Microfluidic platform for omics analysis on single cells with diverse morphology and size: A review.

BACKGROUND: Microfluidic techniques have emerged as powerful tools in single-cell research, facilitating the exploration of omics information from individual cells. Cell morphology is crucial for gene expression and physiological processes. However, there is currently a lack of integrated analysis of morphology and single-cell omics information. A critical challenge remains: what platform technologies are the best option to decode omics data of cells that are complex in morphology and size? RESULTS: This review highlights achievements in microfluidic-based single-cell omics and isolation of cells based on morphology, along with other cell sorting methods based on physical characteristics. Various microfluidic platforms for single-cell isolation are systematically presented, showcasing their diversity and adaptability. The discussion focuses on microfluidic devices tailored to the distinct single-cell isolation requirements in plants and animals, emphasizing the significance of considering cell morphology and cell size in optimizing single-cell omics strategies. Simultaneously, it explores the application of microfluidic single-cell sorting technologies to single-cell sequencing, aiming to effectively integrate information about cell shape and size. SIGNIFICANCE AND NOVELTY: The novelty lies in presenting a comprehensive overview of recent accomplishments in microfluidic-based single-cell omics, emphasizing the integration of different microfluidic platforms and their implications for cell morphology-based isolation. By underscoring the pivotal role of the specialized morphology of different cells in single-cell research, this review provides robust support for delving deeper into the exploration of single-cell omics data.

Clinical diagnostic biomarker "circulating tumor cells" in breast cancer - a meta-analysis.

Objective: Using meta-analysis, we evaluate circulating tumor cells(CTCs) as a potential diagnostic tool for breast cancer. Methods: A document search was conducted using publicly available databases up to May 2021. Specific inclusion and exclusion criteria were formulated and summarize relevant data through literature types, research types, case populations, samples, etc. Subgroup analysis of documents based on regions, enrichment methods, and detection methods. The included research projects were evaluated using DeeKs' bias, and evaluation indicators such as specificity (SPE), sensitivity (SEN), diagnosis odds ratio (DOR) were used as evaluation indicators. Results: 16 studies on the use of circulating tumor cells to diagnose breast cancer were included in our meta-analysis. Overall sensitivity value was 0.50 (95%CI:0.48-0.52), specificity value was 0.93 (95%CI:0.92- 0.95), DOR value was 33.41 (95%CI:12.47-89.51), and AUC value was 0.8129. Conclusion: In meta-regressions and subgroup analysis, potential heterogeneity factors were analyzed, but the source of heterogeneity is still unclear. CTCs, as a novel tumor marker, have a good diagnostic value, but its enrichment and detection methods still need to continue to be developed to improve detection accuracy. Therefore, CTCs can be used as an auxiliary means of early detection, which is helpful to the diagnosis and screening of breast cancer.

Expression of RNA-m6A-related genes correlates with the HIV latent reservoir level and the CD4+ and CD8+T cell profiles of patients with AIDS.

The HIV latent reservoir is the main obstacle to the eradication of AIDS. Recent studies have shown that the RNA m6A is involved in the regulation of HIV-1 replication. However, no relevant study has reported the relationship between RNA m6A and HIV latent reservoir. For this purpose, peripheral blood mononuclear cell (PBMC) was collected from 36 HIV-infected patients at 1, 24, and 48 weeks after treatment initiation. The number of CD4+ and CD8+ T cells was detected by flow cytometry. Amount of HIV DNA in the PBMC samples one week after treatment initiation was detected by Q-PCR. The expression levels of 23 RNA-m6A-related genes were detected by Q-PCR and Pearson's correlation analysis was performed. Results showed that there was a negative correlation between HIV DNA concentration and the number of CD4+ T cells (r=-0.32, p=0.05; r=-0.32, p=0.06) and a positive correlation with the number of CD8+ T cells (r=0.48, p=0.003; r=0.37, p=0.03). Furthermore, a negative correlation was observed between HIV DNA concentration and the CD4+/CD8+ T cell ratio (r=-0.53, p=0.001; r=-0.51, p=0.001). RNAm6A related genes which correlated with HIV DNA concentration includedALKBH5 (r=-0.45, p=-0.006), METTL3 (r=0.73, p=2.76e-7), METTL16 (r=0.71, p=1.21e2.76e-06), YTHDF1 (r=0.47, p=0.004). Moreover, they have different degrees of correlation with numbers ofCD4+ and CD8+ T cell subsets, and the CD4+/CD8+T cell ratio. In addition, the expression of RBM15 was not correlated with HIV DNA concentration but was significantly negatively correlated with the number of CD4+T cells (r=-0.40, p=0.02). In conclusion, the expression of ALKBH5, METTL3, and METTL16 is correlated with the HIV DNA level, the levels of CD4+ and CD8+ T cell counts, and the CD4+/CD8+ T cell ratio. RBM15 is independent of HIV DNA level and negatively correlated with the number of CD4+T cells.

Inspired by game theory: Multi-signal output photoelectrochemical point-of-care immunoassay based on target-triggered organic electronic barriers.

This work presents an integrated photoelectrochemical, impedance and colorimetric biosensing platform for flexible detection of cancer markers based on the targeted response by combining liposome amplification strategies and target-induced non-in situ formation of electronic barriers as the signal transduction modality on carbon-modified CdS photoanodes. Inspired by game theory, the carbon layer modified CdS hyperbranched structure with low impedance and high photocurrent response was firstly obtained by surface modification of CdS nanomaterials. Through a liposome-mediated enzymatic reaction amplification strategy, a large number of organic electron barriers were formed by a biocatalytic precipitation (BCP) reaction triggered by horseradish peroxidase released from cleaved liposomes after the introduction of the target molecule, thereby increasing the impedance characteristics of the photoanode as well as attenuating the photocurrent. The BCP reaction in the microplate was accompanied by a significant color change, which opened up a new window for point-of-care testing. Taking carcinoembryonic antigen (CEA) as a proof of concept, the multi-signal output sensing platform showed a satisfactory sensitive response to CEA with an optimal linear range of 20 pg mL-1-100 ng mL-1. The detection limit was as low as 8.4 pg mL-1. Meanwhile, with the assistance of a portable smartphone and a miniature electrochemical workstation, the electrical signal obtained was synchronized with the colorimetric signal to correct the actual target concentration in the sample, further reducing the occurrence of false reports. Importantly, this protocol provides a new idea for the sensitive detection of cancer markers and the construction of a multi-signal output platform.

CUCUME: An RNA methylation database integrating systemic mRNAs signals, GWAS and QTL genetic regulation and epigenetics in different tissues of Cucurbitaceae.

As an internal modification of transcripts, RNA methylation determines RNA fate by changing RNA-protein binding affinity. In plants, RNA methylation is ubiquitous and is involved in all aspects of RNA post-transcriptional regulation. For instance, long-distance mobile RNAs, strongly influenced by their methylation status, play important roles in plant growth, development and environmental adaptation. Cucumber/pumpkin heterografts are widely used to improve stress tolerance of cucumber and to study mobile RNA signals due to their strong developed vasculature system. Here, we developed the Cucume (Cucurbit RNA methylation, http://cucume.cn/) database for these two important vegetables, cucumber (Cucumis sativus L.) and pumpkin (Cucurbita moschata) with high productivity worldwide. We identified mRNAs harboring 5-methylcytosine (m5C) and N6-methyladenosine (m6A) sites in pumpkin and cucumber at the whole genome level via Methylated RNA Immunoprecipitation sequencing (MeRIP-seq) of different tissues and the vascular exudates. In addition to RNA methylation sites, the Cucume database includes graft-transmissible systemic mRNAs identified in previous studies using cucumber/pumpkin heterografts. The further integration of cucumber genome-wide association analysis (GWAS) and quantitative trait loci (QTL) allows the study of RNA methylation-related genetic and epigenetic regulation in cucurbits. Therefore, the here developed Cucume database will promote understanding the role of cucurbit RNA methylation in RNA mobility and QTL, ultimately benefitting future breeding of agronomic crop germplasms.

Meta-Analysis of Whole Blood Transcriptome Datasets Characterizes the Immune Response of Respiratory Syncytial Virus Infection in Children.

Respiratory syncytial virus (RSV) is the most common and critical viral pathogen causing acute lower respiratory tract infection in infants and young children and has a huge disease burden worldwide. At present, there are many studies on RSV transcriptomics exploring the mechanism of disease, but different studies show different gene expression patterns and results due to different sample collection platforms and data analysis strategies. A meta-analysis was performed on eight whole blood transcriptome datasets containing 436 children with acute RSV infection and 241 healthy children. A total of 319 differentially expressed genes (DEGs) (P value <0.0001) were identified in a meta-analysis using a random effect model. Functional enrichment analysis showed that several pathways related to immunity were significantly altered, including the "chemokine signaling pathway", "natural killer cell mediated cytotoxicity" and "cytokine-cytokine receptor interaction". Immune cell type analysis showed that the proportion of neutrophils in most RSV-infected children was higher than that in healthy children. These immune characteristics may help to provide new insights into RSV infection in children.

Abundance of Lipopolysaccharide Heptosyltransferase I in Human Gut Microbiome and Its Association With Cardiovascular Disease and Liver Cirrhosis.

Lipopolysaccharide (LPS) is a potent endotoxin on the outer membrane of gram-negative bacteria. Heptosyltransferase I (HpeI) takes part in the synthesis of LPS. In this study, we first collected the protein sequences of HpeI homologs from the human microbiome. The collected HpeI sequences was classified based on sequence similarity, and seven clusters of HpeI were obtained. Among these clusters, proteins from Cluster 3 were abundant in the human mouth, while Clusters 1, 6, and 7 were abundant in the human gut. In addition, proteins from Cluster 1 were mainly from the order of Enterobacterales, while Cluster 6 and 7 were from Burkholderiales. The correlation analysis indicated that the total abundance of HpeIs was increased in patients with cardiovascular disease and liver cirrhosis, and HpeI in Cluster 1 contributed to this increase. These data suggest that HpeI homologs in Cluster 1 can be recognized as biomarkers for cardiovascular disease and liver cirrhosis, and that reducing the bacterial load in Cluster 1 may contribute to disease therapy.

Hepatic Remote Ischemic Preconditioning (RIPC) Protects Heart Damages Induced by Ischemia Reperfusion Injury in Mice.

It has been convincingly demonstrated that remote ischemic preconditioning (RIPC) can make the myocardium resistant to the subsequent ischemia reperfusion injury (IRI), which causes severe damages by mainly generating cell death. However, the cardioprotective effects of the hepatic RIPC, which is the largest metabolic organ against I/R, have not been fully studied. The aim of our research is whether remote liver RIPC may provide cardioprotective effects against the I/R-induced injury. Here, we generated an I/R mice model in four groups to analyze the effect. The control group is the isolated hearts with 140-min perfusion. I/R group added ischemia in 30 min following 90-min reperfusion. The third group (sham) was subjected to the same procedure as the latter group. The animals in the fourth group selected as the treatment group, underwent a hepatic RIPC by three cycles of 5-min occlusion of the portal triad and then followed by induction of I/R in the isolated heart. The level of myocardial infarction and the preventive effects of RIPC were assessed by pathological characteristics, namely, infarct, enzyme releases, pressure, and cardiac mechanical activity. Subjected to I/R, the hepatic RIPC minimized the infarct size (17.7 ± 4.96 vs. 50.06 ± 5, p < 0.001) and improved the left ventricular-developed pressure (from 47.42 ± 6.27 to 91.62 ± 5.22 mmHg) and the mechanical activity. Release of phosphocreatine kinase-myocardial band (73.86 ± 1.95 vs. 25.93 ± 0.66 IUL-1) and lactate dehydrogenase (299.01 ± 10.7 vs. 152.3 ± 16.7 IUL-1) was also decreased in the RIPC-treated group. These results demonstrate the cardioprotective effects of the hepatic remote preconditioning against the injury caused by I/R in the isolated perfused hearts.

Meta-Analysis of Peripheral Blood Transcriptome Datasets Reveals a Biomarker Panel for Tuberculosis in Patients Infected With HIV.

Biomarkers are critical for rapid diagnosis of tuberculosis (TB) and could benefit patients with AIDS where diagnosis of TB co-infection is challenging. Meta-analysis is an approach to combine the results of the studies with standard statistical method by weighting each study with different sample size. This study aimed to use meta-analysis to integrate transcriptome datasets from different studies and screen for TB biomarkers in patients who were HIV-positive. Five datasets were subjected to meta-analysis on whole-blood transcriptomes from 640 patients infected with HIV. A total of 293 differentially expressed genes (DEGs) were identified as significant (P<0.0001) using the random effective model to integrate the statistical results from each study. DEGs were enriched in biological processes related to TB, such as "Type I interferon signaling" and "stimulatory C-type lectin receptor signaling". Eighteen DEGs had at least a two-fold change in expression between patients infected with HIV who were TB-positive and those who were TB-negative. GBP4, SERPING1, ATF3 and CDKBN3 were selected as a biomarker panel to perform multivariable logistic regression analysis on TB status and relative gene expression levels. The biomarker panel showed excellent accuracy (AUC>0.90 for HIV+TB) in clinical trial and suggests that meta-analysis is an efficient method to integrate transcriptome datasets from different studies.

Research Progress on Synthesis and Application of Cyclodextrin Polymers.

Cyclodextrins (CDs) are a series of cyclic oligosaccharides formed by amylose under the action of CD glucosyltransferase that is produced by Bacillus. After being modified by polymerization, substitution and grafting, high molecular weight cyclodextrin polymers (pCDs) containing multiple CD units can be obtained. pCDs retain the internal hydrophobic-external hydrophilic cavity structure characteristic of CDs, while also possessing the stability of polymer. They are a class of functional polymer materials with strong development potential and have been applied in many fields. This review introduces the research progress of pCDs, including the synthesis of pCDs and their applications in analytical separation science, materials science, and biomedicine.

Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

Meta-analysis of transcriptome datasets: An alternative method to study IL-6 regulation in coronavirus disease 2019.

In coronavirus disease 2019 (COVID-19) patients, interleukin (IL)-6 is one of the leading factors causing death through cytokine release syndrome. Hence, identification of IL-6 downstream from clinical patients' transcriptome is very valid for analyses of its mechanism. However, clinical study is conditional and time consuming to collect optional size of samples, as patients have the clinical heterogeneity. A possible solution is to deeply mine the relative existing data. Several transcriptome-based studies on other diseases or treatments have revealed different genes to be regulated by IL-6. Through our meta-analysis of these transcriptome datasets, 352 genes were suggested to be regulated by IL-6 in different biological conditions, some of which were related to virus infection and cardiovascular disease. Among them, 232 genes were not identified by current transcriptome studies from clinical research. ICAM1 and PFKFB3 were the most significantly upregulated genes in our meta-analysis and could be employed as biomarkers in patients with severe COVID-19. In general, a meta-analysis of transcriptome datasets could be an alternative way to analyze the immune response and complications of patients suffering from severe COVID-19 and other emergency diseases.

Viral Nanoparticle System: An Effective Platform for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising therapy due to its efficiency and accuracy. The photosensitizer is delivered to the target lesion and locally activated. Viral nanoparticles (VNPs) have been explored as delivery vehicles for PDT in recent years because of their favorable properties, including simple manufacture and good safety profile. They have great potential as drug delivery carriers in medicine. Here, we review the development of PDT photosensitizers and discuss applications of VNP-mediated photodynamic therapies and the performance of VNPs in the treatment of tumor cells and antimicrobial therapy. Furthermore, future perspectives are discussed for further developing novel viral nanocarriers or improving existing viral vectors.

The Application of Prussian Blue Nanoparticles in Tumor Diagnosis and Treatment.

Prussian blue nanoparticles (PBNPs) have attracted increasing research interest in immunosensors, bioimaging, drug delivery, and application as therapeutic agents due to their large internal pore volume, tunable size, easy synthesis and surface modification, good thermal stability, and favorable biocompatibility. This review first outlines the effect of tumor markers using PBNPs-based immunosensors which have a sandwich-type architecture and competitive-type structure. Metal ion doped PBNPs which were used as T1-weight magnetic resonance and photoacoustic imaging agents to improve image quality and surface modified PBNPs which were used as drug carriers to decrease side effects via passive or active targeting to tumor sites are also summarized. Moreover, the PBNPs with high photothermal efficiency and excellent catalase-like activity were promising for photothermal therapy and O2 self-supplied photodynamic therapy of tumors. Hence, PBNPs-based multimodal imaging-guided combinational tumor therapies (such as chemo, photothermal, and photodynamic therapies) were finally reviewed. This review aims to inspire broad interest in the rational design and application of PBNPs for detecting and treating tumors in clinical research.

Near-infrared photothermal immunoassay for pancreatic cancer biomarker CA 19-9 on a digital thermometer.

A near-infrared (NIR) photothermal immunoassay was designed for the sensitive monitoring of CA 19-9 on the capture antibody-coated microplate, coupling a 3D-printed device with a digital thermometer in this work. Prussian blue nanoparticles (PBNPs)-encapsulated CaCO3 microspheres were not only utilized for labeling of detection antibody, but used as the photo-heat conversion materials for the signal amplification. With the sandwiched immunocomplex, the as-released PBNPs under acidic conditions adsorbed and converted NIR-light wavelength to heat under 808-nm laser irradiation, thereby resulting in the temperature change of the detection solution. Under optimum conditions, a linear range from 1.0 U mL-1 to 100 U mL-1 and a detection limit of 0.83 U mL-1 were acquired for the CA 19-9 detection on the portable photothermal immunosensing platform with PBNP-CaCO3-labeling system. Relative standard deviations for reproducibility were ≤9.7% for intra-assay and ≤11.9% for inter-assay. High specificity, long-time storage stability (>10 months) and good accuracy (relative to gold standard with commercial human ELISA kit) with the photothermal immunoassay were encountered for the evaluation of target CA 19-9 in complex system.

Origins and Evolution of the α-L-Fucosidases: From Bacteria to Metazoans.

α-L-fucosidases (EC 3.2.1.51, FUC), belonging to the glycoside hydrolase family 29 (GH29), play important roles in several biological processes and are markers used for detecting hepatocellular carcinoma. In this study, a protein sequence similarity network (SSN) was generated and a subsequent evolutionary analysis was performed to understand the enzymes comprehensively. The SSN indicated that the proteins in the FUC family are mainly present in bacteria, fungi, metazoans, plants, as well as in archaea, but less abundantly. The sequences in bacteria were found to be more diverse than those in other taxonomic groups. The SSN and a phylogenetic tree both supported that the proteins in the FUC family can be classified into 3 subfamilies. FUCs in each subfamily are under the pressure of negative selection. The enzymes from metazoans, fungi, and plants separated into the three subfamilies and shared high similarity with the bacterial homologs. The multiple sequence alignment results indicated that the amino acid residues for binding α-L-fucosidase and catalysis are highly conserved in the 3 subfamilies; however, the evolutionary patterns were different, based on the coevolution analysis in the subfamily of metazoans and bacteria. Finally, gene duplication plays an important role for α-L-fucosidase evolution, not only in metazoans, but also in bacteria and fungi.

Structural Insights for Anti-Influenza Vaccine Design.

Influenza A virus are a persistent and significant threat to human health, and current vaccines do not provide sufficient protection due to antigenic drift, which allows influenza viruses to easily escape immune surveillance and antiviral drug activity. Influenza hemagglutinin (HA) is a glycoprotein needed for the entry of enveloped influenza viruses into host cells and is a potential target for anti-influenza humoral immune responses. In recent years, a number of broadly neutralizing antibodies (bnAbs) have been isolated, and their relative structural information obtained from the crystallization of influenza antigens in complex with bnAbs has provided some new insights into future influenza vaccine research. Here, we review the current knowledge of the HA-targeted bnAbs and the structure-based mechanisms contributing to neutralization. We also discuss the potential for this structure-based approach to overcome the challenge of obtaining a highly desired "universal" influenza vaccine, especially on small proteins and peptides.

Quantitative Comparative Proteomics Reveal Biomarkers for Dengue Disease Severity.

Dengue fever (DF) could develop into dengue haemorrhagic fever (DHF) with increased mortality rate. Since the clinical characteristics and pathogen are same in DF and DHF. It's important to identify different molecular biomarkers to predict DHF patients from DF. We conducted a clinical plasma proteomics study using quantification (TMT)-based quantitative proteomics methodology to found the differential expressed protein in DF patients before they developed into DHF. In total 441 proteins were identified up or down regulated. There proteins are enriched in diverse biological processes such as proteasome pathway, Alanine, aspartate, and glutamate metabolism and arginine biosynthesis. Several proteins such as PLAT, LAMB2, and F9 were upregulated in only DF patients which developed into DHF cases, not in DF, compared with healthy-control. In another way, FGL1, MFAP4, GLUL, and VCAM1 were upregulated in both DHF and DF cases compare with healthy-control. RT-PCR and ELISA were used to validate these upregulated gene expression and protein level in 54 individuals. Results displayed the same pattern as proteomics analysis. All including PLAT, LAMB2, F9, VCAM1, FGL1, MFAP4, and GLUL could be considered as potential markers of predicting DHF since the levels of these proteins vary between DF and DHF. These new founding identified potential molecular biomarkers for future development in precision prediction of DHF in DF patients.

Biochemical Characterization and Phylogenetic Analysis of the Virulence Factor Lysine Decarboxylase From Vibrio vulnificus.

Cadaverine is produced in organisms from the amino acid lysine in a decarboxylation reaction catalyzed by lysine decarboxylase (EC 4.1.1.18). The inducible lysine decarboxylase CadA plays a vital role in acid stress response for enteric bacteria. Vibrio vulnificus is an extremely virulent human pathogen causing gastroenteritis when the acid conditions that prevent survival of V. vulnificus in the stomach or small intestine are overcome. A gene encoding CadA was identified from V. vulnificus. Subsequent analyses showed that CadA from V. vulnificus (VvCadA) is a decamer with a 82-kDa subunit. Homogenous VvCadA was purified from Escherichia coli and used for lysine decarboxylation with an optimal pH of 6.0 and optimal temperature of 37°C. The apparent V max and K m for lysine were 9.45 ± 0.24 µM/min and 0.45 ± 0.05 mM, respectively. Mutation analysis suggested that the amino-acid-binding pyridoxal phosphate, the cofactor of the enzyme, plays a vital role in the reaction. Mutation of the negatively charged residues interacting with lysine also affected the activity of the enzyme to some extent. Quantitative RT-PCR showed that expression of VvcadA was up-regulated under low pH, low salinity, and oxidative stresses. Furthermore, the concentration of cadaverine released to the cell exterior also increased under these stresses. Protein sequence similarity network (SSN) analysis indicated that lysine decarboxylases with ornithine decarboxylases and arginine decarboxylases shared a common ancestor, and that lysine decarboxylases are more conserved during evolution. Our data provide evidence for the biochemical characteristics and important roles of VvCadA under stress conditions.

The relationship between DNA methylation and Reprimo gene expression in gastric cancer cells.

Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting. We found that the RPRM gene promoter region is hypermethylated in gastric cancer tissues (75%, 45/60), plasma samples (86.3%, 44/51) and various cancer cell lines (75%, 3/4), which is correlated with the decrease of RPRM gene expression. The hypermethylation-induced RPRM reduction can be recovered by treating with zebularine, a demethylating agent, and by inhibition of the DNA methyltransferases via RNA interference and CRISPR/Cas9-mediated gene knockout. In addition, we generated RPRM gene-knockout cells and studied the effects of the RPRM deficiency on tumor formation by inoculating these cells in mice. The data show that the loss of RPRM can promote tumorigenesis. These data suggest that the RPRM expression is inhibited by DNA methyltransferases and the RPRM normal function can be restored by treating with DNA methylation inhibitors. The study provides important information regarding the role of RPRM and its methylation related to gastric cancer development.