Construction of a miRNA Signature Using Support Vector Machine to Identify Microsatellite Instability Status and Prognosis in Gastric Cancer.

Background: The specific role and prognostic value of DNA repair and replication-associated miRNAs in gastric cancer (GC) have not been clearly elucidated. Therefore, comprehensive analysis of miRNAs in GC is crucial for proposing therapeutic strategies and survival prediction. Methods: Firstly, clinical information and transcriptome data of TCGA-GC were downloaded from the database. In the entire cohort, we performed differential analysis in all miRNAs and support vector machine (SVM) was used to eliminate redundant miRNAs. Subsequently, we combined survival data and cox regression analysis to construct a miRNA signature in the training cohort. In addition, we used PCA, Kaplan-Meier, and ROC analysis to explore the prognosis value of risk score in the training and testing cohort. It is worth noting that multiple algorithms were used to evaluate difference of immune microenvironment (TME), microsatellite instability (MSI), tumor mutational burden (TMB), and immunotherapy in different risk groups. Finally, we investigated the potential mechanism about miRNA signature. Results: We constructed miRNA signature based on the following 4 miRNAs: hsa-miR-139-5p, hsa-miR-139-3p, hsa-miR-146b-5p, and hsa-miR-181a-3p. Univariate and multivariate Cox regression analyses suggested that risk score is a risk factor and an independent prognostic factor in GC patients. The AUC value of ROC analysis showed a robust prediction accuracy in each cohort. Moreover, significant differences in immune functions, immune cell content, immune checkpoint, MSI status, and TMB score were excavated in different groups distinguished by risk score. Finally, based on the above four miRNA target genes, we revealed that the signature was enriched in DNA repair and replication. Conclusion: We have developed a robust risk-formula based on 4 miRNAs that provides accurate risk stratification and prognostic prediction for GC patients. In addition, different risk subgroups may potentially guide the choice of targeted therapy.

Clinical efficacy of immunotherapy combined with chemotherapy in patients with advanced gastric cancer, its effect on nutritional status and Changes of peripheral blood T lymphocyte subsets.

OBJECTIVES: To evaluate the clinical efficacy of immunotherapy combined with chemotherapy in patients with advanced gastric cancer and its effect on nutritional status and changes of peripheral blood T lymphocyte subsets. METHODS: Sixty patients with locally advanced gastric cancer who were admitted by Affiliated Hospital of Hebei University from March 2020 to February 2021 were enrolled and randomly divided into two groups, with 30 cases in each group. The control group was treated with FOLFOX4 chemotherapy, while the experimental group was additively treated with cindilizumab on the basis of control group. The incidence of adverse reactions, clinical efficacy, improvement of nutritional and physical status, and changes in the levels of T lymphocyte subgroups in the two groups were compared and analyzed. RESULTS: The total effective rate was 70% in the experimental group, which was better than 43.3% of the control group (p=0.04). The improvement rate of performance status (ECOG) score and nutritional indicators in the experimental group was significantly better than that in the control group (p<0.05). Moreover, the indicators of CD3+, CD4+, CD4+/CD8+ in the experimental group were significantly higher than those in the control group after treatment, with statistically significant differences (CD3+, p=0.01; CD4 +, p= 0.02; CD4+/CD8+, p=0.01). CONCLUSION: Immunotherapy combined with chemotherapy has a significant effect on locally advanced gastric cancer patients, with significant improvement in physical strength and nutritional status, significant improvement in T lymphocyte function, and no obvious adverse reactions. It is worth promoting in clinical application.

MicroRNA-575 regulates development of gastric cancer by targeting PTEN.

MicroRNA-575 (miR-575) is oncogene in many tumors. However, the role of miR-575 in the progression of gastric cancer (GC) is still unknown. The aim of this study was to identify whether miR-575 play a role in the development of GC. We obtained GC cell lines, GC tissues from 40 patients to measure the levels of miR-575 and its predicted target PTEN by using RT­PCR, immunohistochemistry or western blot analysis. MGC-803 cells were transfected with miR-575 inhibitor, and cells viability and apoptosis were measured. miR-575 aberrantly up-regulated in GC tissues and GC cell lines compared with corresponding control. In cell lines, MGC­803 expressed highest level of miR-575 among the tested cells. The level of miR-575 was correlated with the tumor size, AJCC stage and prognosis, but not with the other clinical parameters. Knockdown of miR-575 inhibited proliferation and promoted apoptotic death of MGC-803 cells both in vitro and vivo. PTEN levels (both mRNA and protein) were remarkably decreased in cancer tissues compared with the paired-adjacent tissues, and negatively correlated with miR-575 in the tissues. By using luciferase reporter assay, we found PTEN was a direct downstream target of miR-575, and negatively regulated by miR-575 via targeting its 3'UTR. Knockdown or overexpression of miR-575 in MGC-803 cells negatively regulated PTEN expression. Finally, silencing PTEN partially impaired anti-proliferative effects of miR-575 inhibitor. miRNA-575 serves a pivotal role in GC as a cancer promoter gene by targeting PTEN to regulate proliferation and apoptosis of the cancer cells.

[Distribution Characteristics and Ecological Risk Assessment of Organochlorine Pesticides in Surface Sediments of Zhelin Bay in Guangdong Province, China].

The distribution characteristics and pollution degrees of 20 organochlorine pesticides (OCPs) were investigated in surface sediments from Zhelin Bay south of China and the ecological risk of OCPs and integrated ecological risk at the samples stations in sediments were evaluated by risk quotient (RQ) and sediment quality guideline of NOAA. The possible sources of HCHs and DDTs in sediments were preliminarily studied, and the correlation between OCPs and sediment characteristic parameters was discussed. The concentrations of DDTs in surface sediments were found to be higher than those of other OCPs, especially p,p'-DDD, and the concentrations of DDTs were higher than the limited values of Chinese Marine sediment quality criteria. The total concentration of 20 OCPs in surface sediments ranged from 14.14 to 306.88 ng·g-1, with a mean concentration of 78.37 ng·g-1, and the highest total concentration was at site 8(S8). There were inevitable adverse biological effects and high ecological risk of p,p'-DDD and p,p'-DDT, high integrated ecological risk at S8 and S10. The possible sources of HCHs in surface sediments of Zhelin Bay were mainly from the application of Lindane pesticides. At S1,S2,S10,S11 there was input of DDT into mariculture area of Zhelin Bay, and at the other sites the sources of DDT were from the early residue in sediments or the long-term weathering sediments of using pesticides. There was a very significant positive correlation between HCHs, endosulfan, chlordane and dieldrin in sediments, while there was a negative relation between the above OCPs and TOC, which indicated that the presence of TOC could promote their biodegradation. There was a significant positive correlation between p, p'-DDD and Ca, which revealed that DDT used in the early period was deposited with biological carbonate. Increasing size of sediment particles discouraged the accumulation and enrichment of OCPs in sediments.

Proliferative phenotype of pulmonary microvascular endothelial cells plays a critical role in the overexpression of CTGF in the bleomycin-injured rat.

The pathogenesis of idiopathic pulmonary fibrosis (IPF) is not very clear, with evidence for the involvement of both inflammation and aberrant vascular remodeling (associated with angiogenesis). Pulmonary microvascular endothelial cells (PMVECs), which play a major role in inflammation, secrete cytokines that promote the transformation and collagen synthesis of fibroblasts. Moreover, angiogenesis is characterized by PMVEC proliferation. The main aim of this study was to confirm the role of PMVECs in pulmonary fibrosis. Accordingly, we observed the functional changes in PMVECs in bleomycin (BLM)-treated rats (pulmonary fibrosis model) in vivo, and compared them with those of rats with pneumonia. The proliferation phenotype and intracellular ionized calcium concentration ([Ca(2+)]i) of PMVECs from BLM-treated rats were also investigated. The functioning of PMVECs was abnormal in BLM-injured rats, particularly with regard to their proliferation and secretion of connective tissue growth factor (CTGF). [Ca(2+)]i was increased in the proliferated PMVECs from BLM-treated rats. The findings suggest that dysfunction of PMVECs characterized by overexpression of CTGF is critical in rat pulmonary injury induced by BLM, and is probably related with the proliferative phenotype and [Ca(2+)]i overload. It can be concluded from the results that proliferation of PMVECs plays an important role in the pathogenesis of BLM-induced PF.

Notch-RBP-J signaling is required by bone marrow stromal cells for the treatment of acute graft versus host disease.

Recent evidence has shown that bone marrow stromal cells (BMSCs) may exhibit immuno-suppression activities through soluble mediators and direct cell-cell contact, but how these processes are modulated has been poorly understood. In this study, we show that the Notch signaling pathway participates in the modulation of BMSCs to elicit their immuno-suppressive roles. In a murine lethal acute graft versus host disease (aGvHD) model, BMSCs deficient for RBP-J, the critical transcription factor mediating signaling from all four mammalian Notch receptors, failed to delay the development of the disease. RBP-J deficient BMSCs were not able to inhibit the proliferation and activation of allogenic T-cells. Moreover, RBP-J deficient BMSCs could not down-regulate the expression of MHC II and co-stimulation molecules CD80 and CD86 on dendritic cells (DCs). The antigen presentation capacity of DCs co-cultured with RBP-J deficient BMSCs was not impaired in contrast to wild type BMSCs. Furthermore, we showed that the productions of IL-6 and PGE2, two critical molecules mediating the immuno-suppressive activities of BMSCs, were reduced significantly in RBP-J deficient BMSCs. Both of the two molecules were importantly involved in the regulation of BMSCs by Notch signaling. In conclusion, our data suggests that the immuno-suppressive effects of BMSCs in aGvHD are dependent on Notch-RBP-J signaling, which regulates the productions of IL-6 and PGE2.

Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation.

The transcription factor recombination signal binding protein-Jκ (RBP-J) is the critical transcription factor downstream to all four mammalian Notch receptors. Although it has been reported that Notch signaling pathway is involved in bone remodeling, the importance of RBP-J in osteoclastogenesis has not been fully explored. To investigate the role of RBP-J in osteoclastogenesis, we conditionally deleted RBP-J systemically in bone marrow (BM) or specifically in macrophages. We found that disruption of RBP-J in BM resulted in an obvious decrease in trabecular bone mass associated with an increase in osteoclasts, leading to osteopenia. Disruption of RBP-J in macrophages phenocopied the phenotypes of RBP-J deletion in BM with respect to osteoclastogenesis, suggesting that the osteopenia in RBP-J deficient mice is essentially resulted from increased osteoclastogenesis. Furthermore, we found that RBP-J deletion in osteoclasts resulted in a dramatic increase in tartrate-resistant acid phosphatase expression. These findings demonstrate a negatively role of RBP-J in the differentiation of osteoclasts and suggest that Notch pathway may be a new therapeutic target for bone diseases related to increased osteoclastogenesis.

Overexpression of Notch ligand Dll1 in B16 melanoma cells leads to reduced tumor growth due to attenuated vascularization.

Notch signaling plays an important role in vascular development and tumor angiogenesis. It has been shown that disruption of Dll4-triggered Notch signal activation effectively inhibits tumor growth, but this treatment also results in the formation of vascular neoplasms. In this study, we investigate the effects of over-expressing Notch ligand Dll1 in B16 melanoma cells on tumor cell proliferation and tumor growth in vitro and in vivo. Our results showed that over-expression of Dll1 could activate Notch signaling in tumor cells, and promote tumor cell proliferation in vitro. In contrast, growth of Dll1-over-expressing tumors in vivo was reduced, due to abnormal tumor vessel formation. Impaired tumor vasculature enhanced hypoxia and necrosis in tumor tissues, leading to retarded tumor growth. These results suggest that activation of Notch signaling may serve as an anti-angiogenesis strategy in the treatment of malignant tumors.

Notch signaling contributes to the maintenance of both normal neural stem cells and patient-derived glioma stem cells.

BACKGROUND: Cancer stem cells (CSCs) play an important role in the development and recurrence of malignant tumors including glioma. Notch signaling, an evolutionarily conserved pathway mediating direct cell-cell interaction, has been shown to regulate neural stem cells (NSCs) and glioma stem cells (GSCs) in normal neurogenesis and pathological carcinogenesis, respectively. However, how Notch signaling regulates the proliferation and differentiation of GSCs has not been well elucidated. METHODS: We isolated and cultivate human GSCs from glioma patient specimens. Then on parallel comparison with NSCs, we inhibited Notch signaling using γ-secretase inhibitors (GSI) and assessed the potential functions of Notch signaling in human GSCs. RESULTS: Similar to the GSI-treated NSCs, the number of the primary and secondary tumor spheres from GSI-treated GSCs decreased significantly, suggesting that the proliferation and self-renewal ability of GSI-treated GSCs were attenuated. GSI-treated GSCs showed increased differentiation into mature neural cell types in differentiation medium, similar to GSI-treated NSCs. Next, we found that GSI-treated tumor spheres were composed of more intermediate progenitors instead of CSCs, compared with the controls. Interestingly, although inhibition of Notch signaling decreased the ratio of proliferating NSCs in long term culture, we found that the ratio of G2+M phase-GSCs were almost undisturbed on GSI treatment within 72 h. CONCLUSIONS: These data indicate that like NSCs, Notch signaling maintains the patient-derived GSCs by promoting their self-renewal and inhibiting their differentiation, and support that Notch signal inhibitor GSI might be a prosperous candidate of the treatment targeting CSCs for gliomas, however, with GSI-resistance at the early stage of GSCs cell cycle.

Sphincter of Oddi dysfunction in hypercholesterolemic rabbits.

BACKGROUND AND OBJECTIVES: The mechanisms that trigger gallbladder evacuation dysfunction, the key risk factor for gallstone formation, have not yet been fully elucidated. The sphincter of Oddi (SO) plays important roles in the regulation of gallbladder evacuation and maintenance of normal hydraulic pressure of the biliary tract. The aim of our study was to investigate the effects of hypercholesterolemia on the motility function of SO and the underlying mechanisms of SO dysfunction (SOD). METHODS: Forty New Zealand white rabbits were divided randomly into the control group fed with standard chow and the experimental (Ch) group fed with a high-cholesterol diet for 8 weeks. Changes in the maximal gallbladder emptying rate, gallbladder evacuation with cholecystokinin-octapeptide (CCK-8) stimulation and SO functions of both groups were measured in vivo; B ultrasound examination was used for dynamic observation of peristaltic movements in vivo; SO pressure was measured using manometry; morphological characteristics were observed by electronic microscope; laser scanning confocal fluorescence microscopy was used to identify changes in [Ca]i and Ca oscillation in primary SO smooth muscle cells (SMCs). RESULTS: Gallbladder cholestasis was observed during early stages of gallstone formation in Ch rabbits. CCK-8 could not improve the gallbladder cholestatic state in Ch group. Passive dilation of SO significantly improved the cholestatic state in Ch rabbits (P<0.05), although the maximal gallbladder emptying rate was still lower than that of the control group. Manometry data indicted a significant increase in the base pressure of the SO low-pressure ampulla segment and high-pressure segment (P<0.05) in Ch group. laser scanning confocal fluorescence microscopy assay data indicated that [Ca]i in SO cells of Ch group significantly increased and were in a state of overload (P<0.05); Ca oscillation signals in SO cells of Ch group were also abnormal. CONCLUSION: Hypercholesterolemia initially induced SOD, leading to increased gallbladder evacuation resistance and cholestasis. We suggested that [Ca]i overload and/or Ca oscillation abnormality potentially play important roles in the pathogenesis of SOD.

[Effects of infrasound on ultrastructure of testis cell in mice].

OBJECTIVES: To investigate the effects of infrasound on ultrastructure of testis in mouse. METHODS: Twelve male BALB/C mice were randomly divided into three groups according to exposed duration on 1, 7 and 14 day. The mice were separately exposed to infrasound environment under 8 Hz/90 dB, 8 Hz/130 dB, 16 Hz/90 dB, 16 Hz/130 dB 2 hours per day. There was another control group which had three mice were separated into module with no infrasound. All the mice were killed on schedule. Then all the sections of testis were observed under electronic microscope. The alterations of structure and the chromatin were observed. RESULTS: Some acute alteration in one day group was found in testis cell, such as cellular denaturation and necrosis, intercellular edema, mitochondria swelling, liposome hyperplasia. When the infrasound was up to 8 Hz/130 dB, the damage showed seriously. In 7 and 14 day group, the acute alteration was gradually decreased. A plenty of abnormal sperm were found. And other alteration was chromatin condense. The effect of variational frequency was important in ultrastructure. CONCLUSIONS: The infrasound markedly effected to testicular cell morphology and secreting function. Infrasound will lead to the alteration of procreation in mouse.