Study of the stereospecificity in the biotransformation of the four isomers of loxoprofen sodium in rats by chiral HPLC.

Loxoprofen sodium is a chiral drug with two chiral centers. In our previous work, we found that the elimination of its four isomers showed stereospecificity in rats, while how the stereospecific behavior occurred in vivo was unclear. To clarify this issue, each single isomer of loxoprofen sodium was prepared by a chiral semi-preparative high-performance liquid chromatography (HPLC) and then administered to rats. By analysis of each isomer in rat plasma utilizing an analytical chiral HPLC, it was discovered that the chiral inversion occurred only to its (2R)-isomers, one from (1'S,2R)- to (1'S,2S)-isomer and the other from (1'R,2R)- to (1'R,2S)-isomer. The reduction of α-substituted cyclopentanone occurred only to its (1'R)-isomers, with (1'R,2R)-isomer reduced to (2'S,1'R,2R)-trans-alcohol and (1'R,2S)- to (2'S,1'R,2S)-trans-alcohol. Interestingly, both the inversion and the reduction reaction occurred to its (1'R,2R)-isomer due to the special stereo-structure with both (2R)- and (1'R)-configuration, and conversely, neither of them occurred to its (1'S,2S)-isomer, which caused the significantly different elimination rate in vivo. These new findings were meaningful for evaluation of the safety and efficacy of chiral drugs.

Melatonin mitigates PNMC-induced disruption of spindle assembly and mitochondrial function in mouse Oocytes.

3-methyl-4-nitrophenol (PNMC), a degradation product of organophosphorus insecticides and a byproduct of fuel combustion, exerting endocrine-disrupting effects. However, its impact on the meiotic process of oocytes remains unclear. In the present study, we investigated the effects of PNMC on meiotic maturation of mouse oocytes in vitro and related mechanisms. Morphologically, PNMC-exposure affected germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Proteomic analysis suggested that PNMC-exposure altered oocyte protein expression that are associated with cytoskeleton, mitochondrial function and oxidative stress. Further studies demonstrated that PNMC-exposure disrupted spindle assembly and chromosome alignment, caused sustained activation of spindle assembly checkpoint (SAC), and arrested meiosis in oocytes. Specifically, PNMC-exposure interfered with the function of microtubule organizing centers (MTOCs) by significantly reducing phosphorylated mitogen activated protein kinase (p-MAPK) expression and disrupting the localization of Pericentrin and p-Aurora A, leading to spindle assembly failure. Besides, PNMC-exposure also increased α-tubulin acetylation, decreased microtubule stability. Moreover, PNMC-exposure impaired mitochondrial function, evidenced by abnormal mitochondrial distribution, decreased mitochondrial membrane potential and ATP levels, release of Cytochrome C into the cytoplasm, and elevated ROS levels. As a result, exposure to PNMC caused DNA damage and early apoptosis in oocytes. Fortunately, melatonin was able to promote oocyte maturation by removing the excessive ROS and enhancing mitochondrial function. These results highlight the adverse effects of PNMC on meiotic maturation, and underscore the protective role of melatonin against PNMC-induced damage.

Multidisciplinary Team Led by Joint Surgeons to Rapidly Identify Spondyloarthritis.

Context: Spondyloarthritis (SpA) is a group of chronic, inflammatory, rheumatic diseases of which axial SpA and peripheral SpA are the two main types. Patients that predominantly have manifestations of axSpA may have additional peripheral-arthritis symptoms, and vice versa. For these hard-to-diagnose SpA patients, symptoms can be nonspecific and difficult to identify, making it easy to miss a diagnosis or misdiagnosis patients, resulting in disability. Objective: The study intended to evaluate the value of a multidisciplinary team (MDT) led by the joint surgeons to rapidly identify spondyloarthritis (SpA). Design: The research team designed a controlled study that analyzed the clinical data of patients with spondyloarthritis. Setting: The study was conducted in the Department of Joint Surgery at Shandong Second Provincial General Hospital in Jinan, China. Participants: Participants were 113 SpA patients at the hospital between January 2019 and January 2020. Intervention: he research team divided participants into an intervention group, the MDT group that used that model to diagnose 83 participants and the control group with 30 participants, for whom diagnoses occurred using the conventional diagnostic model. Outcome Measures: The research team collected data on participants' number of visits and number of departments visited as well as determined the amount of time that elapsed before a diagnosis occurred. The team also measured C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) and the scores on the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and Bath Ankylosing Spondylitis Functional Index (BASFI) at baseline and after 3 months and 6 months treatment. Results: In the MDT group, diagnoses included: (1) axial SpA (axSpA)-73 participants, and (2) peripheral SpA-10 participants, including three with reactive arthritis, two with uveitis, and five with psoriatic arthritis. Eight participants in that group were HLA-B27 positive, and 14 had complications from a latent tuberculosis infection. In the control group, diagnoses included: (1) axSpA-25 participants; and (2) peripheral SpA-5 participants, including three with psoriatic arthritis and two with reactive arthritis. Six participants in that group were HLA-B27 positive and four had complications from a latent tuberculosis infection. The number of visits, number of departments visited, and time to diagnosis in the MDT group were significantly lower than those in the control group (P < .001). After three and six months of treatment, the MDT group's CRP, ESR, BASDAI, and BASFI were significantly lower than those at baseline (P < .001). Conclusions: The MDT model of spondyloarthritis led by joint surgeons was accurate and efficient, allowing the medical personnel to quickly identify and intervene in SpA and provide effective treatment for patients. It's a diagnosis and treatment model worthy of promotion.

Negative roles of B7-H3 and B7-H4 in the microenvironment of cervical cancer.

Although persistent human papilloma virus (HPV) infection exerts a crucial influence on cervical carcinogenesis, other factors are also involved in its development, such as intraepithelial lesions and cervical cancer. B7-H3 and B7-H4, which have been reported to be co-regulatory ligands in the B7 family, had been found to be overexpressed in cervical cancer and correlated with adverse clinicopathological features and poor prognosis in our previous studies. In this study, we sought to explore the effects of B7-H3 and B7-H4 on the cervical microenvironment. Among several immune cytokines, interleukin-10 (IL-10) and transforming growth factor (TGF) ß1 stand out as important immunosuppressive factors. Our studies found that IL-10 expression increased with pathological change levels and significantly correlated with cervical cancer differentiation (P < 0.05). TGF-ß1 correlated with lymph node metastasis (LNM) (P < 0.01). Expression of B7-H3 and B7-H4 positively correlated with the expression of IL-10 and TGF-ß1. After co-culture, we found that overexpression of B7-H3 and B7-H4 in cervical cancer cell lines resulted in activation of the cell cycle and decreased apoptosis of U-937 cells. In addition, the contents of IL-10 and TGF-ß1, as well as their protein expression levels, increased in co-culture supernatants in U-937 cells, suggesting regulation by the p-JAK2/STAT3 pathway. The in vivo results demonstrated that with the increasing expression of B7-H3/B7-H4, the expression of IL-10 and TGF-ß1 also increased significantly. Overall, the expression of B7-H3 and B7-H4 favored an immunosuppressive microenvironment by promoting the production of IL-10 and TGF-ß1, thereby resulting in progression of cervical carcinogenesis.

Aberrant methylation of EYA4 promotes epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

EYA4, one of the four members of the EYA gene family, is associated with several human cancers. However, its biological functions and molecular mechanisms in the progression of cancer, particularly in esophageal squamous cell carcinoma (ESCC), remain unknown. In the present study, we found that EYA4 was underexpressed and hypermethylated in most of the ESCC cell lines tested (85.7%, 6/7). Treatment with 5-aza-dC and/or trichostatin A (TSA) restored EYA4 expression in ESCC cell lines, which indicates that EYA4 expression was epigenetically regulated. Similarly, EYA4 was aberrantly hypermethylated in ESCC tissues (78%, 39/50) and downregulation of EYA4 occurred in approximately 65% of primary ESCC at protein level where it was associated significantly with TNM stage and lymph node metastases. Knockdown of EYA4 in KYSE30 and KYSE70 ESCC cells using small hairpin RNA increased migration and invasive motility in vitro. Conversely, the overexpression of EYA4 in KYSE180 and KYSE450 promoted an epithelial phenotype, which consisted of decreased migration and invasion abilities and a decrease in TGF-ß1-induced epithelial-mesenchymal transition. Mechanistically, EYA4 overexpression reduced the phosphorylation of Akt and glycogen synthase kinase (GSK) 3ß, which led to the inactivation of slug. In addition, we found that TGF-ß1 decreased EYA4 expression in both a dose-dependent and a time-dependent manner in KYSE30 cells, accompanied by an increase in the expression of DNA methyltransferases, especially DNMT3A. In summary, EYA4 is frequently hypermethylated in ESCC and may function as a tumor suppressor gene in the development of ESCC.

Transcriptome profiling of lncRNA and co-expression networks in esophageal squamous cell carcinoma by RNA sequencing.

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cancer. To identify novel targets for further study in esophageal squamous cell carcinoma (ESCC), we performed a genome-wide analysis of lncRNA expression in 12 ESCC tumor and normal tissues. Publicly available RNA-seq data were downloaded from the NCBI, GEO, and Co-LncRNA databases, and lncRNA and messenger RNA (mRNA) expression profiles were analyzed. In total, 127 lncRNAs were found to be differentially expressed, with a greater than fourfold change in ESCC tumor tissues compared with normal tissues. Among these lncRNAs, 98 were upregulated and 29 downregulated. Moreover, 1469 network nodes and 1720 connection edges between 119 lncRNAs and 1350 coding genes were integrated into the lncRNA and mRNA co-expression network. Bioinformatic analysis using GO terms revealed that these dysregulated lncRNAs are associated with developmental processes, proteinaceous extracellular matrix, and protein binding activity, with ECM-receptor interaction and the PI3K-Akt signaling pathway enrichment. Lastly, qRT-PCR results verified two significantly upregulated lncRNAs and three significantly downregulated lncRNAs in 50 pairs of ESCC tissues and adjacent normal tissues. These results reveal the landscape of ESCC-associated lncRNAs and co-expression networks, providing important insight regarding the lncRNAs involved in ESCC.

Xerophilusin B induces cell cycle arrest and apoptosis in esophageal squamous cell carcinoma cells and does not cause toxicity in nude mice.

Esophageal cancer is the eighth most common cancer in the world and ranks as the sixth leading cause of cancer-related mortality. Esophageal cancer has a poor prognosis partially due to its low sensitivity to chemotherapy agents, and the development of new therapeutic agents is urgently needed. Here, the antitumor activity of a natural ent-kaurane diterpenoid, xerophilusin B (1), which was isolated from Isodon xerophilus, a perennial herb frequently used in Chinese folk medicine for tumor treatment, was investigated. Compound 1 exhibited antiproliferative effects against esophageal squamous cell carcinoma (ESCC) cell lines in a time- and dose-dependent manner with lower toxicity against normal human and murine cell lines. In vivo studies demonstrated that 1 inhibited tumor growth of a human esophageal tumor xenograft in BALB/c nude mice without significant secondary adverse effects, indicating its safety in treating ESCC. Furthermore, 1 induced G2/M cell cycle arrest and promoted apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway in ESCC cell lines. In conclusion, the observations herein reported showed that 1 is a potential chemotherapeutic agent for ESCC and merits further preclinical and clinical investigation for cancer drug development.

LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma.

BACKGROUND: Oesophageal cancer is one of the most deadly forms of cancer worldwide. Long non-coding RNAs (lncRNAs) are often found to have important regulatory roles. OBJECTIVE: To assess the lncRNA expression profile of oesophageal squamous cell carcinoma (OSCC) and identify prognosis-related lncRNAs. METHOD: LncRNA expression profiles were studied by microarray in paired tumour and normal tissues from 119 patients with OSCC and validated by qRT-PCR. The 119 patients were divided randomly into training (n=60) and test (n=59) groups. A prognostic signature was developed from the training group using a random Forest supervised classification algorithm and a nearest shrunken centroid algorithm, then validated in a test group and further, in an independent cohort (n=60). The independence of the signature in survival prediction was evaluated by multivariable Cox regression analysis. RESULTS: LncRNAs showed significantly altered expression in OSCC tissues. From the training group, we identified a three-lncRNA signature (including the lncRNAs ENST00000435885.1, XLOC_013014 and ENST00000547963.1) which classified the patients into two groups with significantly different overall survival (median survival 19.2 months vs >60 months, p<0.0001). The signature was applied to the test group (median survival 21.5 months vs >60 months, p=0.0030) and independent cohort (median survival 25.8 months vs >48 months, p=0.0187) and showed similar prognostic values in both. Multivariable Cox regression analysis showed that the signature was an independent prognostic factor for patients with OSCC. Stratified analysis suggested that the signature was prognostic within clinical stages. CONCLUSIONS: Our results suggest that the three-lncRNA signature is a new biomarker for the prognosis of patients with OSCC, enabling more accurate prediction of survival.

Transglutaminase 3 suppresses proliferation and cisplatin resistance of cervical cancer cells by inactivation of the PI3K/AKT pathway.

Recent studies have shown that dysregulation of transglutaminase 3 (TGM3) is related to the aggressive progression of several cancer types. Our study aimed to determine the function of TGM3 in cervical cancer (CC) tumorigenesis. Gene expression profiles GSE63514, GSE9750, GSE46857 and GSE67522 were obtained from the Gene Expression Omnibus (GEO) database. Overlapping differential expressed genes (DEGs) in CC were screened using GEO2R online tool and Venn diagram software. The Kaplan-Meier plotter was used to determine overall survival. TGM3 expression was analyzed based on GEO and The Cancer Genome Atlas (TCGA) databases, qRT-PCR and western blot analyses. Cell proliferation was evaluated by CCK-8 and EdU incorporation assays. The half-maximal inhibitory concentration (IC50) value of cisplatin and cell apoptosis was assessed by CCK-8 and TUNEL assays, respectively. P-glycoprotein (P-gp) expression and the changes of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway were examined using western blot analysis. We identified 3 overlapping DEGs, including TGM3, glutathione peroxidase 3 (GPX3), and alpha B-crystallin (CRYAB), which were downregulated in CC tissues. TGM3 expression was reduced in CC cells and related to the poor prognosis of CC patients. TGM3 overexpression retarded the proliferation, reduced IC50 value of cisplatin, accelerated cisplatin-induced apoptosis, and inhibited cisplatin-induced P-gp level in CC cells. Furthermore, TGM3 overexpression suppressed the PI3K/Akt pathway in CC cells. Moreover, treatment with 740Y-P, a PI3K activator, abolished the effect of TGM3 overexpression on proliferation and cisplatin resistance in CC cells. In conclusion, overexpression of TGM3 suppressed proliferation and cisplatin resistance in CC cells by blocking the PI3K/Akt pathway.

Stereoselective separation, configuration determination, and quantification of flavanone glycosides in Citrus using chiral HPLC-UV in combination with CD spectra.

Flavanone glucosides, such as naringin and neohesperidin, are present in specific Citrus species and manifest a chiral center at the C-2 position of their flavanone moiety. This study successfully achieved the simultaneous stereoselective separation of the C-2 diastereomers of naringin, neohesperidin and hesperidin, as well as the partial separation of narirutin using a chiral high performance liquid chromatography with ultraviolet detection method with cellulose tris(3,5-dichlorophenylcarbamate) as the stationary phase under normal-phase mode. The mobile phase comprised n-hexane and ethanol (containing 0.25% formic acid) at a proportion of 65 : 35 (v/v) with a flow rate of 0.6 mL min-1. Each single epimer of chiral flavanone glycosides was prepared using chiral semi-preparative chromatography, and the absolute configuration was then characterized by combining the experimental electronic circular dichroism detection and time-dependent density functional theory calculations. The epimer composition of each chiral flavonoid glycoside in Fructus aurantii (Zhiqiao) and Fructus aurantii immaturus (Zhishi) was determined revealing variations among herbs collected from different production regions. Additionally, the epimer composition was found to be related to the harvesting time of the herbs. Considering the safety and efficacy, the existence of epimers of different stereo-configurations should be given more attention in the quality evaluation of natural drugs.

Prognostic significance of C-MYC dysregulation in esophageal squamous cell carcinoma: a meta-analysis.

Aim: The clinicopathological and prognostic significance of C-MYC dysregulation (amplification or overexpression) in esophageal squamous cell carcinoma (ESCC) remains controversial. Therefore, we performed this meta-analysis to elucidate this relationship. Materials & methods: Available studies were retrieved from PubMed, Web of Science, EMBASE and the Cochrane Library, and ten studies with a total of 1432 patients were included in this meta-analysis. Results: Pooled results showed that C-MYC dysregulation was significantly associated with poor overall survival (hazard ratio: 1.405 [95% CI: 1.170-1.639]; p < 0.001) and lymph node metastasis (odds ratio: 1.798 [95% CI: 1.125-2.873]; p = 0.014). Subgroup analysis confirmed the results and more prominent predictive effects were observed in the C-MYC amplification group. Conclusion: C-MYC dysregulation is a promising biomarker for ESCC prognosis.

Metabolic Fingerprinting on Synthetic Alloys for Medulloblastoma Diagnosis and Radiotherapy Evaluation.

Diagnostics is the key in screening and treatment of cancer. As an emerging tool in precision medicine, metabolic analysis detects end products of pathways, and thus is more distal than proteomic/genetic analysis. However, metabolic analysis is far from ideal in clinical diagnosis due to the sample complexity and metabolite abundance in patient specimens. A further challenge is real-time and accurate tracking of treatment effect, e.g., radiotherapy. Here, Pd-Au synthetic alloys are reported for mass-spectrometry-based metabolic fingerprinting and analysis, toward medulloblastoma diagnosis and radiotherapy evaluation. A core-shell structure is designed using magnetic core particles to support Pd-Au alloys on the surface. Optimized synthetic alloys enhance the laser desorption/ionization efficacy and achieve direct detection of 100 nL of biofluids in seconds. Medulloblastoma patients are differentiated from healthy controls with average diagnostic sensitivity of 94.0%, specificity of 85.7%, and accuracy of 89.9%, by machine learning of metabolic fingerprinting. Furthermore, the radiotherapy process of patients is monitored and a preliminary panel of serum metabolite biomarkers is identified with gradual changes. This work will lead to the application-driven development of novel materials with tailored structural design and establishment of new protocols for precision medicine in near future.

The role of low density lipoprotein receptor-related protein 11 as a tumor promoter in cervical cancer.

BACKGROUND: It is unclear whether low density lipoprotein receptor-related protein 11 (LRP11), a newly found lipoprotein receptor regulatory protein, has the carcinogenic effects in cervical cancer. METHODS: Bioinformatics analysis, immunohistochemical (IHC) staining and evaluation, cell proliferation assay, flow cytometry, transwell migration and invasion assays, Western blotting, growth of LRP11-silenced cells in athymic nude mice were performed in this research. RESULTS: We found that LRP11 expression was higher in high-grade squamous intraepithelial lesions (HSIL) and cervical cancer tissue than in normal cervix, and high expression of LRP11 was associated with differentiation degree (P=0.0266), indicating poor prognosis (P=0.0210). The silencing of LRP11 in SiHa and CaSki cell lines inhibited cell proliferation, reduced migration and invasion and suppressed cell growth in nude mice, which possibly related to cell cycle protein regulation of CDK 2/4, cyclin D1/E1, MMP-2/9, and VEGF. Furthermore, LRP11 showed substantial positive correlation with P16 in vivo and in vitro. CONCLUSION: LRP11 plays important roles in proliferation, migration and invasion, with the potential to be a useful prognostic marker and therapeutic target for patients with HSIL and cervical cancer.

Roles of B7-H3 in Cervical Cancer and Its Prognostic Value.

B7-H3, which has been reported to be a co-regulatory ligand of the B7 family, can suppress T cell-mediated immunity and has also been reported to be expressed in many malignancies. In this study, we found that B7-H3 was primarily expressed in the cytoplasm of cervical cancer cells and was associated with deep stromal invasion (P=0.0013). The disease-free survival data showed that cervical cancer patients whose tumours were positive for B7-H3 expression had higher mortality rates compared with patients whose tumours lacked B7-H3 expression (P=0.0317), representing an advantage over P16 (P=0.3486). In contrast, the level of serum B7-H3 was low in cases of cervical intraepithelial neoplasia and cervical cancer. The silencing of B7-H3 in the SiHa, CaSki and H8 cell lines inhibited cell proliferation and enhanced apoptosis, while the over-expression of B7-H3 in HeLa cells showed inverse changes. These changes were partially due to the regulation of cell cycle- and apoptosis-related proteins, such as E2F, P21, P16, PARP-1, Caspase-8, Bax, Bcl-2 and Bcl-xl. The results of in vivo experiments revealed that the knockdown of B7-H3 in tumour cells suppressed SiHa cell growth in nude mice. Overall, B7-H3 is involved in the development and progression of cervical intraepithelial neoplasia and cervical cancer through its effects on the cell cycle and apoptosis, which are mediated via the E7/Rb pathway. B7-H3 also has the potential to be a useful prognostic marker for patients with cervical cancer.

Novel long noncoding RNA NMR promotes tumor progression via NSUN2 and BPTF in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNA) have been implicated in cancer but most of them remain largely unstudied. Here, we identified a novel NSUN2 methylated lncRNA (NMR), which was significantly upregulated in esophageal squamous cell carcinoma (ESCC), functioned as a key regulator of ESCC tumor metastasis and drug resistance. Upregulation of NMR correlated with tumor metastasis and indicated poor overall survival in ESCC patients. Functionally, NMR could promote tumor cell migration and invasion, inhibit cisplatin-induced apoptosis and increase drug resistance in ESCC cells. Mechanistically, transcription of NMR could be upregulated by NF-κB activation after IL-1ß and TNF-α treatment. NMR was methylated by NSUN2 and might competitively inhibit methylation of potential mRNAs. NMR could directly bind to chromatin regulator BPTF, and potentially promote MMP3 and MMP10 expression by ERK1/2 pathway through recruiting BPTF to chromatin. Taken together, NMR functions as an oncogenic gene and may serve as new biomarker and therapeutic target in ESCC.

Decreased 5-hydroxymethylcytosine levels correlate with cancer progression and poor survival: a systematic review and meta-analysis.

Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and then to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), resulting in genomic DNA demethylation. Decreased 5-hmC levels have been reported in a variety of cancers, and loss of 5-hmC might be considered an epigenetic hallmark of cancer. However, the prognostic value of decreased 5-hmC in cancers remain controversial. Here, a systematic review was performed by conducting an electronic search of PubMed, EMBASE, Web of Science and the Cochrane Library. Finally, ten studies with a total of 1736 patients with cancer were included in the present study. Negative/low 5-hmC levels were significantly associated with lymph node metastasis [OR=2.20, 95% CI=1.23-3.96, P=0.008] and advanced TNM stage [OR=2.89, 95% CI=1.21-6.92, P=0.017]. More importantly, negative/low 5-hmC levels were significantly associated with poor prognosis of cancer patients [overall survival: HR=1.76, 95% CI=1.41-2.11, P < 0.001; disease free survival: HR=1.28, 95% CI=0.60-1.96, P < 0.001]. The results of this meta-analysis indicate that decreased 5-hmC levels are an indicator of poor survival of cancer patients. Given variability related to ethnicity, cancer types and detection methods, additional well-designed studies with larger sample sizes are required to further confirm our findings.

Loss of 5-Hydroxymethylcytosine Is an Independent Unfavorable Prognostic Factor for Esophageal Squamous Cell Carcinoma.

Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxylcytosine, which result in genomic DNA demethylation. It was reported that 5-hmC levels were decreased in a variety of cancers and could be regarded as an epigenetic hallmark of cancer. In the present study, 5-hmC levels were detected by immunohistochemistry (IHC) in 173 esophageal squamous cell carcinoma (ESCC) tissues and 91 corresponding adjacent non-tumor tissues; DNA dot blot assays were used to detect the 5-hmC level in another 50 pairs of ESCC tissues and adjacent non-tumor tissues. In addition, the mRNA level of TET1, TET2 and TET3 in these 50 pairs of ESCC tissues was detected by real-time PCR. The IHC and DNA dot blot results showed that 5-hmC levels were significantly lower in ESCC tissues compared with corresponding adjacent non-tumor tissues (P = 0.029). TET2 and TET3 expression was also significantly decreased in tumor tissues compared with paired non-tumor tissues (TET2, P < 0.0001; TET3, P = 0.009), and the decrease in 5-hmC was significantly associated with the downregulation of TET2 expression (r = 0.405, P = 0.004). Moreover, the loss of 5-hmC in ESCC tissues was significantly associated with poor overall survival among patients with ESCC (P = 0.043); multivariate Cox regression analysis showed that the loss of 5-hmC in ESCC tissues was an independent unfavorable prognostic indicator for patients with ESCC (HR = 1.569, P = 0.029). In conclusion, 5-hmC levels were decreased in ESCC tissues, and the loss of 5-hmC in tumor tissues was an independent unfavorable prognostic factor for patients with ESCC.

Integrated bioinformatics analysis of chromatin regulator EZH2 in regulating mRNA and lncRNA expression by ChIP sequencing and RNA sequencing.

Enhancer of zeste homolog 2 (EZH2), a dynamic chromatin regulator in cancer, represents a potential therapeutic target showing early signs of promise in clinical trials. EZH2 ChIP sequencing data in 19 cell lines and RNA sequencing data in ten cancer types were downloaded from GEO and TCGA, respectively. Integrated ChIP sequencing analysis and co-expressing analysis were conducted and both mRNA and long noncoding RNA (lncRNA) targets were detected. We detected a median of 4,672 mRNA targets and 4,024 lncRNA targets regulated by EZH2 in 19 cell lines. 20 mRNA targets and 27 lncRNA targets were found in all 19 cell lines. These mRNA targets were enriched in pathways in cancer, Hippo, Wnt, MAPK and PI3K-Akt pathways. Co-expression analysis confirmed numerous targets, mRNA genes (RRAS, TGFBR2, NUF2 and PRC1) and lncRNA genes (lncRNA LINC00261, DIO3OS, RP11-307C12.11 and RP11-98D18.9) were potential targets and were significantly correlated with EZH2. We predicted genome-wide potential targets and the role of EZH2 in regulating as a transcriptional suppressor or activator which could pave the way for mechanism studies and the targeted therapy of EZH2 in cancer.

MiR-652-3p is upregulated in non-small cell lung cancer and promotes proliferation and metastasis by directly targeting Lgl1.

Our previous study found that miR-652-3p is markedly upregulated in the serum of patients with NSCLC and suggesting that miR-652-3p is a potential biomarker for the early diagnosis of NSCLC. In this study, we detected the expression of miR-652-3p in NSCLC tumor tissues and cell lines and investigated the effect of miR-652-3p on the proliferation and metastasis of NSCLC cells. Our results showed that the expression of miR-652-3p was significantly upregulated in tumor tissues of 50 patients with NSCLC, and it was significantly higher in patients with positive lymph node metastasis, advanced TNM stage and poor prognosis. Using functional analyses by overexpressing or suppressing miR-652-3p in NSCLC cells, we demonstrated that miR-652-3p promoted cell proliferation, migration, invasion and inhibited cell apoptosis. Moreover, the lethal(2) giant larvae 1 (Lgl1) was identified as a direct and functional target of miR-652-3p. Overexpression or knockdown of miR-652-3p led to decreased or increased expression of Lgl1 protein, and the binding site mutation of LLGL1 3'UTR abrogated the responsiveness of the luciferase reporters to miR-652-3p. Overexpression of Lgl1 partially attenuated the function of miR-652-3p. Collectively, these results revealed that miR-652-3p execute a tumor-promoter function in NSCLC through direct binding and regulating the expression of Lgl1.

AJUBA promotes the migration and invasion of esophageal squamous cell carcinoma cells through upregulation of MMP10 and MMP13 expression.

The LIM-domain protein AJUBA has been reported to be involved in cell-cell adhesion, proliferation, migration and cell fate decision by acting as a scaffold or adaptor protein. We previously identified AJUBA as a putative cancer gene in esophageal squamous cell carcinoma (ESCC). However, the function and underlying mechanisms of AJUBA in ESCC remain largely unknown. In the present study, we detected AJUBA levels in ESCC tumor tissues and in corresponding adjacent non-tumor tissues by immunohistochemistry (IHC) and investigated the function and mechanism of AJUBA in ESCC cells. The IHC results showed that AJUBA levels were significantly higher in ESCC tissues compared with corresponding adjacent non-tumor tissues (P < 0.001). Both in vitro and in vivo experiments showed that AJUBA promoted cell growth and colony formation, inhibited cisplatin-induced apoptosis of ESCC cells, and promoted ESCC cell migration and invasion. RNA sequencing was used to reveal the oncogenic pathways of AJUBA that were involved, and MMP10 and MMP13 were identified as two of the downstream targets of AJUBA. Thus, AJUBA upregulates the levels of MMP10 and MMP13 by activating ERK1/2. Taken together, these findings revealed that AJUBA serves as oncogenic gene in ESCC and may serve as a new target for ESCC therapy.