Effect of siRNAs on HSV-1 Plaque Formation and Relative Expression Levels of RR mRNA

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.

Identification of Ubiquitinated Proteins from Human Multiple Myeloma U266 Cells by Proteomics

Objective To identify ubiquitinated proteins from complex human multiple myeloma （MM） U266 cells,a malignant disorder of differentiated human B cells.Methods Employing a globally proteomic strategy combining of immunoprecipitation,LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites,which were identified by detecting signature peptides containing a GG-tag （114.1 Da） and an LRGG-tag （383.2 Da）.Results In total,52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified,respectively.Conclusion Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups.This suggested the involvement of many endogenous proteins in the ubiquitination in MM.

In Vitro Study on the Repair Effect of Fibronectin Lyophilized Powder

Fibronectin has a good repair effect on skin,but its practical application is limited by its easy degradation and difficult preservation.Freeze-drying technology can extend the shelf life of biological products,but may cause damage to some of their biological activities.Therefore,it is necessary to conduct comprehensive and scientific testing of fibronectin lyophilized powder prepared by freeze-drying technology to evaluate its actual efficacy.In this study,by means of in vitro cell experiment,zebrafish animal model experiment and the experiment on the human skin,the safety of fibronectin lyophilized powder prepared by the new formula and the actual efficacy of skin repair were preliminarily and multidimensionally evaluated,so as to provide reference and basis for further research,human clinical trial and application.The results showed that fibronectin lyophilized powder prepared by the new formula had low cytotoxicity,significant ability of promoting cell migration and proliferation,and had stronger skin repair effect.The new Fibronectin lyophilized powder has the characteristics of good safety and strong skin repair effect,which shows that it has good development prospects.These results provide a new strategy for the development of skin repair products.

GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progresses of gastric cancer

Glutamate-ammonia ligase(GLUL, also known as glutamine synthetase) is a crucial enzyme that catalyzes ammonium and glutamate into glutamine in the ATP-dependent condensation. Although GLUL plays a critical role in multiple cancers, the expression and function of GLUL in gastric cancer remain unclear. In the present study, we have found that the expression level of GLUL was significantly lower in gastric cancer tissues compared with adjacent normal tissues, and correlated with N stage and TNM stage, and low GLUL expression predicted poor survival for gastric cancer patients. Knockdown of GLUL promoted the growth, migration, invasion and metastasis of gastric cancer cells in vitro and in vivo, and vice versa, which was independent of its enzyme activity. Mechanistically, GLUL competed with β-Catenin to bind to N-Cadherin, increased the stability of N-Cadherin and decreased the stability of β-Catenin by alerting their ubiquitination. Furthermore, there were lower N-Cadherin and higher β-Catenin expression levels in gastric cancer tissues compared with adjacent normal tissues. GLUL protein expression was correlated with that of N-Cadherin, and could be the independent prognostic factor in gastric cancer. Our findings reveal that GLUL stabilizes N-Cadherin by antagonizing β-Catenin to inhibit the progress of gastric cancer.

Blockade of NMT1 enzymatic activity inhibits N-myristoylation of VILIP3 protein and suppresses liver cancer progression

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors.Identification of the underlying mechanism of HCC progression and exploration of new therapeutic drugs are urgently needed.Here,a compound library consisting of 419 FDA-approved drugs was taken to screen potential anticancer drugs.A series of functional assays showed that desloratadine,an antiallergic drug,can repress proliferation in HCC cell lines,cell-derived xenograft(CDX),patient-derived organoid(PDO)and patient-derived xenograft(PDX)models.N-myristoyl transferase 1(NMT1)was identified as a target protein of desloratadine by drug affinity responsive target stability(DARTS)and surface plasmon resonance(SPR)assays.Upregulation of NMT1 expression enhanced but NMT1 knockdown suppressed tumor growth in vitro and in vivo.Metabolic labeling and mass spectrometry analyses revealed that Visinin-like protein 3(VILIP3)was a new substrate of NMT1 in protein N-myristoylation modification,and high NMT1 or VILIP3 expression was associated with advanced stages and poor survival in HCC.Mechanistically,desloratadine binds to Asn-246 in NMT1 and inhibits its enzymatic activity,disrupting the NMT1-mediated myristoylation of the VILIP3 protein and subsequent NFκB/Bcl-2 signaling.Conclusively,this study demonstrates that desloratadine may be a novel anticancer drug and that NMT1-mediated myristoylation contributes to HCC progression and is a potential biomarker and therapeutic target in HCC.

MiR-194 functions as a tumor suppressor in laryngeal squamous cell carcinoma by targeting Wee1

OBJECTIVE To investigate the clinical and functional association of mi R-194 in human laryngeal squamous cell carcinoma(LSCC).METHODS Cell growth was measured by MTT assay.Cel cycle distribution was detected using PI staining by flow cytometric analysis.Cell migration and invasion were examined by wound healing assay and transwell assay.The 3′-UTR activity was detected by luciferase assay.The expression level of proteins and mR NA were analysed by Western blotting,immunohistochemistry and q RT-PCR.Mouse xenograft model was established to observe the tumor growth in vivo.RESULTS The expression level of miR-194 is significantly lower in clinical LSCC tissues compared with normal tissues,and is correlated with lymph node metastasis and TNM stage.Kaplan-Meier analysis shows that high miR-194 expression predicts a favorable outcome for LSCC patients.Functional assays show that enforced expression of mi R-194 inhibits the growth,migration,invasion and drug-resistance of LSCC cells.Moreover,Wee1 is identified as a novel functional target of mi R-194.Exogenous expression of Wee1 protein in mi R-194-over expressing cells partially reverses the suppressive effects of mi R-194 on LSCC cells.In addition,Wee1 was abnormally overexpressed in clinical LSCC tissues,and its protein levels were inversely correlated with miR-194 expression.High Wee1 protein level was also associated with TNM stage,lymph node metastasis and poor prognosis.CONCLUSION Our study provides new sights into the role of miR-194/Wee1 axis in LSCC,and suggests a novel miR-194/Wee1-based clinical intervention target for LSCC patients.

Corrigendum to“Generation of Leydig-like cells:approaches,characterization,and challenges”

In the published article by Li et al.,1 an error occurred in the Acknowledgments.The funding agency should be“the Guangdong Basic and Applied Basic Research Foundation(No.2021A1515010947)”,not“the Natural Science Foundation of Guangdong Province(No.2021A1515010947)”.The authors sincerely apologize for the error.

Small-molecule agents for cancer immunotherapy

Cancer immunotherapy,exemplified by the remarkable clinical benefits of the immune checkpoint blockade and chimeric antigen receptor T-cell therapy,is revolutionizing cancer therapy.They induce long-term tumor regression and overall survival benefit in many types of cancer.With the advances in our knowledge about the tumor immune microenvironment,remarkable progress has been made in the development of small-molecule drugs for immunotherapy.Small molecules targeting PRR-associated pathways,immune checkpoints,oncogenic signaling,metabolic pathways,cytokine/chemokine signaling,and immune-related kinases have been extensively investigated.Monotherapy of smallmolecule immunotherapeutic drugs and their combinations with other antitumor modalities are under active clinical investigations to overcome immune tolerance and circumvent immune checkpoint inhibitor resistance.Here,we review the latest development of small-molecule agents for cancer immunotherapy by targeting defined pathways and highlighting their progress in recent clinical investigations.

Identification of miR-515-3p and its targets,vimentin and MMP3,as a key regulatory mechanism in esophageal cancer metastasis:functional and clinical significance

Metastasis is the main factor of treatment failure in cancer patients,but the underlying mechanism remains to be elucidated and effective new treatment strategies are urgently needed.This study aims to explore novel key metastasis-related microRNAs(miRNAs)in esophageal squamous cell carcinoma(ESCC).By comparing miRNA profiles of the highly metastatic ESCC cell sublines,we established through serial in vivo selection with the parental cells,we found that the expression level of miR-515-3p was lower in ESCC tumor tissues than adjacent normal tissues,further decreased in metastatic tumors,and moreover,markedly associated with advanced stage,metastasis and patient survival.The in vitro and in vivo assays suggested that miR-515-3p could increase the expression of the epithelial markers as well as decrease the expression of the mesenchymal markers,and more importantly,suppress invasion and metastasis of ESCC cells.Mechanistically,we revealed that miR-515-3p directly regulated vimentin and matrix metalloproteinase-3(MMP3)expression by binding to the coding sequence and 3′untranslated region,respectively.In addition,the data from whole-genome methylation sequencing and methylation-specific PCR indicated that the CpG island within miR-515-3p promoter was markedly hypermethylated in ESCC cell lines and ESCC tumor tissues,which may lead to deregulation of miR-515-3p expression in ESCC.Furthermore,our preclinical experiment provides solid evidence that systemic delivery of miR-515-3p oligonucleotide obviously suppressed the metastasis of ESCC cells in nude mice.Taken together,this study demonstrates that miR-515-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic strategy in ESCC.

Metabolic disorders of fatty acids and fatty acid amides associated with human gastric cancer morbidity

Background Gastric cancer （GC） is one of the most common types of cancer in the world. A change in the metabolism of lipids in tumor cells could lead to the pathogenesis of cancer. In this study, we investigated fatty acid and fatty acid amide metabolic perturbations associated with GC morbidity.

Generation of Leydig-like cells:approaches,characterization,and challenges

Testosterone production by Leydig cells(LCs)plays a crucial role in male reproduction.The functional degeneration of LCs can cause testosterone deficiency,ultimately resulting in primary male hypogonadism.Transplantation of exogenous LCs with the ability to produce testosterone in response to the regulation of the hypothalamus-pituitary-gonad axis could be a promising alternative option to treat male primary hypogonadism.Recent studies have shown that it is possible to generate Leydig-like cells from stem cells by various approaches.In addition,somatic cells,such as embryonic or adult fibroblasts,have also been successfully reprogrammed into Leydig-like cells.In this review,we summarized the recent advances in the generation of Leydig-like cells,with an emphasis on comparing the effectiveness and safety of different protocols used and the cells generated.By further analyzing the characteristics of Leydig-like cells generated from fibroblasts based on small signaling molecules and regulatory factors,we found that although the cells may produce testosterone,they are significantly different from real LCs.For future in vivo applications,it is important that the steroidogenic cells generated be evaluated not only for their steroidogenic functions but also for their overall cell metabolic state by proteomics or transcriptomic tools.

Genome-wide identification of key regulatory lncRNAs in esophageal cancer metastasis

Dear Editor,Metastasis leads to a poor prognosis of patients with esophageal squamous cell carcinoma(ESCC).1-3 but the study on cancer metastasis has been hampered by a lack of reliable cell and animal models.Systematic identification and functional validation of metastasis-associated long non-coding RNAs(lncRNAs)and microRNAs(miRNAs),as well as-their interactions in ESCC are urgently needed.

Turning gray selenium into a nanoaccelerator of tissue regeneration by PEG modification

Selenium(Se)is an essential trace element involved in nearly all human physiological processes but suffers from a narrow margin between benefit and toxicity.The nanoform of selenium has been proven shown to be more bioavailable and less toxic,yet significant challenges remain regarding the efficient and feasible synthesis of biologically active nanoselenium.In addition,although nanoselenium has shown a variety of biological activities,more interesting nanoselenium features are expected.In this work,hydrosoluble nanoselenium termed Nano-Se in the zero oxidation state was synthesized between gray Se and PEG.A zebrafish screen was carried out in zebrafish larvae cocultured with Nano-Se.Excitingly,Nano-Se promoted the action of the FGFR,Wnt,and VEGF signaling pathways,which play crucial roles in tissue regeneration.As expected,Nano-Se not only achieved the regeneration of zebrafish tail fins and mouse skin but also promoted the repair of skin in diabetic mice while maintaining a profitable safe profile.In brief,the Nano-Se reported here provided an efficient and feasible method for bioactive nanoselenium synthesis and not only expanded the application of nanoselenium to regenerative medicine but also likely reinvigorated efforts for discovering more peculiarunique biofunctions of nanoselenium in a great variety of human diseases.

m^(6)A regulator-mediated methylation modification patterns and tumor immune microenvironment characterization in endometrial cancer

Endometrial cancer(EC)is the second most prevalent female reproductive system tumor after cervical cancer and its incidence is rising globally.1 Recently,N6-methyladenosine(m^(6)A)has emerged as an important regulator of a variety of physiological and pathological processes,especially in promoting EC progression.2,3 However,the potential roles of m^(6)A modification in EC and its immune landscape remain unknown.Here,the correlation between m^(6)A modification patterns and tumor microenvironment(TME)cell-infiltrating characteristics was comprehensively evaluated based on 21 m^(6)A regulators of 1301 endometrial cancer samples.We identified four m6Aclasses characterized by distinct TME cell infiltration.We used principal component analysis algorithms to construct m6Ascore to quantify m^(6)A modification patterns of endometrial cancer individuals.Our results showed that lower m6Ascore may be closely related to immune-inflamed phenotype and displayed immune-activated characteristics and better prognosis.

New insights into the interplay between long non-coding RNAs and RNA-binding proteins in cancer

With the development of proteomics and epigenetics,a large number of RNA-binding proteins(RBPs)have been discovered in recent years,and the inter-action between long non-coding RNAs(lncRNAs)and RBPs has also received increasing attention.It is extremely important to conduct in-depth research on the lncRNA-RBP interaction network,especially in the context of its role in the occurrence and development of cancer.Increasing evidence has demonstrated that lncRNA-RBP interactions play a vital role in cancer progression;there-fore,targeting these interactions could provide new insights for cancer drug discovery.In this review,we discussed how lncRNAs can interact with RBPs to regulate their localization,modification,stability,and activity and discussed the effects of RBPs on the stability,transport,transcription,and localization of lncRNAs.Moreover,we explored the regulation and influence of these inter-actions on lncRNAs,RBPs,and downstream pathways that are related to can-cer development,such as N6-methyladenosine(m6A)modification of lncRNAs.In addition,we discussed how the lncRNA-RBP interaction network regulates cancer cell phenotypes,such as proliferation,apoptosis,metastasis,drug resis-tance,immunity,tumor environment,and metabolism.Furthermore,we sum-marized the therapeutic strategies that target the lncRNA-RBP interaction net-work.Although these treatments are still in the experimental stage and various theories and processes are still being studied,we believe that these strategiesmay provide new ideas for cancer treatment.

Advances in targeted therapy for esophageal cancer

Esophageal cancer(EC)is one of the most lethal cancers in the world,and its morbidity and mortality rates rank among the top ten in China.Currently,surgical resection,radiotherapy and chemotherapy are the primary clinical treatments for esophageal cancer.However,outcomes are still unsatisfactory due to the limited efficacy and severe adverse effects of conventional treatments.As a new type of approach,targeted therapies have been confirmed to play an important role in the treatment of esophageal cancer;these include cetuximab and bevacizumab,which target epidermal growth factor receptor(EGFR)and vascular endothelial growth factor(VEGF),respectively.In addition,other drugs targeting surface antigens and signaling pathways or acting on immune checkpoints have been continuously developed.For example,trastuzumab,a monoclonal antibody targeting human epidermal growth factor receptor 2(HER-2),has been approved by the Food and Drug Administration(FDA)as a first-line treatment of HER-2-positive cancer.Moreover,the PD-L1 inhibitor pembrolizumab has been approved as a highly efficient drug for patients with PD-L1-positive or advanced esophageal squamous cell carcinoma(ESCC).These novel drugs can be used alone or in combination with other treatment strategies to further improve the treatment efficacy and prognosis of cancer patients.Nevertheless,adverse events,optimal dosages and effective combinations still need further investigation.In this review,we expound an outline of the latest advances in targeted therapies of esophageal cancer and the mechanisms of relevant drugs,discuss their efficacy and safety,and provide a clinical rationale for precision medicine in esophageal cancer.

Targeting PFKL with penfluridol inhibits glycolysis and suppresses esophageal cancer tumorigenesis in an AMPK/FOXO3a/BIM-dependent manner

As one of the hallmarks of cancer,metabolic reprogramming leads to cancer progression,and targeting glycolytic enzymes could be useful strategies for cancer therapy.By screening a small molecule library consisting of 1320 FDA-approved drugs,we found that penfluridol,an antipsychotic drug used to treat schizophrenia,could inhibit glycolysis and induce apoptosis in esophageal squamous cell carcinoma(ESCC).Gene profiling and Ingenuity Pathway Analysis suggested the important role of AMPK in action mechanism of penfluridol.By using drug affinity responsive target stability(DARTS)technology and proteomics,we identified phosphofructokinase,liver type(PFKL),a key enzyme in glycolysis,as a direct target of penfluridol.Penfluridol could not exhibit its anticancer property in PFKL-deficient cancer cells,illustrating that PFKL is essential for the bioactivity of penfluridol.High PFKL expression is correlated with advanced stages and poor survival of ESCC patients,and silencing of PFKL significantly suppressed tumor growth.Mechanistically,direct binding of penfluridol and PFKL inhibits glucose consumption,lactate and ATP production,leads to nuclear translocation of FOXO3a and subsequent transcriptional activation of BIM in an AMPK-dependent manner.Taken together,PFKL is a potential prognostic biomarker and therapeutic target in ESCC,and penfluridol may be a new therapeutic option for management of this lethal disease.

Targeting m6A modification inhibits herpes virus 1 infection

The latent infection by herpes virus type 1(HSV-1)may be lifelong in trigeminal ganglia and a suspected cause of Alzheimer's Disease(AD)and Amyotrophic lateral sclerosis(ALS).Whether and how N6-methyladenosine(m6A)modification of viral RNAs affects virus infection are poorly understood.Here,we report that HSV-1 infection enhanced the expression of m6A writers(METTL3,METTL14)and readers(YTHDF1/2/3)at the early infection stage and decreased their expression later on,while suppressed the erasers'(FTO,ALBKH5)expression immediately upon infection to facilitate viral replication.Inhibiting m6A modification by 3-deazaadenosine(DAA)significantly decreased viral replication and reduced viral reproduction over 1000 folds.More interestingly,depleting the writers and readers by siRNAs inhibited virus replication and reproduction;whereas depleting the erasers promoted viral replication and reproduction.Silencing YTHDF3 strikingly decreased viral replication by up to 90%,leading to reduction of up to 10-fold viral replication and over 100-fold virus reproduction,respectively.Depletion of m6A initiator METTL3(by 60%–70%)by siRNA correlatedly decreased viral replication 60%–70%,and reduced virus yield over 30-fold.Consistently,ectopic expression of METTL3 largely increased virus yield.METTL3 knockdown suppressed the HSV-1 intermediate early and early genes(ICP0,ICP8 and UL23)and late genes(VP16,UL44,UL49 and ICP47);while ectopic expression of METTL3 upregulated these gene expression.Results from our study shed the lights on the importance for m6A modification to initiate HSV-1 early replication.The components of m6A modification machinery,particularly m6A initiator METTL3 and reader YTHDF3,would be potential important targets for combating HSV-1 infections.

AHA1 upregulates IDH1 and metabolic activity to promote growth and metastasis and predicts prognosis in osteosarcoma

Osteosarcoma(OS)is the most common primary malignant bone tumor in children and adolescents.Although activator of HSP90 ATPase activity 1(AHA1)is reported to be a potential oncogene,its role in osteosarcoma progression remains largely unclear.Since metabolism reprogramming is involved in tumorigenesis and cancer metastasis,the relationship between AHA1 and cancer metabolism is unknown.In this study,we found that AHA1 is significantly overexpressed in osteosarcoma and related to the prognosis of osteosarcoma patients.AHA1 promotes the growth and metastasis of osteosarcoma both in vitro and in vivo.Mechanistically,AHA1 upregulates the metabolic activity to meet cellular bioenergetic needs in osteosarcoma.Notably,we identifed that isocitrate dehydrogenase 1(IDH1)is a novel client protein of Hsp90 AHA1.Furthermore,the IDH1 protein level was positively correlated with AHA1 in osteosarcoma.And IDH1 overexpression could partially reverse the effect of AHA1 knockdown on cell growth and migration of osteosarcoma.Moreover,high IDH1 level was also associated with poor prognosis of osteosarcoma patients.This study demonstrates that AHA1 positively regulates IDH1 and metabolic activity to promote osteosarcoma growth and metastasis,which provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients.

Targeting PI3K/Akt signal transduction for cancer therapy

The phosphatidylinositol 3-kinase(PI3K)/Akt pathway plays a crucial role in various cellular processes and is aberrantly activated in cancers,contributing to the occurrenee and progression of tumors.Examining the upstream and downstream nodes of this pathway could allow full elucidation of its function.Based on accumulating evidenee,strategies targeting major components of the pathway might provide new in sights for can cer drug discovery.Researchers have explored the use of some in hibitors targeti ng this pathway to block survival pathways.However,because oncogenic PI3K pathway activation occurs through various mechanisms,the clinical efficacies of these inhibitors are limited.Moreover,pathway activation is accompanied by the development of therapeutic resista nee.Therefore,strategies involvi ng pathway in hibitors and other can cer treatments in combinati on might solve the therapeutic dilemma.In this review,we discuss the roles of the PI3K/Akt pathway in various cancer phenotypes,review the current statuses of different PI3K/Akt in hibitors,and in troduce combi nation therapies con sisti ng of signaling in hibitors and conven tional cancer therapies.The information presented herein suggests that cascading inhibitors of the PI3K/Akt signaling pathway,either alone or in combi nation with other therapies,are the most effective treatment strategy for can cer.