Investigation of peanut allergen-procyanidin non-covalent interactions: Impact on protein structure and in vitro allergenicity.

Interactions between plant polyphenols and food allergens may be a new way to alleviate food allergies. The non-covalent interactions between the major allergen from peanut (Ara h 2) with procyanidin dimer (PA2) were therefore characterized using spectroscopic, thermodynamic, and molecular simulation analyses. The main interaction between the Ara h 2 and PA2 was hydrogen bonding. PA2 statically quenched the intrinsic fluorescence intensity and altered the conformation of the Ara h 2, leading to a more disordered polypeptide structure with a lower surface hydrophobicity. In addition, the in vitro allergenicity of the Ara h 2-PA2 complex was investigated using enzyme-linked immunosorbent assay (ELISA) kits. The immunoglobulin E (IgE) binding capacity of Ara h 2, as well as the release of allergenic cytokines, decreased after interacting with PA2. When the ratio of Ara h 2-to-PA2 was 1:50, the IgE binding capacity was reduced by around 43 %. This study provides valuable insights into the non-covalent interactions between Ara h 2 and PA2, as well as the potential mechanism of action of the anti-allergic reaction caused by binding of the polyphenols to the allergens.

RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma.

BACKGROUND: As a small G protein of Ras family, Ras-like-without-CAAX-1 (RIT1) plays a critical role in various tumors. Our previous study has demonstrated the involvement of RIT1 in promoting malignant progression of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear. METHODS: Gene set enrichment analysis (GSEA) was conducted in the TCGA LIHC cohort to investigate the underlying biological mechanism of RIT1. Live cell imaging, immunofluorescence (IF) and flow cytometry assays were used to verify biological function of RIT1 in HCC mitosis. Subcutaneous xenografting of human HCC cells in BALB/c nude mice was utilized to assess tumor proliferation in vivo. RNA-seq, co-immunoprecipitation (Co-IP), mass spectrometry analyses, western blot and IF assays were employed to elucidate the mechanisms by which RIT1 regulates mitosis and promotes proliferation in HCC. RESULTS: Our findings demonstrate that RIT1 plays a crucial role in regulating mitosis in HCC. Knockdown of RIT1 disrupts cell division, leading to G2/M phase arrest, mitotic catastrophe, and apoptosis in HCC cells. SMC3 is found to interact with RIT1 and knockdown of SMC3 attenuates the proliferative effects mediated by RIT1 both in vitro and in vivo. Mechanistically, RIT1 protects and maintains SMC3 acetylation by binding to SMC3 and PDS5 during mitosis, thereby promoting rapid cell division and proliferation in HCC. Notably, we have observed an upregulation of SMC3 expression in HCC tissues, which is associated with poor patient survival and promotion of HCC cell proliferation. Furthermore, there is a significant positive correlation between the expression levels of RIT1, SMC3, and PDS5. Importantly, HCC patients with high expression of both RIT1 and SMC3 exhibit worse prognosis compared to those with high RIT1 but low SMC3 expression. CONCLUSIONS: Our findings underscore the crucial role of RIT1 in regulating mitosis in HCC and further demonstrate its potential as a promising therapeutic target for HCC treatment.

Brucine suppresses proliferation and promotes apoptosis of human cholangiacarcinoma cells via the inhibition of COX2 expression.

Aims The aim of this study was to investigate the anti-tumor efficacy of brucine on intrahepatic cholangiocarcinoma (ICC). Methods ICC QBC939 cells were treated with brucine, cell viability, cell cycle and apoptosis were analyzed using CCK-8 and flow cytometry. The expression of COX-2 and apoptosis related proteins Casp3, Bax and Bcl-2 were detected by Western blot analysis. QBC939 cells were subcutaneously transplanted into nude mice and the mice were injected with brucine intraperitoneally. The expression of Ki67, COX-2 and apoptosis related proteins were detected by immunohistochemical staining and Western blot analysis. Results Brucine significantly inhibited the proliferation and cell cycle progression while promoted the apoptosis of QBC939 cells. The expression of the apoptotic proteins Casp3 and Bax was upregulated, while the expression of Bcl-2 and COX-2 was downregulated in QBC939 cells with brucine treatment. Moreover, the overexpression of COX-2 could antagonize the effects of brucine on QBC939 cells. In vivo, brucine inhibited subcutaneous tumor formation in nude mice, and the expression of Ki67, COX-2 and Bcl-2 decreased while the expression of Casp3 and Bax increased in tumor tissues from nude mice with brucine treatment. Conclusions Brucine can significantly inhibit the progression of cholangiocarcinoma in vitro and in vivo, and the mechanism may be related to the inhibition of COX-2 expression.

Macadamia oil-based oleogels as cocoa butter alternatives: Physical properties, oxidative stability, lipolysis, and application.

In this study, macadamia oil-based oleogels were prepared using monoglyceride stearate (MG) as a gelator with a low critical gelation concentration (3.0 wt%). The physical properties of the oleogels were evaluated by polarized light microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, texture and rheological analysis. And the lipid digestion and oxidative stability of the macadamia oil were determined by pH titration and accelerated oxidation test, respectively. The results showed that the hardness, oil binding capacity, and thermal stability of the oleogels increased with increasing MG concentration, which was attributed to the formation of a network of MG crystals held together by van der Waals interactions and hydrogen bonds. Rheological analysis indicated that all the oleogels exhibited a thermally reversible solid-to-liquid transition and viscoelastic behavior at ambient temperatures. Moreover, the formation of oleogels increased fatty acid release during in vitro lipid digestion and improved the oxidative stability of the macadamia oil. In addition, the potential application of these oleogels as replacements for saturated fats in foods was demonstrated by creating a chocolate product where the cocoa butter was replaced with macadamia oil-based oleogels with a high degree of unsaturation. These results can provide guidance for the preparation of macadamia oil-based oleogels, which may increase their application in foods.

The interaction and physicochemical properties of the starch-polyphenol complex: Polymeric proanthocyanidins and maize starch with different amylose/amylopectin ratios.

This study investigated the impact of polymeric proanthocyanidins (PPC) on the physicochemical characteristics of maize starch with varying amylose content, and their potential interaction mechanism. PPC with a lower content (1 %) reduced the viscoelasticity of the high amylose maize starch (HAM) system, inhibited amylose rearrangement, and enhanced its fluidity. However, excessive PPC restrained the interaction between PPC and amylose. In contrast to HAM, PPC improved the gelation ability of waxy maize starch (WAM) as PPC concentration was raised. PPC suppressed the recrystallization of starch during storage, and PPC had a superior inhibition influence on the retrogradation of WAM in comparison to HAM. This indicated that amylopectin was more likely to interact with PPC than amylose. Hydrogen bonds were the main driving force between PPC and starch chains, which was clarified by Fourier transform-infrared, nuclear magnetic resonance, X-ray diffraction, iodine bonding reaction, and dynamic light scattering data. Additionally, the mechanism of interaction between PPC and the two starch components may be similar, and variance in physicochemical attributes can be primarily credited to the percentage of amylose to amylopectin in starch.

Drug Repurposing Flubendazole to Suppress Tumorigenicity via PCSK9-dependent Inhibition and Potentiate Lenvatinib Therapy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal malignant cancers across the world. It has a poor prognosis and lacks effective therapies, especially for patients with advanced-stage cancer, indicating an urgent need for new therapies and novel therapeutic targets. Here, by screening the U.S. Food and Drug Administration drug library against HCC cell lines, we identified that flubendazole, a traditional anthelmintic drug, could prominently suppress HCC cells in vivo and in vitro. RNA sequence analysis and cellular thermal shift assays showed that flubendazole reduced the expression of PCSK9 protein by direct targeting. The increased expression of PCSK9 in HCC tissues was demonstrated to be correlated with poor prognosis, and the inhibitory ability of flubendazole was selectively dependent on PCSK9 expression. PCSK9 knockdown abolished the antitumor effects of flubendazole in HCC. Mechanistically, flubendazole inhibited the Hedgehog signaling pathway induced by PCSK9, resulting in the downregulation of smoothened (SMO) and GLI Family Zinc Finger 1 (Gli1). Moreover, combining flubendazole with lenvatinib was found more effective than administering lenvatinib only for HCC treatment in vivo and in vitro. These findings reveal the therapeutic potential of flubendazole against HCC and provide clues on new repurposed drugs and targets for cancer treatment.

Investigation of bovine ß-lactoglobulin-procyanidin complexes interactions and its utilization in O/W emulsion.

Procyanidins are bioactive polyphenols that have a strong affinity to proteins. Beta-lactoglobulin (BLG) is widely used as an emulsifier in the food and other industries. This study evaluated the interaction between BLG and A-type procyanidin dimer (PA2) using the spectroscopic, thermodynamic, and molecular simulation. PA2 decreased the transmissivity and quenched the intrinsic fluorescence of BLG, suggesting that the two molecules formed a complex. The binding of PA2 reduced the surface hydrophobicity and altered the conformation of BLG with increasing the random coil regions. Thermodynamic and isothermal titration calorimetry analyses suggested that the main driving force of PA2-BLG interaction was hydrophobic attraction. Molecular docking simulations were used to identify the main interaction sites and forces in the BLG-PA2 complexes, which again indicated that hydrophobic interactions dominated. In addition, the influence of PA2 on the ability of BLG to form and stabilize O/W emulsions was analyzed. Emulsions formulated using BLG-PA2 complexes contained relatively small droplets (D4,3 ≈ 0.7 µm) and high surface potentials (absolute value >50 mV). Compared to BLG alone, BLG-PA2 complexes improved the storage stability of the emulsions. This study provides valuable new insights into the formation, properties, and application of protein-polyphenol complexes as functional ingredients in foods.

Sperm associated antigen 4 promotes SREBP1-mediated de novo lipogenesis via interaction with lamin A/C and contributes to tumor progression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor and its progression is associated with altered lipid metabolism in precancerous lesions, such as non-alcoholic fatty liver disease. Here, we identified sperm associated antigen 4 (SPAG4), and explored its oncogenic role in HCC progression. Database analysis and immunohistochemistry indicated increased level of SPAG4 in HCC tissues which was of prognostic value. Gain/loss-of-function experiments showed that SPAG4 exerted oncogenic roles in HCC growth both in vitro and in vivo. RNA sequencing revealed activation of a lipogenic state and SREBP1-mediated pathway following SPAG4 overexpression. Mechanistically, the N-terminal region of SPAG4 bound to lamin A/C, which increased SREBP1 expression, nuclear translocation, and transcriptional activity. Treatment with orlistat, a lipid synthesis inhibitor, reversed SPAG4-mediated oncogenic effects, and its efficacy varied with SPAG4 level. The effect of orlistat was further amplified when combined with sorafenib in tumor xenograft mouse models. Our study provides evidence that SPAG4 mediates HCC progression by affecting lipid metabolism. Administration of orlistat combined with sorafenib reverses SPAG4-mediated oncogenesis in HCC cells and ectopic xenograft tumors in mice, suggesting that this pathway represents a potential target for HCC treatment.

B-Cell Receptor-Associated Protein 31 Promotes Metastasis via AKT/ß-Catenin/Snail Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal cancer worldwide, characterized with high heterogeneity and inclination to metastasize. Emerging evidence suggests that BAP31 gets involved in cancer progression with different kinds. It still remains unknown whether and how BAP31 plays a role in HCC metastasis. Epithelial-mesenchymal transition (EMT) has been a common feature in tumor micro-environment, whose inducer TGF-ß increased BAP31 expression in this research. Elevated expression of BAP31 was positively correlated with tumor size, vascular invasion and poor prognosis in human HCC. Ectopic expression of BAP31 promoted cell migration and invasion while BAP31 knockdown markedly attenuated metastatic potential in HCC cells and mice orthotopic xenografts. BAP31 induced EMT process, and enhanced the expression level of EMT-related factor Snail and decreased contents and membrane distribution of E-cadherin. BAP31 also activated AKT/ß-catenin pathway, which mediated its promotional effects on HCC metastasis. AKT inhibitor further counteracted the activated AKT/ß-catenin/Snail upon BAP31 over-expression. Moreover, silencing Snail in BAP31-overexpressed cells impaired enhanced migratory and invasive abilities of HCC cells. In HCC tissues, BAP31 expression was positively associated with Snail. In conclusion, BAP31 promotes HCC metastasis by activating AKT/ß-catenin/Snail pathway. Thus, our study implicates BAP31 as potential prognostic biomarker, and provides valuable information for HCC prognosis and treatment.

Downregulation of miR-98-5p expression induces interleukin-6 expression in rheumatoid fibroblast-like synoviocytes.

AIM: The increased level of interleukin-6 (IL-6) plays a significant role in the pathogenesis of rheumatoid arthritis (RA). Specific blockade of IL-6 or its receptor has been used successfully in treating RA. MicroRNAs can regulate gene expression and act as regulators of target genes. Manipulation of specific microRNAs provides a novel therapeutic strategy for treating/preventing diseases. This study explored the role of miR-98-5p in the regulation of IL-6 expression in rheumatoid fibroblast-like synoviocytes (RA-FLSs). METHODS: Real-time PCR was used to detect miR-98-5p expression in RA-FLSs and normal human fibroblast-like synovial cells (HFLSs). Site-directed gene mutagenesis and reporter gene assay were performed to identify the interaction between miR-98-5p and IL-6. Manipulation of miR-98-5p expression in RA-FLS used transfection with miR-98-5p mimic or inhibitor. Stimulation of FLSs with IL-1ß induced IL-6 production. Enzyme-linked immunosorbent assay was used to detect the level of IL-6 secreted into the RA-FLS culture supernatant. RESULTS: Compared with HFLSs, the expression of miR-98-5p in RA-FLSs was significantly downregulated, and was negatively correlated with DAS28 scores and rheumatoid factor. In patients with anti-keratin antibody-positive RA, the expression level of miR-98-5p was lower. miR-98-5p negatively regulated the expression of IL-6 in RA-FLSs. After IL-1ß stimulation, the expression of miR-98-5p decreased and the level of IL-6 protein was upregulated during IL-6 secretion. CONCLUSION: These data suggest that manipulation of miR-98-5p, which negatively modulates IL-6 expression, may be a potential clinical approach in RA.

MicroRNA-101-3p inhibits fibroblast-like synoviocyte proliferation and inflammation in rheumatoid arthritis by targeting PTGS2.

OBJECTIVE: Rheumatoid arthritis (RA) is the most frequently occurring inflammatory arthritis. The present study was performed to characterize the role of microRNA-101-3p (miR-101-3p) and prostaglandin-endoperoxide synthase 2 (PTGS2) in inflammation and biological activities of fibroblast-like synoviocytes (FLSs) in RA. METHODS: Initially, miR-101-3p and PTGS2 expression in RA tissues of RA patients and RA rats was detected by qRT-PCR and Western blot analysis. Rat model of type II collagen-induced arthritis (CIA) was adopted to simulate RA, followed by injection of miR-101-3p mimics or siRNA against PTGS2. Next, the apoptosis in synovial tissue and the levels of tumor necrosis factor (TNF)-α, IL-1ß and IL-6 were identified. Subsequently, FLSs in RA (RA-FLSs) were isolated, after which in vitro experiments were conducted to analyze cell proliferation, apoptosis, migration and invasion upon treatment of up-regulated miR-101-3p and silenced PTGS2. Furthermore, the relationship of miR-101-3p and PTGS2 was determined by bioinformatics prediction and luciferase activity assay. RESULTS: We identified poorly expressed miR-101-3p and highly expressed PTGS2 in synovial tissues of RA patients and RA rats, which showed reduced synoviocyte apoptosis and enhanced inflammation. In response to miR-101-3p mimics and si-PTGS2, the RA-FLSs were observed with attenuated cell proliferation, migration and invasion, corresponding to promoted apoptosis. Down-regulation of PTGS2 could rescue the effect of inhibited miR-101-3p in synovial injury and phenotypic changes of FLS in RA rats. Notably, miR-101-3p was found to negatively regulate PTGS2. CONCLUSION: Taken together, miR-101-3p reduces the joint swelling and arthritis index in RA rats by down-regulating PTGS2, as evidenced by inhibited FLS proliferation and inflammation.

RNA-binding protein KHSRP promotes tumor growth and metastasis in non-small cell lung cancer.

BACKGROUND: KH-type splicing regulatory protein (KHSRP) plays an important role in cancer invasion, but the relevant mechanism is not well known. In the present study, we investigated the function and potential molecular mechanism of KHSRP in non-small cell lung cancer (NSCLC) metastasis and elucidated its clinical significance. METHODS: Isobaric tags for relative and absolute quantitation and the SWATH™ approach were combined with nanoliquid chromatography-tandem mass spectrometry analysis to identify metastasis-associated nucleoproteins in NSCLC. Real-time PCR and Western blot were used to screen for metastasis-associated candidate molecules. Gene knockdown and overexpression were used to investigate their functions and molecular mechanisms in lung cancer cells. Coimmunoprecipitation (Co-IP) experiments were performed to identify the interactions between candidate molecules and their interacting proteins. Gene expression and its association with multiple clinicopathologic characteristics were analyzed by immunohistochemistry (IHC) and Western blot in human lung cancer specimens. RESULTS: KHSRP was identified as a metastasis-associated candidate molecule. In NSCLC cell lines, knockdown of KHSRP significantly reduced lung cancer cell proliferation, migration, and invasion in vitro and in vivo, whereas overexpression of KHSRP did the opposite. Mechanistically, the protein heterogeneous nuclear ribonucleoprotein C (C1/C2) (HNRNPC) was identified to interact with KHSRP using Co-IP experiments. In NSCLC cell lines, overexpression of HNRNPC significantly promoted lung cancer cell proliferation, migration, and invasion in vitro and in vivo. KHSRP and HNRNPC may induce human lung cancer cell invasion and metastasis by activating the IFN-α-JAK-STAT1 signaling pathway. Drastically higher expression levels of KHSRP and HNRNPC were observed in lung cancer tissues compared to those in adjacent noncancerous tissues. Increased KHSRP and HNRNPC expression was significantly associated with advanced tumor stages and metastasis (both lymph node and distant). Kaplan-Meier survival analysis showed that patients with high KHSRP and HNRNPC expression levels were predicted to have the shortest survival times and to have a poor prognosis. CONCLUSIONS: KHSRP plays an important role in NSCLC metastasis and may serve as a potential prognostic marker and novel therapeutic target for lung cancer metastasis treatment.

OTUB2 stabilizes U2AF2 to promote the Warburg effect and tumorigenesis via the AKT/mTOR signaling pathway in non-small cell lung cancer.

Increasing evidence has confirmed that deubiquitinating enzymes play an important role in lung cancer progression. In the current study, we investigated the expression profile of deubiquitinating enzymes in non-small cell lung cancer (NSCLC) tissues and identified OTUB2 as an upregulated deubiquitinating enzyme. The role of OTUB2 in NSCLC is unknown. Methods: Quantitative, real-time PCR and Western blot were used to detect OTUB2 and U2AF2 expression in NSCLC tissues. The correlations between OTUB2 and U2AF2 expression and clinicopathologic features were then analyzed. We used In vitro Cell Counting Kit-8 (CCK-8) , colony formation , and trans-well invasion assays to investigate the function of OTUB2 and U2AF2 in tumorigenesis. The regulation of glycolysis by OTUB2 and U2AF2 was assessed by determining the extracellular acid ratio, glucose consumption, and lactate production. The mechanism of OTUB2 was explored through co-immunoprecipitation and mass spectrometry analyses. A xenograft model was also used to study the tumorigenesis role of OTUB2 In vivo. Results: OTUB2 expression was significantly upregulated in primary NSCLC tissues and greatly associated with metastasis, advanced tumor stages, poor survival, and recurrence. In NSCLC cell lines, OTUB2 promoted cell growth, colony formation, migration, and invasive activities. Mechanistic investigations showed that OTUB2 stimulated the Warburg effect and induced the activation of the serine/threonine kinase/mechanistic target of rapamycin kinase (AKT/mTOR) pathway in different NSCLC cells. More importantly, OTUB2 promoted NSCLC progression, which was largely dependent on the direct binding to and deubiquitination of U2AF2, at least in NSCLC cells. U2AF2 expression was also significantly upregulated in primary NSCLC tissues and dramatically associated with metastasis, advanced tumor stages, poor survival, and recurrence. Importantly, a positive correlation between the protein expression of OTUB2 and U2AF2 in NSCLC tissues was found. In vivo experiments indicated that OTUB2 promoted xenograft tumor growth of NSCLC cell. In addition, our results suggest that high expression of OTUB2, U2AF2 and PGK1 is significantly associated with worse prognosis in NSCLC patients. Conclusion: Taken together, the present study provides the first evidence that OTUB2 acts as a pivotal driver in NSCLC tumorigenesis by stabilizing U2AF2 and activating the AKT/mTOR pathway and the Warburg effect. It may serve as a new potential prognostic indicator and therapeutic target in NSCLC.

Physcion 8­O­ß­glucopyranoside extracted from Polygonum cuspidatum exhibits anti­proliferative and anti­inflammatory effects on MH7A rheumatoid arthritis­derived fibroblast­like synoviocytes through the TGF­ß/MAPK pathway.

The present study aimed to investigate the anti­arthritic effect of physcion 8­O­ß­glucopyranoside (POGD) and its possible mechanisms. The anti­proliferative effects of POGD on MH7A cells were detected using a CCK­8 assay, and the release of pro­inflammatory cytokines, interleukin (IL)­1ß, IL­6, IL­8, IL­12 and IL­17A, were determined by ELISA. A type II collagen­induced arthritis (CIA) rat model was established to evaluate the anti­arthritic effect of POGD in vivo. The paw volumes, arthritis indices and serum levels of tumor necrosis factor (TNF)­α, IL­1ß, IL­6, IL­8, IL­17A were determined by ELISA. The mRNA expression levels of matrix metalloproteinase (MMP)­2, MMP­3, MMP­9, vascular endothelial growth factor and cyclooxygenase­2 were determined by reverse transcription­quantitative polymerase chain reaction analysis, and the expression levels of transforming growth factor (TGF)­ß1, small mothers against decapentaplegic (Smad)4, Smad7, c­Jun N­terminal kinase (JNK), phosphorylated (p­)JNK, p­P38, P38, p­extracellular signal­regulated kinase (ERK)1/2, ERK1/2, nuclear factor (NF)­κB p65 in the nucleus (N), cytosolic NF­κB p65 (C), and inhibitor of NF­κB (IκB) were determined by western blot analysis. The results indicated that POGD significantly inhibited MH7A cell growth. POGD markedly inhibited paw swelling and the arthritis indices of the CIA rats, and POGD may also inhibit the release of pro­inflammatory cytokines. Furthermore, POGD downregulated the expression levels of TGF­ß1, Smad4, NF­κB p65 (N), p38, p­p38, p­ERK1/2, JNK, p­JNK, TGF­ß1, Smad4, p­JNK, JNK, p­P38, P38, p­ERK1/2, ERK1/2 and NF­κB p65 (N), and upregulated the Smad7, NF­κB p65 (C) and IκB in TNF­α induced MH7A cells. In conclusion, the results suggested that POGD is a promising potential anti­inflammatory drug, and that POGD may decrease the expression of pro­inflammatory cytokines and mediators via inhibiting the TGF­ß/NF­κB/mitogen­activated protein kinase pathways.

LncRNA HOTAIR alleviates rheumatoid arthritis by targeting miR-138 and inactivating NF-κB pathway.

Rheumatoid arthritis (RA) is a chronic and autoimmune-mediated inflammatory disease. We aimed to investigate the regulation of lncRNA HOTAIR in LPS-treated chondrocytes and RA mouse. Our results showed that HOTAIR expression was significantly reduced in LPS-treated chondrocytes. The HOTAIR was then over-expressed in chondrocytes by transfecting recombinant lentivirus carrying sequences encoding HOTAIR. The LPS-induced reduction of cell proliferation rate and production of two inflammatory factors interleukin (IL)-17, IL-23 were markedly inhibited. Enforced expression of HOTAIR also led to the upregulation of proliferation-related protein Ki67 and proliferating cell nuclear antigen (PCNA). Moreover, a negative correlation was detected between the expression of HOTAIR and microRNA (miR)-138, and the expression of miR-138 was significantly increased in LPS-induced chondrocytes. The effects of HOTAIR over-expression on the proliferation and inflammation were partly reversed by miR-138 overexpression. Furthermore, the overexpression of HOTAIR significantly inhibited the activation of nuclear transcription factor-κB (NF-κB) in LPS-treated chondrocytes by suppressing p65 to cell nucleus, resulting in the down-regulation of IL-1ß and tumor necrosis factor (TNF)-α. In addition, the in vivo experiments exhibited that overexpression of HOTAIR increased cell proliferation and inhibited inflammation in RA rats, which were demonstrated by upregulation of Ki67 and PCNA, reduced CD4+IL-17+,CD4+IL-23+ cells, and down-regulation of p-p65, IL-1ß and TNF-α. In summary, our study suggests HOTAIR plays a protective role in RA by increasing proliferation rate and inhibiting inflammation, which may be related with the regulation of miR-138 expression and NF-κB signaling pathway. These results suggest that the regulation of HOTAIR may be a promising therapeutic strategy for RA.

Expression of Dicer in rheumatoid arthritis is associated with disease activity and balances the production of TNF­α.

Gene expression can be altered through RNA interference (RNAi), including microRNA (miRNA) or small interfering RNA. Alterations of miRNAs in rheumatoid arthritis (RA) have been reported, however, the components of the RNAi machinery in RA remain to be fully elucidated. The aim of the present study was to detect the levels of Dicer, Argonaute2 and Drosha, components of the RNAi machinery, in the peripheral blood mononuclear cells of patients with RAusingreverse transcription­quantitative polymerase chain reaction, and to compare the results with disease activity and clinical features. Disease activity was assessed using the Disease Activity Score 28 (DAS28). Transfection and stimulation of cultured cells were conducted to determine the biological function of Dicer. ELISA was used to test tumor necrosis factor (TNF)­α protein levels. It was found that the mRNA expression levels of Dicer and Drosha were upregulated in patients with RA, and that the increased level of Dicer was correlated with disease activity in patients with RA. Dicer and TNF­α were activated in the serum of patients with RA. The activation of Dicer suppressed the production of TNF­α. These results suggested that Dicer can balance the production of TNF­α, and thus may serve as a regulator of the immune response in patients with RA.

Establishment of a highly metastatic model with a newly isolated lung adenocarcinoma cell line.

Lung cancer is the leading cause of malignancy-related death worldwide, and metastasis always results in a poor prognosis. However, therapeutic progress is hampered by a deficiency of appropriate pre-clinical metastatic models. To bridge this experimental gap, we developed an in vivo metastatic model via subcutaneous (s.c.) injection. The original cell line (XL-2) adopted in this model was newly isolated from the ascites of a patient with extensive metastases of lung adenocarcinoma, thereby avoiding any alteration of its initial molecular biology features from artificial serial cultivation. After comprehensive phenotypical and histological analysis, it was identified as a lung adenocarcinoma cell line. Additionally, the drug test showed that XL-2 cell line was sensitive to docetaxel, and resistant to doxorubicin, indicating it might serve as a cell line model of drug resistance for identifying mechanisms of tumors resistant to doxorubicin. Through this s.c. model, we further obtained a highly metastatic cell line (designated XL-2sci). The metastatic rate of mice in XL-2 group was 3/10, in contrast to the rate of 9/10 in XL-2sci group. Optical imaging, micro-computed tomography (micro-CT) scanning and Transwell assays were further applied to identify the enhanced metastatic capacity of Xl-2sci cells both in vivo and in vitro. Compared with XL-2 cells, ITRAQ labeled proteomics profiling study showed that some tumor metastasis-associated proteins were upregulated in XL-2sci cells, which also indicated the reliability of our model. Proliferation ability of XL-2 and XL-2sci were also evaluated. Results showed that highly metastatic XL-2sci possessed a decreased proliferation capacity versus XL-2, which demonstrated that its increased metastatic activity was not facilitated by a faster growth rate. In conclusion, we successfully developed an in vivo metastatic model using a newly established lung adenocarcinoma cell line, which will be beneficial to further investigations of lung cancer metastasis and to the development of anti-metastasis drugs.

Quantitative proteomic analysis of the metastasis-inhibitory mechanism of miR-193a-3p in non-small cell lung cancer.

BACKGROUND: microRNAs can repress the expression of target genes by destabilizing their mRNAs or by inhibiting their translation. Our previous findings suggested that miR-193a-3p inhibited the progression of NSCLC both in vitro and in vivo. However, the biological processes and molecular pathways through which this miRNA exerts its positive effects are unknown. METHODS: To explore the molecular mechanisms by which miR-193a-3p inhibited NSCLC metastasis, we investigated the changes in the protein profile of SPC-A-1sci (highly metastatic) cells in response to the up-regulation of miR-193a-3p expression using a proteomics approach (iTRAQ combined with NanoLC-MS/MS). Changes in the profiles of the expressed proteins were verified using western blotting and were analyzed using the DAVID and STRING programs. RESULTS: In the two replicated experiments, 4962/4946 proteins were identified, and the levels of expression of 4923/4902 proteins were quantified. In total, 112 of these proteins were differentially expressed. Among them, the up-regulated levels of expression of two of the 62 proteins with up-regulated expression (PPP2R2A and GSN) and the down-regulated levels of expression four of the 50 proteins with down-regulated expression (LMNB2, UHRF1, G3BP1, and HNRNPU) were verified using western blotting. The bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. CONCLUSION: miR-193a-3p inhibited the metastasis of lung cancer cells by deregulating the expression of tumor-related proteins. These findings may improve the understanding of the molecular mechanisms underlying the metastatic-inhibitory effect of miR-193a-3p on lung cancer cells.

[Establishing the nude mice bone metastasis model of lung adenocarcinoma and applying MicroCT into the observation].

BACKGROUND AND OBJECTIVE: 50%-70% of patients with advanced lung cancer will develop bone metastases. The aim of this study is to establish the nude mice bone metastasis model of lung adenocarcinoma using A549, H1299, SPC-A-1 and XL-2, all of which own different invasion and migration abilities in vitro and supervise the bone metastases by MicroCT. METHODS: fifty BALB/C-nu/nu nude mice were grouped into five groups on average randomly. Cells of the four cell lines were injected into the left cardiac ventricle of mice in the four experimental groups (0.2 mL/mouse) respectively; meanwhile, mice in the control group were injected with normal saline (0.2 mL/mouse) in the same manner. Periodical radiological examination was carried out to supervise the variation of the mice since the second week after injection. When mice in each group became thin obviously, end the experiment of this group. Before the end, pathological sections of bone tissues were made. We classified the bone metastatic sites into axial skeleton and limb bone, in order to compare the metastatic rates of these two different parts. The bone metastatic abilities of the four cell lines was statistically analyzed by comparing the average time cost in the appearance of bone metastases and the percentage of bone metastases among the experimental groups. RESULTS: Different metastatic sites which had been identified both by MicroCT and pathological sections appeared in each group of the four experimental groups. By contrast, no metastasis was observed in the control group. The percentage of cancer metastasizing to axial skeleton was remarkably higher than the percentage of tumor metastasizing to the limb bone in each experimental group, which was consistent with the clinical regularity and characteristics of skeletal metastases with lung cancer. Thus, the model has been established triumphantly. However, there were no statistical differences in the average time consumed and skeletal metastatic rate among the four experimental groups. CONCLUSIONS: The disruption in the bone can be clearly detected by MicroCT, which is benefit to supervise the osseous metastasis. We successfully developed the nude mice bone metastasis model of lung adenocarcinoma, which will pave the way for exploring novel prevention and therapy strategies clinically. The four cell lines varied in invasion and migration abilities in vitro, but there was no statistical difference in the metastatic ability in vivo, and the reason need to be explored further in future.

Quantitative proteomic analysis identifies CPNE3 as a novel metastasis-promoting gene in NSCLC.

To discover metastasis-associated proteins within cancer cells, we used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis to identify proteins that were differentially expressed between lung adenocarcinoma cancer cell lines SPC-A-1sci cells with high metastatic potential and parent SPC-A-1 cells with low metastatic potential. By employing biological and technical replicates, we identified 5818 nonredundant proteins and quantified 5443 proteins, 256 of which were differentially expressed in the two cell lines. Through si-RNA-mediated functional screens, Myosin heavy chain 9 (MYH9) and Copine III (CPNE3) were indicated as positively correlating with the migration and invasion properties of SPC-A1sci cells, and the same function of CPNE3 was confirmed in another lung cancer cell line, H1299. Furthermore, overexpressing CPNE3 promoted nonsmall-cell lung cancer (NSCLC) cell line (SPC-A-1 and XL-2) migration and invasion in vitro. Moreover, the targeted knock-down of CPNE3 inhibited the in vivo metastatic abilities of H1299 cells in mouse models. Lastly, immunohistochemistry revealed that the CPNE3 expression level was positively correlated with the clinical stage and TNM classification in NSCLC patients. Taken together, our results indicate that CPNE3 could play a critical role in NSCLC metastasis.