Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM.

Compact Cas9 nucleases hold great promise for therapeutic applications. Although several compact Cas9 nucleases have been developed, many genomic loci still could not be edited due to a lack of protospacer adjacent motifs (PAMs). We previously developed a compact SlugCas9 recognizing an NNGG PAM. Here we demonstrate that SlugCas9 displays comparable activity to SpCas9. We developed a simple phage-assisted evolution to engineer SlugCas9 for unique PAM requirements. Interestingly, we generated a SlugCas9 variant (SlugCas9-NNG) that could recognize an NNG PAM, expanding the targeting scope. We further developed a SlugCas9-NNG-based adenine base editor and demonstrated that it could be delivered by a single adeno-associated virus to disrupt PCSK9 splice donor and splice acceptor. These genome editors greatly enhance our ability for in vivo genome editing.

Bispecific GPC3/PD­1 CAR­T cells for the treatment of HCC.

Constantly stimulated by the tumor microenvironment (TME), programmed death 1 (PD­1) is elevated, and it interacts with PD ligand 1 (PD­L1), rendering chimeric antigen receptor (CAR)­T cells dysfunctional. Hence, CAR­T cells immune to PD­1­induced immunosuppression were constructed to improve the function of CAR­T cells in hepatocellular carcinoma (HCC). Double­target CAR­T cells, targeting glypican­3 (GPC3) [a tumour-associated antigen (TAA)] and hindering PD­1­PD­L1 binding, were established. The expression of GPC3, PD­L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR­T cells were determined using lactate dehydrogenase release assay, enzyme­linked immunosorbent assay, and flow cytometry, respectively. HCC cells were targeted and eliminated by double­target CAR­T cells. These double­target CAR­T cells limit PD­1­PD­L1 binding and sustain cytotoxicity to PD­L1+ HCC cells. The relatively low IR expression and differentiation level in double­target CAR­T cells in tumour tissues induced tumour­suppression and extended survival in PD­L1+ HCC TX models, as opposed to their single­target counterparts. The results of the present study suggested that the newly constructed double­target CAR­T cells exhibit stronger tumour­suppressing effects in HCC than their single­target counterparts, which are common, suggesting the potential of strengthening CAR­T cell activity in HCC treatment.

LMP1 mediates tumorigenesis through persistent epigenetic modifications and PGC1ß upregulation.

Latent membrane protein 1 (LMP1), which is encoded by the Epstein­Barr virus (EBV), has been considered as an oncogene, although the detailed mechanism behind its function remains unclear. It has been previously reported that LMP1 promotes tumorigenesis by upregulation of peroxisome proliferator­activated receptor­Î³ coactivator­1ß (PGC1ß). The present study aimed to investigate the potential mechanism for transient EBV/LMP1 exposure­mediated persistent PGC1ß expression and subsequent tumorigenesis through modification of mitochondrial function. Luciferase reporter assay, chromatin immunoprecipitation and DNA mutation techniques were used to evaluate the PGC1ß­mediated expression of dynamin­related protein 1 (DRP1). Tumorigenesis was evaluated by gene expression, oxidative stress, mitochondrial function and in vitro cellular proliferation assays. The potential effects of EBV, LMP1 and PGC1ß on tumor growth were evaluated in an in vivo xenograft mouse model. The present in vitro experiments showed that LMP1 knockdown did not affect PGC1ß expression or subsequent cell proliferation in EBV­positive tumor cells. PGC1ß regulated DRP1 expression by coactivation of GA­binding protein α and nuclear respiratory factor 1 located on the DRP1 promoter, subsequently modulating mitochondrial fission. Transient exposure of either EBV or LMP1 in human hematopoietic stem cells caused persistent epigenetic changes and PGC1ß upregulation after long­term cell culture even in the absence of EBV/LMP1, which decreased oxidative stress, and potentiated mitochondrial function and cell proliferation in vitro. Enhanced tumor growth and shortened survival were subsequently observed in vivo. It was concluded that PGC1ß expression and subsequent cell proliferation were independent from LMP1 in EBV­positive tumor cells. PGC1ß modulated mitochondria fission by regulation of DRP1 expression. Transient EBV/LMP1 exposure caused persistent PGC1ß expression, triggering tumor growth in the absence of LMP1. The present study proposes a novel mechanism for transient EBV/LMP1 exposure­mediated tumorigenesis through persistent epigenetic changes and PGC1ß upregulation, uncovering the reason why numerous forms of lymphoma exhibit upregulated PGC1ß expression, but are devoid of EBV/LMP1.

Nucleos(t)ide analogues altered quasispecies composition of hepatitis B virus (HBV)-resistant mutations in serum HBV DNA and serum HBV RNA.

Serum hepatitis B virus (HBV) RNA is a new serological indicator reflecting viral replication with good clinical application prospects. This study aimed to clarify the dynamic changes of serum HBV RNA levels and the quasispecies of HBV RNA virus-like particles in nucleos(t)ide analogues (NAs)-experienced chronic hepatitis B (CHB) patients harboring NAs-resistant mutations and their identifiable effects on NAs resistance. We included CHB patients who were on long-term NAs treatment and with HBV DNA rebound. The longitudinally dynamics of serum HBV RNA levels were quantitatively detected, and the quasispecies differences between serum HBV DNA and serum HBV RNA were compared by high-throughput sequencing. The effect of NAs concentration pressure on altering the resistance mutations quasispecies proportion of HBV DNA and HBV RNA in cell supernatant was analyzed in vitro. A total of 447 serum samples from 36 CHB patients treated with NAs were collected. The median follow-up period was 47 months (about 4 years), and the longest follow-up period was 117 months (about 10 years). Our results showed that HBV RNA could reflect virological breakthrough in 23 (64%, 23/36) patients, and serum HBV RNA rebound earlier than HBV DNA in 12 (52%, 12/23) patients. However, serum HBV RNA remained at a consistently high level and did not fluctuate significantly with the HBV DNA rebound in 6 of 36 patients. In addition, serum HBV RNA was not consistently detectable in 7 of the 36 patients, and their serum HBV RNA was undetectable even after HBV DNA had rebounded. The proportion of drug-resistant mutations in HBV DNA was higher than that of HBV RNA by high-throughput sequencing. The results of in vitro experiments showed that the viral strains with drug-resistant mutation in HBV DNA in cell supernatants gradually become the dominant strains with the increase of NAs concentrations. Serum HBV RNA levels can reflect virological breakthrough in most NAs- treated CHB patients, but there are certain limitations. NAs alter the quasispecies composition of serum HBV DNA and serum HBV RNA, resulting in a higher detection rate of drug-resistant mutations in serum HBV DNA than in serum HBV RNA.

Silencing of TMED5 inhibits proliferation, migration and invasion, and enhances apoptosis of hepatocellular carcinoma cells.

BACKGROUND: Transmembrane P24 trafficking protein 5 (TMED5) is highly expressed in cervical and bladder cancer cell lines. Moreover, TMED5 promotes nuclear autophagy and the malignant behavior of cervical cancer cells. However, the role of TMED5 in hepatocellular carcinoma (HCC) has not been extensively reported. OBJECTIVES: To investigate the role of TMED5 in HCC cells. MATERIAL AND METHODS: Bioinformatics was used to analyze the messenger-ribonucleic acid (mRNA) expression of TMED5 in HCC and its relationship with overall survival and disease-free interval of HCC patients. After TMED5 was decreased in SMMC-7721 and Hep3B cells, they were assayed for proliferation, cell cycle, apoptosis, migration, and invasion. RESULTS: The expression of TMED5 mRNA in HCC tissues was higher than in adjacent normal tissues, and the overall survival of HCC patients with high TMED5 transcription levels was worse. Moreover, the overexpression of TMED5 was associated with HCC progression. The downregulation of TMED5 suppressed cell proliferation, migration and invasion, and enhanced apoptosis. Therefore, TMED5 may be involved in the regulation of the cell cycle, the mammalian target of rapamycin signaling pathway, and the transforming growth factor beta (TGF-ß) signaling pathway. CONCLUSIONS: The TMED5 has the potential to promote HCC progression. Therefore, lowering TMED5 levels could represent a potential strategy for the treatment of HCC.

Anti-aging effect of ß-carotene through regulating the KAT7-P15 signaling axis, inflammation and oxidative stress process.

BACKGROUND: Research on aging is growing as the elderly make up a greater share of the population, focusing on reversing and inhibiting the aging process. The exhaustion and senescence of stem cells are the fundamental drivers behind aging. ß-Carotene has been depicted to have many biological functions, and we speculate that it may have an anti-aging effect. METHODS: Firstly, the anti-aging property of ß-carotene was investigated in vitro using mesenchymal stem cells (MSCs) induced by H2O2. The anti-aging effect was characterized using Western-bloting, confocal laser scanning microscopy, indirect immunofluorescence, and immunohistochemistry. The anti-aging property was also tested in vivo using aged mice. RESULTS: The in vitro experiment revealed that ß-carotene could relieve the aging of MSCs, as evidenced by a series of aging marker molecules such as p16 and p21. ß-Carotene appeared to inhibit aging by regulating the KAT7-P15 signaling axis. The in vivo experiment revealed that ß-carotene treatment has significantly down-regulated the aging level of tissues and organs. CONCLUSIONS: In this work, we explored the anti-aging effect of ß-carotene in vivo and in vitro. The experimental results indicate that ß-carotene may be an important potential anti-aging molecule, which can be used as a drug or in functional food to treat aging in the future.

Characteristics of HBV Novel Serum Markers across Distinct Phases in Treatment-Naïve Chronic HBV-Infected Patients.

Methods: A total of 111 patients in total from different disease phases were recruited, including 21 in immune-tolerant (IT) phase, 49 in immune-clearance (IC) phases, 29 in immune-control or low replicative (LR) phase, and 12 in reactivation phases. Serum HBV RNA, anti-HBc, HBcrAg, and intrahepatic covalently closed circular DNA (cccDNA) were quantified and each of these indicator's correlation with liver inflammation was analyzed. Results: HBeAg-positive individuals had significant higher serum levels of HBV RNA and HBcrAg than those who were HBeAg negative, similar to that of serum HBV DNA. Comparatively, HBV RNA (r =0.79, P < 0.01) and HBcrAg (r =0.78, P < 0.01) had almost same higher overall correlation with the cccDNA, as that of HBV DNA (r =0.81, P < 0.01). Serum anti-HBc level (r = -0.52, P < 0.05) is negatively correlated with cccDNA level at IT phase rather than the other three phases. When set the cutoff value at 4.00 log10 IU/mL, serum anti-HBc showed potential to indicate liver inflammation, with AUC as 0.79 and the specificities as 78.85% for HBeAg positive, and with AUC as 0.72 and the specificities as 62.16% for HBeAg-negative patients, respectively. Conclusions: In treatment-naïve patients, levels of serological markers HBV RNA and HBcrAg could mirror intrahepatic cccDNA level, but were not superior to HBV DNA level. Serum anti-HBc level had certain potential to be used as a predicting marker for liver inflammation.

Exosomes derived from NGF-overexpressing bone marrow mesenchymal stem cell sheet promote spinal cord injury repair in a mouse model.

Cell transplantation has been an appealing way to improve the recovery of motor, sensory, and autonomic functions following spinal cord injury (SCI). Herein, we sought to elucidate the function of bone marrow mesenchymal stem cells (BMSCs) sheet in the progression of SCI and its underlying mechanism. BMSCs were extracted from bone marrow of femur and tibia collected from C57BL/6 mice, and the BMSC sheet was prepared when cells grew to 100% confluence after approximately 14 days. Exosomes (Exos) derived from BMSCs were isolated and characterized. The expression of NGF in the isolated Exos and neural stem cells (NSCs) was quantified. NSCs were co-cultured with Exos derived from the BMSC sheet that was treated with overexpressed NGF (oe-NGF) (Exos-oe-NGF). NSC differentiation, axonal regeneration and locomotor function were detected in vitro and in vivo. The BMSC sheet was successfully prepared and exerted a promoting effect on NSC differentiation into neuronal cells and axonal regeneration after SCI by releasing Exos. Co-culture data showed that NGF was highly expressed in the BMSC sheet-loaded Exos and facilitated neuronal differentiation of NSCs and axonal regeneration. In vivo experimental results unveiled that transplantation of BMSC sheet-loaded Exos-oe-NGF into SCI mice displayed enhanced functional recovery. Collectively, Exo-oe-NGF loaded on the BMSC sheet can accelerate NSC differentiation, axonal regeneration and SCI repair, therefore offering us with a potential therapeutic target for treating SCI.

MicroRNA-139-5p negatively regulates NME1 expression in hepatocellular carcinoma cells.

BACKGROUND: High expression of NME1 is associated with hepatocellular carcinoma (HCC) progression and poor prognosis. However, there are few reports on the association between NME1 and microRNAs (miRNAs) in HCC progression. OBJECTIVES: To explore miRNAs that regulate NME1 expression in HCC. MATERIAL AND METHODS: Data from the Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), TargetScan, starBase, and mirDIP were used to analyze the expression pattern of NME1 in HCC tissues, the relationship between NME1 level and the progression of HCC or patient prognosis, miRNAs targeting NME1, and the biological processes that may be regulated by NME1. The regulation of miRNAs to NME1 was assessed using the dual-luciferase reporter assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. The cell cycle and cell proliferation were detected using propidium iodide (PI) staining and EdU assay, respectively. RESULTS: Highly expressed NME1 in HCC was associated with HCC progression and prognosis. The miR-139-5p and miR-335-5p were weakly expressed in HCC samples and negatively correlated with NME1. The downregulation of miR-139-5p in HCC patients resulted in worse overall survival (OS) and disease-free interval (DFI); however, the level of miR-335-5p was not significantly correlated with OS and DFI in patients with HCC. In vitro experiments verified that the level of miR-139-5p was lower and NME1 expression was higher in HCC cell lines compared to L-02. Moreover, miR-139-5p negatively regulates the expression of NME1 in HCC cell lines. The NME1 may regulate cell cycle, DNA replication, oxidative phosphorylation, and the pentose phosphate pathway. The miR-139-5p inhibited cell proliferation by negatively regulating NME1 expression. CONCLUSIONS: The upregulation of NME1 in HCC indicates a poor prognosis. The NME1 is negatively regulated by miR-139-5p to inhibit cell proliferation.

Different isoforms of growth hormone (20 kD-GH and 22 kD-GH) shows different biological activities in mesenchymal stem cell (MSC).

There are two main types of growth hormone (GH) in the circulatory system. One is 22 kD-GH, which is the predominant isoform in the circulating system, 90% GH is present as a 22 kD protein, and 10% of GH is present as a 20 kD protein. Amino acid sequences are identical between 20 kD-GH and 22 kD-GH protein, except that 20 kD-GH lacks 15 amino acid residues 32 to 46. Studies have shown that GH has many important biological effects on mesenchymal stem cells (MSCs). However, so far, the cellular characteristics of the two types of GH have not been studied in BM-MSCs. Furthermore, the biological activity of 20 kD-GH has not been explored in BM-MSCs. For this, in the current work, BM-MSCs were used as in vitro cell model. We have carried out the current research using a series of experimental techniques (such as Western-blot and indirect immunofluorescence). Firstly, we explored the cell behavior of two types of GH in the Bm-MSC model and found that they showed different biological characteristics; Secondly, we investigated the biological characteristics of 20 kD-GH and 22 kD-GH, and results showed that 22 kD-GH and 20 kD-GH exhibited different signaling profiles; Thirdly, we found that the 20 kD-GH and 22 kD-GH Gexhibited different regulatory effects on the osteogenic differentiation of BM-MSCs. The current research lays a solid foundation for further studies on the regulatory effects of GH on MSCs.

microRNA-378a-3p regulates the progression of hepatocellular carcinoma by regulating PD-L1 and STAT3.

Programmed death ligand 1 (PD-L1) plays an essential role in the development or progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression during normal and pathophysiological events. Here, we explored the functions and detailed mechanisms of miR-378a-3p and PD-L1 in HCC progression. First, miR-378a-3p was selected by analyzing miRNA levels in two HCC Gene Expression Omnibus datasets. We found that miR-378a-3p levels exhibited a downward trend in HCC and were negatively correlated with PD-L1 levels. Additionally, a dual luciferase assay predicted that miR-378a-3p directly targets PD-L1. Moreover, the transfection of miR-378a-3p mimics into Li-7 and HuH-7 cells effectively decreased the PD-L1 mRNA and protein expression levels, and inhibited Treg differentiation in co-culture models by modulating the expression levels of certain cytokines. Furthermore, the overexpression of miR-378a-3p hindered cell proliferation and migration but facilitated apoptosis by repressing STAT3 signaling in HCC cells. In conclusion, miR-378a-3p appears to inhibit HCC tumorigenesis by regulating PD-L1 and STAT3 levels. Thus, miR-378a-3p may be a potential target for HCC therapy.

LINC02362 attenuates hepatocellular carcinoma progression through the miR-516b-5p/SOSC2 axis.

Hepatocellular carcinoma (HCC) is one of the most death-related cancers worldwide. Identifying cancer-associated genes and uncovering the vital molecular mechanisms of HCC progression contribute greatly to the prognosis and novel therapeutic strategies for HCC patients. Although lncRNAs have been proved to be critical modulators of various cellular processes, the functions of lncRNAs in HCC progression are just emerging. Here, we found that a long non-coding RNA (lncRNA) named LINC02362, whose biological effects have yet been unveiled in cancers, was associated with a better prognosis in patients with HCC. Gain-of-function analyses showed that LINC02362 inhibited the survival, migration, invasion and epithelial-to-mesenchymal transition (EMT) of HCC cells. Moreover, miR-516b-5p was enriched as a target of LINC02362, which functioned as a sponge to regulate the endogenous levels of miR-516b-5p. Furthermore, we confirmed that SOSC2 served as a downstream target gene which was negatively controlled by miR-516b-5p. Importantly, a series of rescue experiments indicated that the tumor-suppressive effects of LINC02362 were achieved through the modulation of the miR-516b-5p/SOSC2 axis. In summary, we identified LINC02362 as a candidate tumor-inhibitory lncRNA that might serve as a biomarker for the prognosis of HCC and a promising therapeutic agent for patients with HCC.

Objective to identify and verify the regulatory mechanism of DTNBP1 as a prognostic marker for hepatocellular carcinoma.

Although the overall survival of hepatocellular carcinoma (HCC) patients has been significantly improved, prognostic clinical evaluation remains a substantial problem owing to the heterogeneity and complexity of tumor. A reliable and accurate predictive biomarker may assist physicians in better monitoring of patient treatment outcomes and follow the overall survival of patients. Accumulating evidence has revealed that DTNBP1 plays functional roles in cancer prognosis. Therefore, the expression and function of DTNBP1in HCC was systematically investigated in our study. The expression and prognostic value of DTNBP1 were investigated using the data from Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) cohorts and clinical samples. A series of cellular function assays were performed to elucidate the effect of DTNBP1 on cellular proliferation, apoptosis and metastasis. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment and Protein-protein interaction (PPI) network construction were performed to screen the genes with highest interaction scores with DTNBP1. Finally, the underlying mechanism was also analyzed using Gene Set Enrichment Analysis (GSEA) and confirmed using RT-qPCR and western blotting. DTNBP1 was upregulated in many types of cancers, especially in HCC. The DTNBP1 expression levels is associated with clinicopathologic variables and patient survival status. The differential expression of DTNBP1 could be used to determine the risk stratification of patients with HCC. DTNBP1 deficiency inhibited cell proliferation and metastasis, but promoted cell apoptosis. Mechanistically, DTNBP1 regulated the cell cycle progression through affecting the expression of cell cycle-related genes such as CDC25A, CCNE1, CDK2, CDC20, CDC25B, CCNB1, and CDK1. DTNBP1, which regulates the cell cycle progression, may be used as a prognostic marker for HCC.

Uridine alleviates carbon tetrachloride-induced liver fibrosis by regulating the activity of liver-related cells.

At present, liver fibrosis is a major challenge of global health. When hepatocyte regeneration cannot compensate for hepatocyte death, it will develop into liver fibrosis in chronic liver disease. Initially, collagen produced by myofibroblasts plays a role in maintaining liver integrity, but excessive collagen accumulation can inhibit the residual liver function, leading to liver failure. At present, many scientists are actively looking for drugs to alleviate liver fibrosis. In the current study, we investigated the potential role of uridine in the treatment of liver fibrosis (uridine is a plant/animal-derived pyrimidine nucleoside, therefore uridine can also be ingested and absorbed by the body, accompanied by the process of food intake). For this, we systematically studied the effect of uridine on CCl4-induced liver fibrosis in vitro and in vivo through a series of technologies, such as Western blot, laser confocal scanning microscope, ELISA and immunohistochemistry. The experimental results showed that uridine can effectively reduce the accumulation of collagen in liver. Furthermore, uridine can improve the activity of liver cells and alleviate CCl4-induced liver injury. Furthermore, uridine can significantly alleviate the risk factors caused by hepatic stellate cell activation, uridine treatment significantly down-regulated the expression of α-SMA, collagen type-I and fibronectin. In conclusion, the current research shows that uridine can alleviate CCl4-induced liver fibrosis, suggesting that uridine can be used as a potential drug to alleviate liver fibrosis.

22-kD growth hormone-induced nuclear GHR/STAT5/CyclinD1 signaling pathway plays an important role in promoting mesenchymal stem cell proliferation.

Growth hormone (GH) exhibited the important biological activities in the mesenchymal stem cell (MSCs). However, the cellular behavior and properties of GH/GHR in MSCs remain unclear. A series of experiments (such as confocal laser scanning microscope [CLSM] and Western-blot) were performed to systematically investigate the cellular behavior of GH/GHR in MSCs, and the results showed that GH/GHR not only internalized into the cytoplasm, but also transported into the cell nuclei of MSCs. Furthermore, we studied the molecular mechanism by which GH/GHR internalized into cell, and the results indicated that clathrin plays more important role in the process of GHR internalization. More importantly, it can be found that nuclear-targeted GHR has the important biological functions in MSCs, which could promote MSCs proliferation. We further revealed the molecular mechanism by which nuclear-localized GHR regulates MSCs proliferation, and found that nuclear-targeted GHR enhanced the phosphorylation of STAT5, and the activated STAT5 initiates the transcription of CyclinD1, after which, the complex of CyclinD1 and CDK4 further phosphorylates Rb, and the activated Rb releases E2F1, the released E2F1 ultimately realizes the biological function of GH promoting cell proliferation. In short, in the current study，we used MSCs as a model to study the cellular behavior and properties of GH/GHR, and found that GH/GHR can internalize into cell cytoplasm and transport into the cell nuclei. Further work showed that nuclear GHR could drive cell proliferation via GHR/STAT5/CyclinD1 signaling pathway. The current research has laid an important foundation for further study on the regulatory effect of GH on MSCs.

A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells.

CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells.

Repositioning Azelnidipine as a Dual Inhibitor Targeting CD47/SIRPα and TIGIT/PVR Pathways for Cancer Immuno-Therapy.

Strategies boosting both innate and adaptive immunity have great application prospects in cancer immunotherapy. Antibodies dual blocking the innate checkpoint CD47 and adaptive checkpoint PD-L1 or TIGIT could achieve durable anti-tumor effects. However, a small molecule dual blockade of CD47/SIRPα and TIGIT/PVR pathways has not been investigated. Here, an elevated expression of CD47 and PVR was observed in tumor tissues and cell lines analyzed with the GEO datasets and by flow cytometry, respectively. Compounds approved by the FDA were screened with the software MOE by docking to the potential binding pockets of SIRPα and PVR identified with the corresponding structural analysis. The candidate compounds were screened by blocking and MST binding assays. Azelnidipine was found to dual block CD47/SIRPα and TIGIT/PVR pathways by co-targeting SIRPα and PVR. In vitro, azelnidipine could enhance the macrophage phagocytosis when co-cultured with tumor cells. In vivo, azelnidipine alone or combined with irradiation could significantly inhibit the growth of MC38 tumors. Azelnidipine also significantly inhibits the growth of CT26 tumors, by enhancing the infiltration and function of CD8+ T cell in tumor and systematic immune response in the tumor-draining lymph node and spleen in a CD8+ T cell dependent manner. Our research suggests that the anti-hypertensive drug azelnidipine could be repositioned for cancer immunotherapy.

METTL3 facilitates the progression of hepatocellular carcinoma by modulating the m6A level of USP7.

OBJECTIVE: To explore whether METTL3 was involved in the pathogenesis of hepatocellular carcinoma (HCC) by modulating the m6A level of USP7. METHODS: We performed qRT-PCR and western blot assays to detect the expression level of METTL3 in HCC tissues and paired adjacent normal tissues, as well as HCC cell lines. The level of m6A in HCC tissues and cells was quantitatively analyzed by m6A RNA Methylation Quantitative Kit. We examined the effect of METTL3 on cell proliferation ability by CCK-8 and EdU assays, and examined cell migration and cell invasion ability by Transwell assay. It was predicted via bioinformatics tool that USP7 may undergo methylation in HCC. Subsequently, we performed qRT-PCR assay to detect the expression level of USP7 in HCC tissues and analyzed its correlation with the expression level of METTL3. We verified the regulatory relationship between METTL3/USP7 and transfected USP7 siRNA in cells to detect its effects on cell invasion, migration and proliferation. The regulatory effect of METTL3 on USP7 in HCC was analyzed by corresponding experiments. RESULTS: The qRT-PCR results indicated that METTL3 was highly expressed in HCC tissues and cell lines. The level of m6A was remarkably increased in HCC tissues and cell lines. Besides, the elevated METTL3 expression was related to worse overall survival. The abilities of cell invasion, migration and proliferation were remarkably attenuated by down-regulation of METTL3 expression. Through bioinformatics analysis, it was found that USP7 might be regulated by METTL3 to undergo methylation modification. The qRT-PCR results showed that the USP7 was highly expressed in HCC tissues, and was positively correlated with the level of METTL3. Further experiments showed that down-regulation of USP7 could reduce cell proliferation, migration, and invasion. METTL3 could positively regulate the malignant phenotype of USP7 in HCC. CONCLUSION: METTL3 might regulate the expression of USP7 through m6A methylation and facilitate the invasion, migration and proliferation of HCC cells.