WTAP promotes proliferation of esophageal squamous cell carcinoma via m6A-dependent epigenetic promoting of PTP4A1.

Esophageal squamous cell carcinoma (ESCC) represents a frequently seen malignancy with high prevalence worldwide. Although current studies have shown that Wilms' tumor 1-associated protein (WTAP), a major part in the methyltransferase complex, is involved in various tumor pathological processes, its specific role in ESCC remains unclear. Therefore, the present work focused on exploring WTAP's function and mechanism in ESCC progression using clinical ESCC specimens, ESCC cells, and mammalian models. Firstly, we proved WTAP was significantly upregulated within ESCC, and WTAP mRNA expression showed a good diagnostic performance for ESCC. Functionally, WTAP positively regulated in-vivo and in-vitro ESCC cells' malignant phenotype through the AKT-mTOR signaling pathway. Meanwhile, WTAP positively regulated the N6-methyladenosine (m6A) modification levels in ESCC cells. Protein tyrosine phase type IVA member 1 (PTP4A1) was confirmed to be the m6A target of WTAP, and WTAP positively regulated the expression of PTP4A1. Further study revealed that PTP4A1 showed high expression within ESCC. Silencing PTP4A1 inhibited the AKT-mTOR signaling pathway to suppress ESCC cells' proliferation. Rescue experiments showed that silencing PTP4A1 partially reversed the WTAP-promoting effect on ESCC cells' proliferation ability. Mechanistically, WTAP regulated PTP4A1 expression to activate the AKT-mTOR pathway, promoting the proliferation of ESCC cells. Our study demonstrated that WTAP regulates the progression of ESCC through the m6A-PTP4A1-AKT-mTOR signaling axis and that WTAP is a potential target for diagnosing and treating ESCC.

Emerging roles of circular RNAs in tumorigenesis, progression, and treatment of gastric cancer.

With an estimated one million new cases reported annually, gastric cancer (GC) ranks as the fifth most diagnosed malignancy worldwide. The early detection of GC remains a major challenge, and the prognosis worsens either when patients develop resistance to chemotherapy or radiotherapy or when the cancer metastasizes. The precise pathogenesis underlying GC is not well understood, which further complicates its treatment. Circular RNAs (circRNAs), a recently discovered class of noncoding RNAs that originate from parental genes through "back-splicing", have been shown to play a key role in various biological processes in both eukaryotes and prokaryotes. CircRNAs have been linked to cardiovascular diseases, diabetes, hypertension, Alzheimer's disease, and the occurrence and progression of tumors. Prior studies have established that circRNAs play a crucial role in GC, impacting tumorigenesis, diagnosis, progression, and therapy resistance. This review aims to summarize how circRNAs contribute to GC tumorigenesis and progression, examine their roles in the development of drug resistance, discuss their potential as biotechnological drugs, and summarize their response to therapeutic drugs and microorganism in GC.

Establishment and validation of the autophagy-related ceRNA network in irreversible pulpitis.

BACKGROUND: The molecular mechanisms underlying the onset and progression of irreversible pulpitis have been studied for decades. Many studies have indicated a potential correlation between autophagy and this disease. Against the background of the competing endogenous RNA (ceRNA) theory, protein-coding RNA functions are linked with long noncoding RNAs (lncRNAs) and microRNAs (miRNAs). This mechanism has been widely studied in various fields but has rarely been reported in the context of irreversible pulpitis. The hub genes selected under this theory may represent the key to the interaction between autophagy and irreversible pulpitis. RESULTS: Filtering and differential expression analyses of the GSE92681 dataset, which contains data from 7 inflamed and 5 healthy pulp tissue samples, were conducted. The results were intersected with autophagy-related genes (ARGs), and 36 differentially expressed ARGs (DE-ARGs) were identified. Functional enrichment analysis and construction of the protein‒protein interaction (PPI) network of DE-ARGs were performed. Coexpression analysis was conducted between differentially expressed lncRNAs (DElncRNAs) and DE-ARGs, and 151 downregulated and 59 upregulated autophagy-related DElncRNAs (AR-DElncRNAs) were identified. StarBase and multiMiR were then used to predict related microRNAs of AR-DElncRNAs and DE-ARGs, respectively. We established ceRNA networks including 9 hub lncRNAs (HCP5 and AC112496.1 ↑; FENDRR, AC099850.1, ZSWIM8-AS1, DLX6-AS1, LAMTOR5-AS1, TMEM161B-AS1 and AC145207.5 ↓), which were validated by a qRT‒PCR analysis of pulp tissue from patients with irreversible pulpitis. CONCLUSION: We constructed two networks consisting of 9 hub lncRNAs based on the comprehensive identification of autophagy-related ceRNAs. This study may provide novel insights into the interactive relationship between autophagy and irreversible pulpitis and identifies several lncRNAs that may serve as potential biological markers.

Chloroquine Sensitizes Esophageal Carcinoma EC109 Cells to Paclitaxel by Inhibiting Autophagy.

As an autophagy inhibitor, chloroquine (CQ) showed anti-tumor effect on several types of cancer and paclitaxel (PTX) is widely used in the treatment of esophageal carcinoma patients, but chemoresistance remains a major hurdle for PTX application due to the cytoprotective autophagy. Therefore, the aim of this study was to investigate whether CQ could elevate the anti-tumor effect of PTX on esophageal carcinoma cell line EC109 and explore the potential molecular mechanisms. We confirmed the suppressive effect of PTX on EC109 by MTT, scratch test, transwell and soft agar assay. And, we detected the key proteins in Akt/mTOR pathway, as well as the autophagy marker LC3 and p62 through Western Blot. In addition, GFP-LC3 plasmid was transfected into EC109 cells to monitor the autophagosome after CQ and PTX treatment. Ultimately, we observed the alterations in the proliferation and colony formation abilities of EC109 after knocking down mTOR by shRNA. We confirmed PTX could suppress the proliferation, migration and colony formation (all P < 0.05) abilities of EC109, and CQ could sensitize the inhibition effect of PTX by inhibiting autophagy through Akt/mTOR pathway. Furthermore, inhibiting Akt/mTOR pathway initiated autophagy and enhanced the sensitivity of EC109 to CQ and PTX. In summary, we suggest CQ could be used as a potential chemosensitizer for PTX in esophageal carcinoma treatment.

TPP1 Inhibits DNA Damage Response and Chemosensitivity in Esophageal Cancer.

TPP1, as one of the telomere-protective protein complex, functions to maintain telomere stability. In this study, we found that TPP1 was significantly upregulated in esophageal cancer (EC). We found that the proliferation and migration ability were significantly inhibited, while the results of flow cytometry assay indicated that the growth was hindered in the G1 phase after TPP1 knockdown. However, the proliferative viability and migratory ability were reversed after TPP1 overexpression in EC cells. Then, we found a significant increase in ß-galactosidase positivity following TPP1 knockdown and the opposite following TPP1 overexpression in EC cells. Furthermore, TPP1 knockdown increased DNA damage and upregulated expression of the γ-H2AXS139 in the cell nucleus. Correspondingly, DNA damage was reversed after TPP1 overexpression in EC cells. Similarly, we found that the expression of ATM/ATR pathway proteins were upregulated after TPP1 knockdown, while the expression of the above proteins was downregulated after TPP1 overexpression in EC cells. TPP1 knockdown significantly inhibited the growth of transplanted tumors and upregulated the expression of ATM/ATR pathway proteins in transplanted tissues, whereas TPP1 overexpression significantly promoted their proliferation and downregulated the expression of the above proteins in vivo. Strikingly, we found that TPP1 could reduce the chemosensitivity of EC cells to cisplatin, which may have a potential link to clinical chemoresistance. In conclusion, TPP1 regulates the DNA damage response through the ATM/ATR-p53 signaling pathway and chemoresistance and may be a new target for improving the efficacy of chemotherapy in the treatment of EC.

Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage.

BACKGROUND: Isothermal amplification is considered to be one of the most promising tools for point-of-care testing molecular diagnosis. However, its clinical application is severely hindered by nonspecific amplification. Thus, it is important to investigate the exact mechanism of nonspecific amplification and develop a high-specific isothermal amplification assay. METHODS: Four sets of primer pairs were incubated with Bst DNA polymerase to produce nonspecific amplification. Gel electrophoresis, DNA sequencing, and sequence function analysis were used to investigate the mechanism of nonspecific product generation, which was discovered to be nonspecific tailing and replication slippage mediated tandem repeats generation (NT&RS). Using this knowledge, a novel isothermal amplification technology, bridging primer assisted slippage isothermal amplification (BASIS), was developed. RESULTS: During NT&RS, the Bst DNA polymerase triggers nonspecific tailing on the 3'-ends of DNAs, thereby producing sticky-end DNAs over time. The hybridization and extension between these sticky DNAs generate repetitive DNAs, which can trigger self-extension via replication slippage, thereby leading to nonspecific tandem repeats (TRs) generation and nonspecific amplification. Based on the NT&RS, we developed the BASIS assay. The BASIS is carried out by using a well-designed bridging primer, which can form hybrids with primer-based amplicons, thereby generating specific repetitive DNA and triggering specific amplification. The BASIS can detect 10 copies of target DNA, resist interfering DNA disruption, and provide genotyping ability, thereby offering 100% accuracy for type 16 human papillomavirus detection. CONCLUSION: We discovered the mechanism for Bst-mediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity.

Hysteroscopic Fenestration with Precise Incision of the Cavity Septum: A Novel Minimally Invasive Surgery of Complete Septate Uterus with Double Cervix.

STUDY OBJECTIVE: This study aimed to develop and describe a novel surgical procedure that involves hysteroscopic fenestration with precise incision of the complete uterine septum and double cervix preservation after magnetic resonance imaging (MRI) evaluation in patients and to evaluate its efficacy. DESIGN: A prospective consecutive clinical study. SETTING: A university teaching hospital. PATIENTS: Twenty-four patients with complete septate uterus and double cervix. INTERVENTIONS: Three-dimensional reconstruction of uterus was performed with pelvic MRI and three-dimensional SPACE sequence scanning. Hysteroscopic fenestration with precise incision of the cavity septum and double cervix preservation was performed in patients. Three months after operation, follow-up pelvic MRI and second-look hysteroscopy were performed conventionally. MEASUREMENTS AND MAIN RESULTS: Operating time, blood loss, operative complications, MRI and hysteroscopic changes of uterus, symptoms improvement, and reproductive outcomes were assessed. The surgery was successfully completed without any intraoperative complications in all patients. Operating time was 21.71 ± 8.28 minutes (range, 10-40 minutes) and blood loss was 9.92 ± 7.14 mL (range, 5-30 mL). Postoperative MRI showed the uterine anteroposterior diameter (3.66 cm vs 3.92 cm; p <.05) was increased. Postoperative MRI and the second-look hysteroscopy showed the cavity shape and uterine volume were expanded to the normal. Symptoms of dysmenorrhea, abnormal uterine bleeding, and dyspareunia were ameliorated after the surgery in 70% of patients (7 of 10), 60% of patients (3 of 5), and 1 patient, respectively. The preoperative spontaneous abortion rate was 80% (4 of 5) and the postoperative spontaneous abortion rate was 11.11% (1 of 9). After the surgery, there were 2 ongoing pregnancies and 6 pregnancies ended in term births. Two live births were delivered by cesarean section and 4 by vaginal delivery without cervical incompetence during pregnancy. CONCLUSIONS: Hysteroscopic fenestration with precise incision of the uterine septum and double cervix preservation is an effective surgical procedure.

Multi-Omics Analysis Reveals the Pathogenesis of Growth-Disordered Raccoon Dog.

Microorganisms of the genus Eperythrozoon are a zoonotic chronic infectious disease with wide distribution. We found that raccoons infected with Eperythrozoon showed obvious stunting, which seriously affected the economic benefits of raccoon dogs. To investigate the pathogenesis of the raccoon dog, we used transcriptome and proteome sequencing to analyze the changes in mRNA, miRNA, and protein expression in raccoon dogs infected with Eperythrozoon and normal raccoons. The results showed that the expression levels of genes related to immunity, metabolism, and enzyme activity were significantly changed. Among these, ERLIN1, IGF1R, CREB3L1, TNS1, TENC1, and mTOR play key roles. Additionally, the miR-1268, miR-125b, miR-10-5p, and miR-10 as central miRNAs regulate the expression of these genes. Integrated transcriptomic and proteomic analyses revealed consistent trends in mRNA and protein changes in MYH9, FKBP1A, PRKCA, and CYP11B2. These results suggest that Eperythrozoon may contribute to the slow development of raccoons by affecting the expression of mRNAs and miRNAs, reducing their immunity and causing metabolic abnormalities.

Analysis of Hypoxia Inducible Factor-1α Expression and Its Effects on Glycolysis of Esophageal Carcinoma.

We investigated the regulatory effects of hypoxia-inducible factor-1a (HIF-1α) on glycolysis metabolism in esophageal carcinoma (ESCA) cells. A series of bioinformatics databases and tools were used to investigate the expression and role of HIF-1α in ESCA. The expression of HIF-1a in ESCA tissues and adjacent tissues was validated by real-time PCR. Small interfering RNA (siRNA) was used to inhibit HIF-1α-related genes in human ESCA cells (Eca109 and KYSE150). Cell proliferation was detected by the CCK-8 assay. The expression of HIF-1α and glycolytic enzymes were investigated by real-time PCR and Western blot. HIF-1α is highly expressed in ESCA and is involved in many biological processes such as cell hypoxia reaction, glucose metabolic process. Further in vitro experiments showed that expression of HIF-1α in Eca109 and KYSE150 significantly increased under hypoxia compared with normoxia conditions. Also, the glucose uptake and lactate production under hypoxia were higher. The expression levels of hexokinase 2 (HK2) and pyruvate dehydrogenase kinase 1 (PDK1), glycolysis-related genes, were significantly increased under hypoxia. After siRNA knockdown of HIF-1a in Eca109 and KYSE150, the glucose uptake and lactate production, as well as cell proliferation were significantly decreased under hypoxia, and HK2 and PDK1 were significantly downregulated. HIF-1α promotes glycolysis of ESCA cells by upregulating the expression of HK2 and PDK1 under hypoxia.

Metabolome and transcriptome profiling unveil the mechanisms of light-induced anthocyanin synthesis in rabbiteye blueberry (vaccinium ashei: Reade).

BACKGROUND: Blueberry is one of the most important fruit crops worldwide. Anthocyanin is an important secondary metabolites that affects the appearance and nutritive quality of blueberries. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation induced by light intensity in blueberries. RESULTS: The metabolic analysis revealed that there were 134 significantly changed metabolites in the natural light compared to the control, and flavone, flavonol, and anthocyanins were the most significantly increased. Transcriptome analysis found 6 candidate genes for the anthocyanin synthesis pathway. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the light intensity-treatment increased the accumulation of delphinidin-3-O-arabinoside compared to under the shading-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted that the gene of VcF3'5'H4 may be a candidate gene for anthocyanin accumulation and is highly expressed in light intensity-treated fruit. Through the co-expression analysis of transcription factors and anthocyanin synthesis pathway genes, we found that the VcbHLH004 gene may regulate VcF3'5'H4, and then we transformed VcbHLH004 heterologously into tomato to verify its function. CONCLUSION: These results provide novel insights into light intensity regulation of blueberry anthocyanin accumulation and represent a valuable data set to guide future functional studies and blueberry breeding.

Impaired AT2 to AT1 cell transition in PM2.5-induced mouse model of chronic obstructive pulmonary disease.

BACKGROUND: Particular matter 2.5 (PM2.5) is one of the most important air pollutant, and it is positively associated with the development of chronic obstructive pulmonary disease (COPD). However, the precise underlying mechanisms through which PM2.5 promotes the development of COPD remains largely unknown. METHODS: Mouse alveolar destruction were determined by histological analysis of lung tissues and lung function test. Alveolar type II cells (AT2) to alveolar type I cells (AT1) transition in PM2.5-induced COPD mouse model was confirmed via immunofluorescence staining and qPCR analysis. The differentially expressed genes in PM2.5-induced COPD mouse model were identified by RNA-sequencing of alveolar epithelial organoids and generated by bioinformatics analysis. RESULTS: In this study, we found that 6 months exposure of PM2.5 induced a significantly decreased pulmonary compliance and resulted in pulmonary emphysema in mice. We showed that PM2.5 exposure significantly reduced the AT2 to AT1 cell transition in vitro and in vivo. In addition, we found a reduced expression of the intermediate AT2-AT1 cell process marker claudin 4 (CLDN4) at day 4 of differentiation in mouse alveolar organoids treated with PM2.5, suggesting that PM2.5 exposure inhibited AT2 cells from entering the transdifferentiation process. RNA-sequencing of mouse alveolar organoids showed that several key signaling pathways that involved in the AT2 to AT1 cell transition were significantly altered including the Wnt signaling, MAPK signaling and signaling pathways regulating pluripotency of stem cells following PM2.5 exposure. CONCLUSIONS: In summary, these data demonstrate a critical role of AT2 to AT1 cell transition in PM2.5-induced COPD mouse model and reveal the signaling pathways that potentially regulate AT2 to AT1 cell transition during this process. Our findings therefore advance the current knowledge of PM2.5-induced COPD and may lead to a novel therapeutic strategy to treat this disease.

The M6A methyltransferase METTL3 regulates proliferation in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal human cancers with a lower 5-year survival rate. N6-methyladenosine (m6A) methylation, an important epigenetic modification, has been reported to associate with physiological and pathological processes of cancers. However, its role in ESCC remains unclear. In this work, we found that the m6A levels were elevated in ESCC cancer tissues and ESCC cells. The PPI network demonstrated that METTL3, METTL14, WTAP, RBM15, and KIAA1429 were all significantly associated with each other. Moreover, we found a significant upregulation of METTL3 mRNA and protein amounts in ESCC tissues. The METTL3 mRNA expression level of tissues had associations with ESCC differentiation extent and sex (p < 0.05). The METTL3 mRNA expression level of tissues, sensitivity for diagnosing ESCC was 75.00%, specificity was 72.06% and area under the ROC curve was 0.8030. Depletion of METTL3 markedly diminished m6A levels in human ESCC cell lines and METTL3 overexpression restored the reduction in m6A levels. These results suggested that METTL3 is the primary enzyme that modulates m6A methylation and a critical regulatory factor in ESCC. Additionally, METTL3 knockdown significantly suppressed the ESCC cell proliferation, while METTL3 overexpression markedly promoted ESCC cell proliferation both in cell and animal models. These results demonstrated that METTL3 promotes ESCC development. Furthermore, METTL3 may modulate the cell cycle of ESCC cells through a p21-dependent pattern. METTL3-guided m6A modification may contribute to the progression of ESCC via the p21-axis. Our study is the first investigation to report that METTL3-mediated m6A methylation plays a crucial role in ESCC oncogenesis and highlights that METTL3 might be a potential biomarker and therapeutic target for ESCC patients.

c-Abl-Mediated Tyrosine Phosphorylation of PARP1 Is Crucial for Expression of Proinflammatory Genes.

Poly(ADP-ribosyl)ation is a rapid and transient posttranslational protein modification mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP1). Fundamental roles of activated PARP1 in DNA damage repair and cellular response pathways are well established; however, the precise mechanisms by which PARP1 is activated independent of DNA damage, and thereby playing a role in expression of inflammatory genes, remain poorly understood. In this study, we show that, in response to LPS or TNF-α exposure, the nonreceptor tyrosine kinase c-Abl undergoes nuclear translocation and interacts with and phosphorylates PARP1 at the conserved Y829 site. Tyrosine-phosphorylated PARP1 is required for protein poly(ADP-ribosyl)ation of RelA/p65 and NF-κB-dependent expression of proinflammatory genes in murine RAW 264.7 macrophages, human monocytic THP1 cells, or mouse lungs. Furthermore, LPS-induced airway lung inflammation was reduced by inhibition of c-Abl activity. The present study elucidated a novel signaling pathway to activate PARP1 and regulate gene expression, suggesting that blocking the interaction of c-Abl with PARP1 or pharmaceutical inhibition of c-Abl may improve the outcomes of PARP1 activation-mediated inflammatory diseases.

A Method To Prevent SARS-CoV-2 IgM False Positives in Gold Immunochromatography and Enzyme-Linked Immunosorbent Assays.

We set out to investigate the interference factors that led to false-positive novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM detection results using gold immunochromatography assay (GICA) and enzyme-linked immunosorbent assay (ELISA) and the corresponding solutions. GICA and ELISA were used to detect SARS-CoV-2 IgM in 86 serum samples, including 5 influenza A virus (Flu A) IgM-positive sera, 5 influenza B virus (Flu B) IgM-positive sera, 5 Mycoplasma pneumoniae IgM-positive sera, 5 Legionella pneumophila IgM-positive sera, 6 sera of HIV infection patients, 36 rheumatoid factor IgM (RF-IgM)-positive sera, 5 sera from hypertensive patients, 5 sera from diabetes mellitus patients, and 14 sera from novel coronavirus infection disease 19 (COVID-19) patients. The interference factors causing false-positive reactivity with the two methods were analyzed, and the urea dissociation test was employed to dissociate the SARS-CoV-2 IgM-positive serum using the best dissociation concentration. The two methods detected positive SARS-CoV-2 IgM in 22 mid-to-high-level-RF-IgM-positive sera and 14 sera from COVID-19 patients; the other 50 sera were negative. At a urea dissociation concentration of 6 mol/liter, SARS-CoV-2 IgM results were positive in 1 mid-to-high-level-RF-IgM-positive serum and in 14 COVID-19 patient sera detected using GICA. At a urea dissociation concentration of 4 mol/liter and with affinity index (AI) levels lower than 0.371 set to negative, SARS-CoV-2 IgM results were positive in 3 mid-to-high-level-RF-IgM-positive sera and in 14 COVID-19 patient sera detected using ELISA. The presence of RF-IgM at mid-to-high levels could lead to false-positive reactivity of SARS-CoV-2 IgM detected using GICA and ELISA, and urea dissociation tests would be helpful in reducing SARS-CoV-2 IgM false-positive results.

Bioinformatics Analysis of Key Candidate Genes and Pathways in Ulcerative Colitis.

Ulcerative colitis (UC) is chronic, idiopathic disease that affects the colon and the rectum and the underlying pathogenesis of UC remains to be known. The clinical drugs are mainly work based on anti-inflammation and immune system. However, most of them are expensive and have severe side effects. Therefore, identification of novel targets and exploring new drugs are urgently needed. In this study, several bioinformatics approaches were used to discover key genes and further in order to explore the pathogenesis of UC. Two microarray datasets, GSE38713 and GSE9452 were selected from NCBI-Gene Expression Omnibus database. Differentially expression genes (DEGs) were identified by using LIMMA Package of R. Then, we filtered clustered candidate genes into Gene Ontology (GO) and pathway enrichment analysis with the Database for Annotation, Visualization and Integrated Discovery (DAVID), KEGG pathway based on functions and signaling pathways with significant enrichment analysis. The protein-protein interaction (PPI) network was constructed by the Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis, and visualized by Cytoscape and further analyzed by Molecular Complex Detection. Lastly, 353 up-regulated and 145 down-regulated genes were than recognized. After consulting a number of references and network degree analysis, four hub genes, namely FCGR2A, C3, INPP5A, and ACAA1 were identified, and these genes were mainly enriched in complement and coagulation cascades, mineral absorption, and Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathways. In conclusion, this study would provide new clues for the pathogenesis and identification of drug targets of UC in the near future.

Ethnic, geographic, and seasonal differences of vitamin D status among adults in south-west China.

BACKGROUND: There are limited data on vitamin D status of Sichuan province, and no investigation has been carried out on the correlations of 25(OH)D and BTMs between healthy Hans and Tibetans of Sichuan province. This study aimed to examine 25(OH)D levels around Sichuan province and to assess differences by ethnicity, age, gender, sunlight exposure, geographic location, and seasons. METHODS: Blood samples from 2317 healthy adults aged of 18 to 75 years and of Han and Tibetan ethnicities were collected in six regions and during four seasons. Serum 25(OH)D2 and 25(OH)D3 levels were measured by LC-MS/MS method. Serum total P1NP and ß-CTX were measured by immunoassay. RESULTS: Participants aged 18-40 years showed significantly lower 25(OH)D levels than participants aged 41-75 years old (P < .0001). The median serum 25(OH)D level for males was significantly higher than that of females (P < .0001). Serum 25(OH)D levels among four seasons and different districts varied significantly (P < .0001). In addition, the 25(OH)D level of Tibetans was significantly lower than that of Hans, while the serum total P1NP and ß-CTX levels of Tibetans were significantly higher than those of Hans (P < .0001). CONCLUSION: Adult population was more common to have vitamin D deficiency/insufficiency among Tibetans, females, north regions and in spring and winter.

Identification and validation of key genes associated with non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is one of the main causes of death induced by cancer globally. However, the molecular aberrations in NSCLC patients remain unclearly. In the present study, four messenger RNA microarray datasets (GSE18842, GSE40275, GSE43458, and GSE102287) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between NSCLC tissues and adjacent lung tissues were obtained from GEO2R and the overlapping DEGs were identified. Moreover, functional and pathway enrichment were performed by Funrich, while the protein-protein interaction (PPI) network construction were obtained from STRING and hub genes were visualized and identified by Cytoscape software. Furthermore, validation, overall survival (OS) and tumor staging analysis of selected hub genes were performed by GEPIA. A total of 367 DEGs (95 upregulated and 272 downregulated) were obtained through gene integration analysis. The PPI network consisted of 94 nodes and 1036 edges in the upregulated DEGs and 272 nodes and 464 edges in the downregulated DEGs, respectively. The PPI network identified 46 upregulated and 27 downregulated hub genes among the DEGs, and six (such as CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M) of that have not been identified to be associated with NSCLC so far. Moreover, the expression differences of the mentioned hub genes were consistent with that in lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database. Further analysis showed that all the six hub genes were associated with tumor staging except MYH11, while only the upregulated DEG CENPE was associated with the worse OS of patients with NSCLC. In conclusion, the current study showed that CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M might be the key genes contributed to tumorigenesis or tumor progression in NSCLC, further functional study is needed to explore the involved mechanisms.

Betaine-assisted recombinase polymerase assay with enhanced specificity.

Recombinase polymerase amplification (RPA) is a widespread isothermal amplification method and regarded as an excellent candidate to replace polymerase chain reaction. However, the specificity of RPA is not always satisfactory when the sample contains amounts of background DNA. Herein, we report a novel RPA method named betaine-assisted RPA (B-RPA) that uses inexpensive betaine to avoid nonspecific amplification effectively. Result show that nonspecific amplification is prone to occur in RPA if the primers have not been rigorously refined, especially in detecting samples with large amounts of background DNA. This problem has been addressed by adding betaine to the RPA reactions. Our data show that the addition of 0.8 M betaine can significantly increase specificity and efficiency simultaneously. This B-RPA method is also used to detect hepatitis B virus DNA in clinical plasma samples, thereby demonstrating the clinical practicability of B-RPA.

[Molecular genetic diagnosis of a carrier with rare α-thalassemia mutations].

OBJECTIVE: To explore the cause of inconsistent genotypes for an α-thalassemia carrier by using two commercial genotyping kits. METHODS: GAP-PCR and PCR-reverse dot blotting (PCR-RDB) were employed to determine the genotype of the carrier, while Sanger sequencing was used to verify the results. RESULTS: Sequencing analysis demonstrated that the subject has carried a α1 globin gene with a 3.7 kb heterozygous deletion. In addition, two novel mutations, IVS-II-55(T>G) and IVS-II-119(G>TCGGCCC), were found in intron 2 of α2 globin gene. CONCLUSION: The two mutations located in the binding regions of PCR primers have caused failure of PCR amplification and misreading of the genotype. Combination of clinical and hematological phenotypes is indispensible to infer the genotype of carriers for accurate diagnosis.

MiR-27a Promotes Hemin-Induced Erythroid Differentiation of K562 Cells by Targeting CDC25B.

BACKGROUND/AIMS: MicroRNAs (miRNAs) play a crucial role in erythropoiesis. MiR-23a∼27a∼24-2 clusters have been proven to take part in erythropoiesis via some proteins. CDC25B (cell division control Cdc2 phosphostase B) is also the target of mir-27a; whether it regulates erythropoiesis and its mechanism are unknown. METHODS: To evaluate the potential role of miR-27a during erythroid differentiation, we performed miR-27a gain- and loss-of-function experiments on hemin-induced K562 cells. We detected miR-27a expression after hemin stimulation at different time points. At the same time, the γ-globin gene also was measured via real-time PCR. According to the results of the chips, we screened the target protein of miR-27a through a dual-luciferase reporter assay and identified it via Western blot analyses. To evaluate the function of CDC25B, benzidine staining and flow cytometry were employed to detect the cell differentiation and cell cycle. RESULTS: We found that miR-27a promotes hemin-induced erythroid differentiation of human K562 cells by targeting cell division cycle 25 B (CDC25B). Overexpression of miR-27a promotes the differentiation of hemin-induced K562 cells, as demonstrated by γ-globin overexpression. The inhibition of miR-27a expression suppresses erythroid differentiation, thus leading to a reduction in the γ-globin gene. CDC25B was identified as a new target of miR-27a during erythroid differentiation. Overexpression of miR-27a led to decreased CDC25B expression after hemin treatment, and CDC25B was up-regulated when miR-27a expression was inhibited. Moreover, the inhibition of CDC25B affected erythroid differentiation, as assessed by γ-globin expression. CONCLUSION: This study is the first report of the interaction between miR-27a and CDC25B, and it improves the understanding of miRNA functions during erythroid differentiation.