miR-199b-3p contributes to acquired resistance to cetuximab in colorectal cancer by targeting CRIM1 via Wnt/ß-catenin signaling.

BACKGROUND: Despite advances in the development of efficient chemotherapy, the treatment of colorectal cancer (CRC) remains a challenge due to acquired chemoresistance. It has been reported that microRNAs (miRNAs) dysregulation is associated with the development of chemoresistance. Recently, the expression of miR-199b-3p has been found to be significantly different between cetuximab (CTx)-resistant and -sensitive CRC cells. However, its role and the underlying mechanisms in acquired chemoresistance to CTx in CRC are still obscure. METHODS: Here we report that miR-199b-3p is significantly up-regulated in both CTx-resistant (CTxR) CRC tissues and cell lines. RESULTS: Functional assays showed that suppressing miR-199b-3p could improve the sensitivity of CRC-CTxR cells to CTx, thereby reducing cell proliferation, migration and invasion, and enhancing cell apoptosis. Mechanistic studies revealed that CRIM1 is a direct target of miR-199b-3p in CRC-CTxR cells; and the effect of miR-199b-3p on CTx-resistance was exerted by regulating the Wnt/ß-catenin signaling pathway via CRIM1. Furthermore, mice xenograft models were established and confirmed that down-regulating miR-199b-3p restores the inhibition effect of CTx on tumor growth in CRC-CTxR. Collectively, our data suggest that silencing miR-199b-3p could enhance the anti-tumor effects of CTx on CTx-resistant CRC in vitro and in vivo by activating Wnt/ß-catenin signaling via the down-regulation of CRIM1. CONCLUSIONS: Our findings suggest miR-199b-3p might serve as a promising therapeutic target against CTx resistant CRC, and provide scientific information for exploring novel strategies of improving the efficacy of CTx for CRC patients.

Apatinib exhibits synergistic effect with pyrotinib and reverses acquired pyrotinib resistance in HER2-positive gastric cancer via stem cell factor/c-kit signaling and its downstream pathways.

BACKGROUND: Recently, progress has been made in the development of targeted therapies for human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC). However, drug resistance has severely limited the efficacy of anti-HER2 therapies. Pyrotinib is a novel pan-HER inhibitor. Although it is effective in HER2-positive GC treatment, its efficacy in combination with apatinib and associated resistance mechanisms in HER2-positive GC remains unclear. METHODS: In this study, the combination effects of pyrotinib and apatinib were examined in two pyrotinib-sensitive GC cells and xenografts. The RNA sequencing was used to determine the underlying mechanisms of acquired pyrotinib resistance. The role of imatinib and apatinib in reversing pyrotinib resistance was tested in pyrotinib-resistant cells and xenografts. RESULTS: Here, we reported that a combination of pyrotinib and apatinib exhibits synergistic effect in HER2-positive NCI-N87 xenografts, and showed enhanced antitumor efficacy in HER2-positive GC, both in vitro and in vivo. Moreover, up-regulation of the stem cell factor (SCF) levels, and the PI3K/AKT and MAPK pathways was associated with acquired pyrotinib resistance in HER2-positive GC. Mechanistically, we demonstrated that the activation of the SCF/c-kit signaling and its downstream PI3K/AKT and MAPK pathways mediated pyrotinib resistance by promoting cell survival and proliferation. Imatinib and apatinib augmented the sensitivity of pyrotinib-resistant cells and xenografts to pyrotinib, by blocking SCF/c-kit signaling. CONCLUSION: These results highlight the effectiveness of pyrotinib combined with apatinib in HER2-positive GC and acquired pyrotinib resistance, thus providing a theoretical basis for new treatment methods.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanidin Biosynthesis of Grape Hyacinth.

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth.

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

Clinical Analysis of 122 Cases of Langerhans Cell Histiocytosis.

Objective To investigate the features of Langerhans cell histiocytosis (LCH). Method Skin lesions,systemic involvement,imaging characteristics,laboratory tests,immunophenotying,treatment response,and survival of 122 LCH patients treated at our center from February 1983 to August 2013 were retrospectively analyzed. Results LCH was associated with diverse skin lesions. Lung was the most involved organ,followed by bone,skin,lymph nodes,liver,spleen,oral cavity,and thyroid. Multisystem LCH was more common than single-system LCH. Single-system LCH was mostly treated by surgery,whereas multisystem LCH by combined chemotherapy. Conclusion LCH has diverse clinical manifestations,with lungs being the most often involved organ. Surgery or chemotherapy is the mainstream treatment.

miR-214 promotes apoptosis and sensitizes breast cancer cells to doxorubicin by targeting the RFWD2-p53 cascade.

miR-214 is involved in numerous physiological and pathological processes including tumorigenesis. However, the function of miR-214 in the development and treatment of breast cancer remains elusive. In this study, we report that miR-214 is strikingly down-regulated in breast cancer cell lines and clinical samples, particularly, in the doxorubicin resistant tumor tissues. Remarkably, restoration of miR-214 expression induces apoptosis and sensitizes the MCF7 cells sustaining wild-type p53, but not the p53 null MDA-MB-157 cells, to doxorubicin. Furthermore, we reveal that miR-214 directly down-regulates the expression of RFWD2, also known as COP1, an E3 ligase targeting the tumor suppressor p53 for proteasomal degradation. In addition, RFWD2 protein levels are reversely correlated with miR-214 expression levels in breast cancer tissues. Moreover, ectopic expression of RFWD2 markedly abolishes miR-214-triggered apoptosis of MCF7 cells. In conclusion, miR-214 functions as a tumor suppressor by regulating the RFWD2-p53 cascade, thus delivery of miR-214 analogs could be a potential adjunct therapy in breast cancer harboring wild type p53.

MiR-330-5p regulates tyrosinase and PDIA3 expression and suppresses cell proliferation and invasion in cutaneous malignant melanoma.

BACKGROUND: Increasing evidence has suggested that miR-330-5p can function as a tumor suppressor in different types of cancers. However, the effects and underlying mechanisms of miR-330-5p in the development of cutaneous malignant melanoma (CMM) remain largely unknown. The aim of the present study was to investigate the role of miR-330-5p in CMM and to determine the molecular mechanisms underlying its action. MATERIALS AND METHODS: The expression level of miR-330-5p was detected in 26 cases of primary CMM tissues and cell lines by real-time quantitative polymerase chain reaction. We also assessed whether overexpression of miR-330-5p influences in vitro cell proliferation, invasion, and migration. Western blotting analysis was used to detect the influence of miR-330-5p on the targets, and Pearson analysis was used to calculate the correlation between the expression of targets gene and miR-330-5p in CMM tissues. RESULTS: Our study showed that miR-330-5p was downregulated in CMM tissues (P = 0.010) and cell lines (P < 0.05), and patients with high mitotic activity showed lower miR-330-5p expression levels (P = 0.002). Enforced expression of miR-330-5p inhibits malignant CMM cells proliferation and migration and led to downregulation of the TYR and PDIA3 protein. Moreover, the expression level of miR-330-5p in CMM tissues showed inverse relationship with the expression level of TYR and PDIA3 protein. CONCLUSIONS: In conclusion, our findings suggested that miR-330-5p represents a potential tumor-suppressive miRNA and plays an important role in CMM progression by suppressing TYR and PDIA3 expression.

Adaptive sliding mode fault-tolerant attitude control for flexible satellites based on T-S fuzzy disturbance modeling.

This paper investigates the fault tolerance problem of flexible satellites subject to actuator faults and multiple disturbances. An adaptive sliding mode fault tolerant control (ASMFTC) approach based on Takagi-Sugeno (T-S) fuzzy disturbance observer (TSFDO) is presented for attitude control system (ACS) under loss of actuator effectiveness, environmental disturbance torque and elastic modal generated by flexible appendages. Compared with the traditional disturbance observer based control (DOBC) methods, the T-S fuzzy technology is applied to estimate the unknown nonlinear elastic modal. Then, the energy bounded disturbance is eliminated by designing an adaptive sliding mode controller. The proposed ASMFTC design can guarantee the sliding surface to approach zero. Finally, the effectiveness of the control method proposed in this paper is further verified by comparative simulation.

Pan-HER Tyrosine Kinase Inhibitor Pyrotinib Enhances Radiosensitivity via ERK1/2 Pathway in HER2-Positive Gastric Cancer.

BACKGROUND: Radiotherapy has an unsatisfactory effect on gastric cancer. The purpose of this study was to investigate the effect of pyrotinib, a highly effective human epidermal growth factor receptor (HER) family inhibitor, on the radiosensitivity of HER2-positive gastric cancer and its mechanism in vivo and in vitro. METHODS: NCI-N87 and SNU-216 were HER2-positive gastric cancer cell lines; these cell lines were treated with or without 0.01 µM pyrotinib 12 h before irradiation. The proliferation capacity was determined by CCK8, and clone formation experiments were used to test the radiosensitization effect of pyrotinib. The expression of HER2, γ-H2AX, apoptosis protein, senescence-associated proteins, and AKT/ERK signaling pathway changes were determined by Western blot. Cell cycle and apoptosis were established by flow cytology. Immunofluorescence was utilized to detect the expression of HER2 and γ-H2AX. Senescence cells were stained by ß-galactosidase staining method. Pathway enrichment analysis was tested by RNA sequencing. Next, a xenograft tumor model was constructed in nude mice for verification in vivo. RESULTS: The clone formation experiment indicated the radiosensitivity of pyrotinib. The combined treatment can inhibit the entry of HER2 protein into the nucleus and reduce the phosphorylation of the ERK signaling pathway caused by irradiation. Pyrotinib also enhances DNA damage; induces apoptosis, G2/M phase arrest; and promotes senescence. In the xenograft model, the tumor inhibition rate was significantly stronger in the combined treatment group. CONCLUSIONS: Our results demonstrated that pyrotinib can induce radiosensitivity in HER2-positive gastric cancer in vivo and in vitro. This effect is through reducing irradiation-induced HER2 entry into the nucleus and inhibiting ERK1/2 signaling pathway.

Fusobacterium nucleatum promotes liver metastasis in colorectal cancer by regulating the hepatic immune niche and altering gut microbiota.

Liver metastasis is the major cause of death in colorectal cancer (CRC) patients. Nevertheless, the underlying mechanisms remain unknown. Gut microbiota intricately affect the initiation and progression of CRC by instigating immune response through the secretion of pro-inflammatory cytokines. In this study, we investigated the contribution of Fusobacterium nucleatum (F.nucleatum) to the microbiota-liver axis of CRC in mice, focusing on the correlation between liver immunity and gut microbiota alterations. When F. nucleatum was orally administered to mice, CRC liver metastasis was evidently exaggerated and accompanied by noticeable deleterious effects on body weight, cecum weight, and overall survival time. Further evaluation of the immune response and cytokine profiles revealed a substantial increase in the levels of pro-inflammatory cytokines such as IL6, IL12, IL9, IL17A, CXCL1, MCP-1, TNF-α, and IFN-γ in the plasma of mice treated with F. nucleatum as compared to that in the untreated control mice. Besides, hepatic immune response was also modulated by recruitment of myeloid-derived suppressor cells, reduction in the infiltration of natural killer (NK) and T helper-17 (Th17) cells, as well as increase in regulatory T cell accumulation in the liver. Additionally, sustained F. nucleatum exposure abridged the murine gut microbiota diversity, inducing an imbalanced and restructured intestinal microflora. In particular, the abundance of CRC-promoting bacteria such as Enterococcus and Escherichia/Shigella was evidently elevated post F. nucleatum treatment. Thus, our findings suggest that F. nucleatum might be an important factor involved in promoting CRC liver metastasis by triggering of liver immunity through the regulation of gut microbiota structure and composition.

Drought-responsive WRKY transcription factor genes IgWRKY50 and IgWRKY32 from Iris germanica enhance drought resistance in transgenic Arabidopsis.

Drought greatly affects the growth and development of garden plants and affects their ornamental value. WRKY transcription factors make up one of the largest transcription factor families in plants and they play an important role in the plant response to drought stress. However, the function of the WRKY gene in response to drought stress in Iris germanica, which is commonly used in landscaping, has not been studied. In this study, we isolated two WRKY transcription factor genes from Iris germanica, IgWRKY50 and IgWRKY32, which belong to Group II and Group III of the WRKY family, respectively. IgWRKY50 and IgWRKY32 could be induced by PEG-6000, high temperature and ABA in Iris germanica. IgWRKY50 and IgWRKY32 could quickly respond to drought and they peaked at 3 h after PEG-6000 treatment (19.93- and 23.32-fold). The fusion proteins IgWRKY50-GFP and IgWRKY32-GFP were located in the nucleus of mesophyll protoplasts of Arabidopsis. The overexpression of the IgWRKY50 and IgWRKY32 genes improved the osmotic tolerance of transgenic Arabidopsis, mainly exhibited by the transgenic plants having a higher germination rate and a longer total root length on 1/2 MS medium containing mannitol. Under PEG-6000 stress, the transgenic plants had higher stomatal closure than the wild type (WT). Under natural drought stress, the water loss rate of the isolated leaves of transgenic Arabidopsis was lower than that of WT, the contents of proline (Pro) and soluble protein (SP) and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the transgenic plants were higher, but the content of malondialdehyde (MDA) was lower. Furthermore, the expression of several stress-related genes (RD29A, DREB2A, PP2CA, and ABA2) was significantly increased in IgWRKY50- and IgWRKY32- overexpressing transgenic Arabidposis plants after drought treatment. These results suggest that IgWRKY50 and IgWRKY32, as two positive regulators, enhance the drought resistance of transgenic Arabidopsis by mediating the ABA signal transduction pathway. IgWRKY50 and IgWRKY32 can be used as candidate genes for molecular breeding of drought resistance in Iris.

Targeting polarized phenotype of microglia via IL6/JAK2/STAT3 signaling to reduce NSCLC brain metastasis.

Tumor-associated macrophages have emerged as crucial factors for metastases. Microglia are indispensable components of the brain microenvironment and play vital roles in brain metastasis (BM). However, the underlying mechanism of how activated microglia promote brain metastasis of non-small cell lung cancer (NSCLC) remains elusive. Here, we purified cell lines with brain-metastatic tropism and employed a co-culture system to reveal their communication with microglia. By single-cell RNA-sequencing and transcriptome difference analysis, we identified IL6 as the key regulator in brain-metastatic cells (A549-F3) to induce anti-inflammatory microglia via JAK2/STAT3 signaling, which in turn promoted the colonization process in metastatic A549-F3 cells. In our clinical samples, patients with higher levels of IL6 in serum showed higher propensity for brain metastasis. Additionally, the TCGA (The Cancer Genome Atlas) data revealed that NSCLC patients with a lower level of IL6 had a longer overall survival time compared to those with a higher level of IL6. Overall, our data indicate that the targeting of IL6/JAK2/STAT3 signaling in activated microglia may be a promising new approach for inhibiting brain metastasis in NSCLC patients.

MiR-186 inhibits cell proliferation and invasion in human cutaneous malignant melanoma.

AIMS: MicroRNA-186 (miR-186) has been shown to be involved in various types of cancer. The purpose of this study was to investigate the expression level and functional role of miR-186 in human cutaneous malignant melanoma cells. SUBJECTS AND METHODS: Expression of miR-186 was analyzed in human cutaneous malignant melanoma (CMM) cell lines SK-MEL-1, G-361, A375 and A875, and human normal epidermal melanocytes cell line HEMn-LP by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Additionally, the functional role of miR-186 on melanoma cells was investigated by transfection of miR-186 mimic followed by analyses of cell proliferation, apoptosis, and metastasis. RESULTS: We found that the expression levels of miR-186 were decreased in CMM cell lines compared with normal epidermal melanocytes cell line. Moreover, overexpression of miR-186 inhibited cells proliferation through abrogating the G1-S transition, and reduced cells migration and invasion. CONCLUSIONS: Our findings suggested that miR-186 exhibit an inhibitory effect on CMM cell proliferation, migration, and invasion; thus, may serve as a potential therapeutic target for human CMM intervention.

Association of E-cadherin methylation with risk of nasopharyngeal cancer: A meta-analysis.

BACKGROUND: Various studies have assessed the association between E-cadherin methylation and risk of nasopharyngeal cancer (NPC) but the conclusion remains unclear. This meta-analysis was conducted to evaluate the effects of E-cadherin methylation on the incidence and clinicopathological characteristics of NPC. METHODS: Ten studies published up to June 30, 2016, were collected. Odds ratios (ORs) with corresponding confidence intervals (CIs) were calculated and summarized, respectively. RESULTS: The E-cadherin methylation in NPC was significantly higher than those in normal groups (OR 16.23; 95% CI 8.34-31.60; P < .001). Ethnicity-stratified analysis indicated that E-cadherin methylation was strongly correlated with NPC among both Asians (OR 16.98; 95% CI 8.45-34.14; P < .001) and North Africans (OR 10.67; 95% CI 1.21-93.72; P = .033). However, further analysis showed that E-cadherin methylation was not strongly associated with clinicopathological feathers in patients with NPC. CONCLUSION: The E-cadherin methylation is strongly associated with the incidence of NPC, which can serve as an effective biomarker for early detection of NPC.

Snail-mediated cancer stem cell-like phenotype in human CNE2 nasopharyngeal carcinoma cell.

BACKGROUND: Cancer stem cell (CSC)-like phenotype, which has been proven to play a critical role in invasion and metastasis of many kinds of cancers, has also been reported to be associated with epithelial-mesenchymal transition. Snail, a potent repressor of E-cadherin expression, was found to have a function to regulate the aforementioned processes. METHODS: In the current study, expression of putative CSCs biomarkers and the ratio of CSC-like CNE2 (cancer cell line) in total CNE2 were measured, and CSC-like characteristics were analyzed with tumor-sphere self-renewal and colony-forming assays. Migration and invasion properties were determined by using transwell and wound healing assays. Xenograft tumor assays in vivo were done to evaluate the function of Snail and radiation in the tumor forming ability. RESULTS: In human nasopharyngeal carcinoma (NPC) cells, overexpression of Snail mediates a CSC-like phenotype, which enhances the initiation, invasion, and migration ability of cancer cells. CONCLUSION: Thus, Snail is a potential therapeutic target in NPC.

Twist1 promotes radioresistance in nasopharyngeal carcinoma.

With the development of advanced imaging and radiation technologies, radiotherapy has been employed as the principal treatment approach for nasopharyngeal carcinoma (NPC). So far, a number of patients still suffer from the failure of this treatment due to the acquired radioresistance, but the underlying mechanisms are still poorly defined. In this study, we found that Twist1, participating in a variety of cell biological process, was associated with the malignancy of NPC and could induce NPC radioresistance in vitro and in vivo. Mechanically, Twist1 could promote the accumulation of DNA damage repair and inhibit the apoptosis of NPC cells. Therefore, our study not only elucidates the significant role of Twist1 in radioresistance of NPC, but also highlights Twist1 as a potential therapeutic target for NPC.

Health related management plans improve sleep disorders of patients with chronic liver disease.

BACKGROUNDS: Sleep disorders (SDs) are commonly occurred in patients with chronic liver disease (CLD) and always bring with uncomfortable experience Lavender hot-bathing, foot-soaking, or progressive relaxation have been widely used to provide comfortable feeling for CLD patients and promote their sleep quality. AIMS: Thus, the aim of present study is to investigate effective intervention from above mentioned in the managements of SD and promote sleep quality for CLD patients. METHODS: This study was conducted in People's Liberation Army No. 302 Hospital. A total of 317 subjects joined in our research. Initially, 197 CLD patients were enrolled and divided randomly into four groups for receiving lavender hot-bathing and foot-soaking, progressive relaxation, or the combination of both methods, and controls. After that, all of enrolled subjects were given sleep state questionnaires to assess their sleep qualities and other associated factors. Self-rating scores of sleep (SRSS) was sued to assess sleep disorder. Furthermore, another cohort with 120 CLD patients were also investigated for further confirming related findings. RESULTS: The SRSS scores were significantly higher in the patients with CLD (62.94%) than those of domestic common model and internal medicine inpatients. However, all three methods of intervention were effectively decreased SRSS scores. The four mostly influencing factors of sleep states were short sleep, difficulty falling asleep, staying asleep and early morning awakening. Besides, age was identified as one of associating with sleep states. 44.67% of patients suffered from polyuria, abdominal distention or itch of skin. And those factors contributed to major risk factors of sleep disorder. Furthermore, sleep states also influenced by environmental interference (37.06%). CONCLUSION: The health managements of health education could reduce risk factors and implement intervention strategies, effectively decreased occurrence of sleep disorder related symptoms.

Aberrant miR-215 expression is associated with clinical outcome in breast cancer patients.

Dysregulation of microRNA plays critical roles in various malignancies. However, whether the aberrant expression of miR-215 in breast cancer is associated with malignancy, metastasis, or prognosis remains unknown. In this study, we demonstrated that the relative level of miR-215 expression was lower in cancer tissues compared with adjacent non-malignant tissues (p < 0.001). Low-miR-215 expression was observed to be closely correlated with higher tumor grade (p = 0.008), human epidermal growth factor receptor 2 (HER2) positivity (p = 0.006), HER2 positive breast cancer subtype (p = 0.016), and lymph node metastasis (p = 0.039). Moreover, patients with low-miR-215 expression showed shorter 5-year disease-specific survival (DSS) than the high-miR-215 expression group (p = 0.007). Multivariate analysis results revealed that miR-215 downexpression was an unfavorable prognostic factor for DSS in addition to tumor size, ER, and lymph node metastasis. Our results support the potential of miR-215 as a prognostic predictor for breast cancer with its high expression in cancer tissues and its relationship with other clinicopathologic factors and survival.