Interferon-γ in the tumor microenvironment promotes the expression of B7H4 in colorectal cancer cells, thereby inhibiting cytotoxic T cells.

The bioactivity of interferon-γ (IFN-γ) in cancer cells in the tumor microenvironment (TME) is not well understood in the current immunotherapy era. We found that IFN-γ has an immunosuppressive effect on colorectal cancer (CRC) cells. The tumor volume in immunocompetent mice was significantly increased after subcutaneous implantation of murine CRC cells followed by IFN-γ stimulation, and RNA sequencing showed high expression of B7 homologous protein 4 (B7H4) in these tumors. B7H4 promotes CRC cell growth by inhibiting the release of granzyme B (GzmB) from CD8+ T cells and accelerating apoptosis in CD8+ T cells. Furthermore, interferon regulatory factor 1 (IRF1), which binds to the B7H4 promoter, is positively associated with IFN-γ stimulation-induced expression of B7H4. The clinical outcome of patients with CRC was negatively related to the high expression of B7H4 in cancer cells or low expression of CD8 in the microenvironment. Therefore, B7H4 is a biomarker of poor prognosis in CRC patients, and interference with the IFN-γ/IRF1/B7H4 axis might be a novel immunotherapeutic method to restore the cytotoxic killing of CRC cells.

Synthesis of plant-derived cholesterol from bisnoralcohol.

Currently, the market demand of the non-animal-derived cholesterol is increasing. A novel synthetic route of producing cholesterol was developed through multiple reactions from plant-sourced and commercially available bisnoralcohol (BA). The key reaction conditions, including solvents, reaction temperatures, bases and reducing agents of the route were investigated and optimized. In this straightforward synthetic pathway of cholesterol, most of the reaction steps possess high conversions with average yields of 94%, and the overall yield is up to 74% (5 steps) from the BA. The epicholesterol and were also synthesized. This promising route offers economical and efficient strategies for potential large-scale production of plant-derived cholesterol.

Cdc42 subcellular relocation in response to VEGF/NRP1 engagement is associated with the poor prognosis of colorectal cancer.

Microscopic indications of malignancy and hallmark molecules of cancer are pivotal to determining cancer patient prognosis and subsequent medical intervention. Here, we found that compared to apical expression of Cdc42, which indicated that basal expression of Cdc42 occurred at the migrating cell front, glandular basal expression of Cdc42 (cell division cycle 42) in tissues indicated poorer prognoses for colorectal cancer (CRC) patients. The current study shows that activated Cdc42 was rapidly recruited to the migrating CRC cell front after VEGF stimulation through engagement of membrane-anchored neuropilin-1 (NRP1). When VEGF signalling was blocked with NRP1 knockdown or ATWLPPR (A7R, antagonist of VEGF/NRP1 interaction), Cdc42 activation and relocation to the cell front was attenuated, and filopodia and invadopodia formation was inhibited. The VEGF/NRP1 axis regulates directional migration, invasion, and metastasis through Cdc42 activation and relocation resulting from actin filament polymerisation of the extensions of membrane protrusions. Collectively, the immuno-micromorphological pattern of subcellular Cdc42 at the cell front indicated aggressive behaviours and predicted poor prognosis in CRC patients. Disruption of the intra- and extracellular interactions of the VEGF/NRP1 axis or Cdc42 relocation could be performed in clinical practice because it might inhibit cancer cell motility and metastasis.

Dysregulated NF-κB signal promotes the hub gene PCLAF expression to facilitate nasopharyngeal carcinoma proliferation and metastasis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is common in Southern China. The molecular mechanism underlying NPC genesis and progression has been comprehensively investigated, but the key gene (s) or pathway (s) pertaining to NPC are unidentified. METHODS: We explored some key genes and pathways involved in NPC through using meta-analysis of deposited expression of microarray data of NPC. The expression of proliferating cell nuclear antigen clamp associated factor (PCLAF) was determined by real-time PCR and western blots. CCK-8 assay, colony formation assay, transwell migration assay, cell wound healing assay, cell cycle analysis and cell apoptosis were carried out to assess biological behaviors caused by downregulation and overexpression of PCLAF in vitro. CHIP was utilized to determine the direct upstream regulatory transcription factors of PCLAF. RESULTS: PCLAF was the key gene of NPC, which was significantly up-regulated in NPC cell line compared to the normal nasopharyngeal cell line. Additionally, in vitro assay has demonstrated the down-regulation and overexpression of PCLAF, resulted in significantly suppressed and enhanced NPC proliferation, metastasis and invasion respectively. Furthermore, the up-regulation of PCLAF in NPC is induced by direct binding of dysregulated NF-κB p50/RelB complex to the promoter of PCLAF. CONCLUSION: Our results offer a strategy for re-using the deposited data to find the key genes and pathways involved in pathogenesis of cancer. Our study has provided evidence of supporting the role of PCLAF in NPC genesis and progression.

High expression of PRKDC promotes breast cancer cell growth via p38 MAPK signaling and is associated with poor survival.

BACKGROUND: DNA-Dependent Protein Kinase Catalytic Subunit (PRKDC), a key component of the DNA damage repair pathway, is associated with chemotherapy resistance and tumor progression. METHODS: Here we analyzed transcriptome data of ~2,000 breast cancer patients and performed functional studies in vitro to investigate the function of PRKDC in breast cancer. RESULTS: Our results revealed overexpression of PRKDC in multiple breast cancer subtypes. Consistent with patients' data, overexpression of PRKDC was also observed in breast cancer cell lines compared to normal breast epithelial cells. Knockdown of PRKDC in MCF-7 and T47D breast cancer cell lines resulted in proliferation inhibition, reduced colony formation and G2/M cell cycle arrest. Furthermore, we showed that PRKDC knockdown induced proliferation inhibition through activation of p38 MAPK, but not ERK MAPK, signaling pathway in breast cancer cells. Blockage of p38 MAPK signaling could largely rescue proliferation inhibition and cell cycle arrest induced by PRKDC knockdown. Moreover, we analyzed gene expression and clinical data from six independent breast cancer cohorts containing ~1,000 patients. In all cohorts, our results consistently showed that high expression of PRKDC was significantly associated with poor survival in both treated and untreated breast cancer patients. CONCLUSION: Together, our results suggest that high expression of PRKDC facilitates breast cancer cell growth via regulation of p38 MAPK signaling, and is a prognostic marker for poor survival in breast cancer patients.

PRKDC is a prognostic marker for poor survival in gastric cancer patients and regulates DNA damage response.

A hallmark of gastric cancer is the high rate of genomic instability associated with deregulation of DNA damage repair pathways. DNA-Dependent Protein Kinase Catalytic Subunit (PRKDC) is a key component of the non-homologous end-joining (NHEJ) pathway. By reanalyzing transcriptome data of 80 pairs of gastric cancer tumors and the adjacent normal tissues from non-treated patients, we identified PRKDC as the top upregulated DNA damage repair genes in gastric cancer. High expression of PRKDC is associated with poor survival of gastric cancer patients, and genomic amplification of the gene is frequently observed across most gastric cancer subtypes. Knockdown of PRKDC in gastric cell lines resulted in reduced proliferation and cell cycle arrest. Furthermore, we showed that loss of PRKDC induced DNA damage and enhanced gastric cancer cell chemosensitivity to DNA-damaging reagents. Together, our results suggest that PRKDC is a prognostic marker of poor survival and is a putative target to overcome chemoresistance in gastric cancer.

Application whole exome sequencing for the clinical molecular diagnosis of patients with Duchenne muscular dystrophy; identification of four novel nonsense mutations in four unrelated Chinese DMD patients.

BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common form of inherited muscular dystrophy. Germline mutations in dystrophin (DMD) gene cause DMD, with a X-linked recessive mode of inheritance. Patients with DMD are usually characterized by weakness of muscle, usually started since childhood and gradually the patient will unable to stand and walk. METHODS: In our present study, we identified four unrelated Chinese patients with DMD from four Chinese families. Whole exome sequencing was performed for genetic molecular analysis for these probands. RESULTS: Whole exome sequencing and confirmatory Sanger sequencing identified four novel nonsense mutations in these four unrelated Chinese patients, respectively. All these four mutations lead to the formation of a truncated DMD protein by formation of a premature stop codon. According to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG), these four novel nonsense mutations are categorized as "likely pathogenic" variants. CONCLUSION: Our present finding not only identified four novel loss-of-function mutations in dystrophin (DMD) gene but also strongly emphasized the significance of whole exome sequencing as the most efficient way of identifying the candidate genes and mutations which enables us for easy and rapid clinical diagnosis, follow-up, and management of DMD patients.

ERp29 counteracts the suppression of malignancy mediated by endoplasmic reticulum stress and promotes the metastasis of colorectal cancer.

Endoplasmic reticulum protein 29 (ERp29), an endoplasmic reticulum (ER) protein, participates in ER stress (ERS), but little is known about the association of ERp29 with ERS in the metastasis and prognosis of cancerous diseases. The present study revealed that ERp29 was important to ERS and interfered with the malignant behaviors of colorectal cancer (CRC). Experiments in in vitro and in animal models revealed that ERS inhibited the cell growth and suppressed the metastatic capacity of CRC cells, but ERp29 counteracted these effects. Furthermore, it was demonstrated that ERp29 recovered the migration and metastatic behaviors of CRC cells suppressed by ERS, mediated only when it combined with cullin5 (CUL5). ERp29 also relied on CUL5 to promote epithelial­mesenchymal transition. From the immunohistochemical examination of CRC tissues, the high expression of ERp29 was revealed to predict the poor prognosis of 457 CRC cases. The retrospective analysis of the clinicopathological data of patients with CRC was consistent with the results of the in vitro and in vivo experiments. Thus, ERp29 protected CRC cells from ERS­mediated reduction of malignancy to promote metastasis and may be a potential target of medical intervention for CRC therapy.

Telepathology consultation for frozen section diagnosis in China.

BACKGROUND: Telepathology (TP) provides remote pathology services for primary diagnosis practices, including intraoperative consultation of surgical pathology; it has not been widely implemented in China. In this study, the results of an implementation were reported, which lasted for two and a half years, and demonstrated the experience of the diagnosis of the intraoperative frozen sections by using TP consultation platform of Southern Medical University and Guangzhou Huayin Medical Laboratory Center (SMU-HUAYIN TP) in China. METHODS: The SMU-HUAYIN TP consultation platform connects 71 participating basic hospitals and 11 senior pathologists. Nanfang Hospital is a high-level hospital located in a large city in China. This retrospective study summarizes the experience and results of TP for frozen section diagnosis by comparing the data of the platform and Nanfang Hospital over a period of 2.5 years from January 2015 to June 2017. RESULTS: A total of 5233 cases were submitted to the platform, including 1019 cases in 2015, 2320 cases in 2016, and 1894 cases in 2017. The most common cases were breast (30.42%), followed by thyroid (29.05%) and gynecological (24.86%). Average turn-around time (TAT) of the cases from the platform in 2015 and 2016 was controlled within 30 min. In most TP cases (90.31%) and cases from Nanfang Hospital (86.14%), a definitive diagnosis was provided. The coincidence rate was 99.77% in the TP cases and 99.35% in the cases from Nanfang Hospital. The false positive and false negative rates of TP cases were 0.04 and 0.19%, respectively and no significant difference was found among different senior pathologists (P = 0.974, P = 0.989, P > 0.05). Similarly, there was no significant difference between TP cases and cases from Nanfang Hospital that were diagnosed by the same senior pathologist (P > 0.05). CONCLUSIONS: Our results indicate that TP in frozen section diagnosis could improve patient care and solve the problem of unevenly distributed pathology resources in China. We believe that in the near future, TP in frozen section diagnosis will become an important component of telemedicine and will play a significant role in health care reform in China.

Cysteinyl leukotriene receptor 1 regulates glucose-stimulated insulin secretion (GSIS).

Insulin resistance is an important pathological hallmark of type 2 diabetes mellitus. Glucose-stimulated insulin secretion (GSIS) plays a key role in maintaining blood glucose levels within normal range. Impaired GSIS has been associated with type 2 diabetes, however, the underlying molecular mechanisms remain largely unknown. Cysteinyl leukotriene receptor 1 (cysLT1R) is an important G protein-coupled receptor mediating the biological functions of cysteinyl leukotrienes (cys-LTs). Little is known about the effects of cysLT1R in insulin secretion and pathogenesis of T2DM. In the present study, we aimed to define the physiological functions of cysLT1R in GSIS in MIN6 ß-cells. Using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, we found that cysLT1R was expressed in pancreatic MIN6 ß-cells. We also reported that glucose increased the expression of cysLT1R in MIN6 cells. Additionally, the cysLT1R antagonist montelukast promoted GSIS in a dose dependent manner, however, the cysLT1R agonist LD4 inhibited GSIS, suggesting an antagonistic effect of cysLT1R on GSIS. Silencing of cysLT1R by transfection with cysLT1R siRNA enhanced GSIS while overexpression of cysLT1R reduced GSIS in pancreatic MIN6 ß-cells. Mechanistically, we found that the Arf6/Cdc42/Rac1 pathway was involved in this process. Collectively, our findings highlight the essential role of cysLT1R in suppressing pancreatic insulin secretion, and potentially provided a new insight into understanding the mechanical regulation of glucose homeostasis.

Interleukin-31 expression and relation to disease severity in human asthma.

Interleukin 31 (IL-31) is a novel T helper type 2 effector cytokine that plays an important role in the pathogenesis of allergic diseases. However, its role in human asthma remains unclear. The aim of this study was to measure IL-31 levels in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of asthmatics and healthy subjects, and identify its possible correlation to disease severity. We quantified IL-31 levels in the serum of patients with asthma (n = 44), as well as in controls (n = 22). Of these subjects, 9 asthmatics and five controls underwent bronchoscopy with endobronchial biopsy and BALF collection. Our data showed that serum and BALF IL-31 levels were significantly elevated in patients with asthma compared with controls. Expressions of IL-31 and IL-31 receptor (IL-31RA and OSMR) were more prominent in the bronchial tissue in severe compared to mild asthma and controls. Serum IL-31 levels correlated positively with Th2 related cytokines (IL-5, IL-13, and TSLP), asthma severity or total serum immunoglobulin E (IgE), and inversely with asthma control and the forced expiratory volume in 1 second (FEV1). The current data may provide insight into the underlying pathogenesis of asthma, in which IL-31 has an important pathogenic role.

YKL-40 expression in chronic obstructive pulmonary disease: relation to acute exacerbations and airway remodeling.

BACKGROUND: Recent studies suggest that YKL-40, also called chitinase-3-like-1 protein, has been implicated in the pathogenesis of various inflammatory diseases. It is currently unknown, however, whether YKL-40 plays a role in acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and airway remodeling. METHODS: We evaluated serum YKL-40 levels in patients with AECOPD (n = 37) and stable COPD (n = 44), as well as in controls (n = 47). The association between YKL-40 expression and airway remodeling was analyzed. The effects of YKL-40 on collagen synthesis of primary human lung fibroblasts were also evaluated. RESULTS: Serum YKL-40 levels were elevated at AECOPD onset as compared to stable disease (median [interquartile range], 78.6 [52.3-122.2] ng/ml versus 46.7 [31.2-75.5] ng/ml; p = 0.0005). The ideal cutoff point for distinguishing patients with AECOPD from those with stable COPD was 64.7 ng/ml (AUC: 0.71; 95%CI: 0.596 to 0.823). YKL-40 expression correlated with airflow obstruction, C-reactive protein, and collagen deposition. Stimulation with YKL-40 promoted collagen production in lung fibroblasts through ERK- and p38-dependent mechanisms. CONCLUSIONS: YKL-40 expression is up-regulated in patients with COPD and correlates with exacerbation attacks and may contribute to airway remodeling by acting on lung fibroblasts. The current data may provide insight into the underlying pathogenesis of COPD, in which YKL-40 has an important pathogenic role. TRIAL REGISTRATION: ChiCTR-OCC-13003567.

GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

The aim of the study was to investigate the tumor-suppressor effect of GALC in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) and determine whether GALC was downregulated by promoter hypermethy-lation in the NPC cell line, CNE-2Z. Forty-one archival NPC biopsy specimens were compared with 15 chronic nasopharyngitis specimens. EBV-encoded RNA (EBER) was verified by in situ hybridization and GALC protein expression was analyzed by immunohistochemistry. Promoter methylation in CNE-2Z cells was analyzed by bisulfite sequencing polymerase chain reaction. The functional role of GALC in NPC was investigated by restoring GALC expression in CNE-2Z cells via treatment with the DNA-deme-thylating agent 5-Aza-2'-deoxycytidine (5-Aza­dC). EBER was expressed in 92.68% NPC specimens but no chronic nasopharyngitis specimens (P<0.01). GALC protein was present in 60% of chronic nasopharyngitis specimens and 24.39% NPC specimens (P<0.05). GALC protein expression was present significantly more frequently in tumors without lymph node metastasis than in those with metastasis (P<0.05). Logistic regression showed that GALC protein expression protected against lymph node metastasis (P<0.05). GALC protein expression was not correlated with age, gender and TNM stage (P>0.05). Treatment of GALC-negative CNE-2Z cells with 5-Aza-dC reduced GALC promoter methylation and restored GALC expression in a dose-dependent manner (P<0.05). The re-expression of GALC in CNE-2Z cells reduced cell proliferation and migration compared to the controls (P<0.05). GALC was downregulated by promoter hypermethylation and contributed to the pathogenesis of EBV-associated NPC. The findings showed the putative tumor-suppressor effect of GALC in NPC.

A quantum dot-based microRNA nanosensor for point mutation assays.

We have developed a quantum dot-based microRNA nanosensor for point mutation assays using primer generation-mediated rolling circle amplification. The proposed method exhibits high sensitivity with a detection limit of as low as 50.9 aM and a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Importantly, this method can be further applied to analyze the point mutation of mir-196a2 in the lung tissues of non small-cell lung cancer patients.