.05) and that in the uterus of the two needling groups were significantly decreased (P<0.01), as well as that in the hypothalamus was obviously increased in the two needling groups (P<0.05). The expression levels of AVPR1A protein and mRNA in the uterus were significantly down-regulated in the two needling groups (P<0.01, P<0.05) and AVPR1B protein in the hypothalamus of the perpendicular needling group was up-regulated (P<0.05). Moreover, no significant differences were found between the two needling groups in regulating the related indexes mentioned above (P>0.05). CONCLUSION: Both perpendicular and subcutaneous transverse needling at SP6 have an immediate analgesic effect in cold-stasis type dysmenorrhea rats, which may be related to their effects in regulating AVP levels and its receptor expression in the uterine and hypothalamus.