TGFß1, SMAD and ß-Catenin in pulmonary arteries of smokers, patients with small airway disease and COPD: potential drivers of EndMT.

We previously reported pulmonary arterial remodelling and active endothelial to mesenchymal transition (EndMT) in smokers and patients with early COPD. In this study, we aimed to evaluate the role of different drivers of EndMT. Immunohistochemical staining for EndMT drivers, TGF-ß1, pSMAD-2/3, SMAD-7, and ß-catenin, was performed on lung resections from 46 subjects. Twelve were non-smoker-controls (NC), six normal lung function smokers (NLFS), nine patients with small-airway diseases (SAD), nine mild-moderate COPD-current smokers (COPD-CS) and ten COPD-ex-smokers (COPD-ES). Histopathological measurements were done using Image ProPlus softwarev7.0. We observed lower levels of total TGF-ß1 (p<0.05) in all smoking groups than in the non-smoking control (NC). Across arterial sizes, smoking groups exhibited significantly higher (p <0.05) total and individual layer pSMAD-2/3 and SMAD-7 than in the NC group. The ratio of SAMD-7 to pSMAD-2/3 was higher in COPD patients compared to NC. Total ß-catenin expression was significantly higher in smoking groups across arterial sizes (p <0.05), except for COPD-ES and NLFS groups in small and medium arteries, respectively. Increased total ß-catenin was positively correlated with total S100A4 in small and medium arteries (r= 0.35, 0.50; p=0.02, 0.01, respectively), with vimentin in medium arteries (r=0.42, p=0.07), and with arterial thickness of medium and large arteries (r= 0.34, 0.41, p=0.02, 0.01, respectively). This is the first study uncovering active endothelial SMAD pathway independent of TGF-ß1 in smokers, SAD, and COPD patients. Increased expression of ß-catenin indicates its potential interaction with SMAD pathway, warranting further research to identify the deviation of this classical pathway.

Airway inflammatory changes in the lungs of patients with asthma-COPD overlap (ACO): a bronchoscopy endobronchial biopsy study.

BACKGROUND: Although asthma and chronic obstructive pulmonary disease (COPD) are two distinct chronic airway inflammatory diseases, they often co-exist in a patient and the condition is referred to as asthma-COPD overlap (ACO). Lack of evidence regarding the inflammatory cells in ACO airways has led to their poor prognosis and treatment. The objective of this endobronchial biopsy (EBB) study was to enumerate inflammatory cellular changes in the airway wall of ACO compared with asthma, COPD current smokers (CS) and ex-smokers (ES), normal lung function smokers (NLFS), and non-smoker controls (HC). METHODS: EBB tissues from 74 patients were immunohistochemically stained for macrophages, mast cells, eosinophils, neutrophils, CD8+ T-cells and CD4+ T-cells. The microscopic images of stained tissues were evaluated in the epithelium, reticular basement membrane (RBM) cells/mm RBM length, and lamina propria (LP) cells/mm2 up to a depth of 120 µM using the image analysis software Image-Pro Plus 7.0. The observer was blinded to the images and disease diagnosis. Statistical analysis was performed using GraphPad Prism v9. RESULTS: The tissue macrophages in ACO were substantially higher in the epithelium and RBM than in HC (P < 0.001 for both), COPD-ES (P < 0.001 for both), and -CS (P < 0.05 and < 0.0001, respectively). The ACO LP macrophages were significantly higher in number than COPD-CS (P < 0.05). The mast cell numbers in ACO were lower than in NLFS (P < 0.05) in the epithelium, lower than COPD (P < 0.05) and NLFS (P < 0.001) in RBM; and lower than  HC (P < 0.05) in LP. We noted lower eosinophils in ACO LP than HC (P < 0.05) and the lowest neutrophils in both ACO and asthma. Furthermore, CD8+ T-cell numbers increased in the ACO RBM than HC (P < 0.05), COPD-ES (P < 0.05), and NLFS (P < 0.01); however, they were similar in number in epithelium and LP across groups. CD4+ T-cells remained lower in number across all regions and groups. CONCLUSION: These results suggest that the ACO airway tissue inflammatory cellular profile differed from the contributing diseases of asthma and COPD with a predominance of macrophages.

Targeting the P2Y13 Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

Differential airway remodeling changes were observed in patients with asthma COPD overlap compared to patients with asthma and COPD alone.

Management of patients with asthma COPD overlap (ACO) is clinically challenging due to insufficient evidence of pathological changes in these patients. In this cross-sectional study, we evaluated airway remodeling in endobronchial biopsies from a total of 90 subjects, which included 12 ACO, 14 patients with asthma, 12 COPD exsmokers (ES), 11 current smokers (CS), 28 healthy controls (HC), and 13 normal lung function smokers (NLFS). Tissue was stained with Masson's trichrome. Epithelium, goblet cells, reticular basement membrane (RBM), cellularity, lamina propria (LP), and smooth muscle (SM) changes were measured using Image-Pro Plus v7 software. Differential airway remodeling pattern was seen in patients with ACO. A limited change was noted in the ACO epithelium compared with other pathological groups. RBM was substantially thicker in patients with ACO than in HC (P < 0.0002) and tended to be thicker than in patients with asthma and NLFS. The total RBM cells were higher in ACO than in the HC (P < 0.0001), COPD-CS (P = 0.0559), -ES (P = 0.0345), and NLFS (P < 0.0002), but did not differ from patients with asthma. Goblet cells were higher in the ACO than in the HC (P = 0.0028) and COPD-ES (P = 0.0081). The total LP cells in ACO appeared to be higher than in HC, COPD-CS, and NLFS but appeared to be lower than in patients with asthma. Finally, SM area was significantly lower in the ACO than in patients with asthma (P = 0.001), COPD-CS (=0.0290), and NLFS (P = 0.0011). This first comprehensive study suggests that patients with ACO had distinguishable tissue remodeling that appeared to be more severe than patients with asthma and COPD. This study will help in informed decision-making for better patient management in clinical practice.

Adverse roles of mast cell chymase-1 in COPD.

BACKGROUND: COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase (hCMA)1 and its orthologue mCMA1/mouse mast cell protease (mMCP)5 are exocytosed from activated mast cells and have adverse roles in numerous disorders, but their role in COPD is unknown. METHODS: We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5-deficient (-/-) mice to evaluate this protease's role and potential for therapeutic targeting in CS-induced experimental COPD. In addition, we used ex vivo/in vitro studies to define mechanisms. RESULTS: The levels of hCMA1 mRNA and CMA1+ mast cells were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated mast cell numbers and mMCP5 protein were increased in lung tissues of wild-type mice with experimental COPD. mmcp5 -/- mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of mast cells from wild-type, but not mmcp5 -/- mice with wild-type lung macrophages increased in tumour necrosis factor (TNF)-α release. It also caused the release of CMA1 from human mast cells, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD. CONCLUSION: CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.

Dysregulation of endocytic machinery and ACE2 in small airways of smokers and COPD patients can augment their susceptibility to SARS-CoV-2 (COVID-19) infections.

Lungs of smokers and chronic obstructive pulmonary disease (COPD) are severely compromised and are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attack. The dangerous combination of enhanced SARS-CoV-2 attachment receptor protein ACE2 along with an increase in endocytic vacuoles will enable viral attachment, entry, and replication. The objective of the study was to identify the presence of SARS-CoV-2 host attachment receptor angiotensin-converting enzyme-2 (ACE2) along with endocytic vacuoles, early endosome antigen-1 (EEA1), late endosome marker RAB7, cathepsin-L, and lysosomal associated membrane protein-1 (LAMP-1) as lysosome markers in the airways of smokers and COPD patients. The study design was cross-sectional and involved lung resections from 39 patients in total, which included 19 patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I or GOLD stage II COPD, of which 9 were current smokers with COPD (COPD-CS) and 10 were ex-smokers with COPD (COPD-ES), 10 were normal lung function smokers, and 10 were never-smoking normal controls. Immunostaining for ACE2, EEA1, RAB7, and cathepsin-L was done. A comparative description for ACE2, EEA1, RAB7, and cathepsin-L expression pattern is provided for the patient groups. Furthermore, staining intensity for LAMP-1 lysosomes was measured as the ratio of the LAMP-1-stained areas per total area of epithelium or subepithelium, using Image ProPlus v7.0 software. LAMP-1 expression showed a positive correlation to patient smoking history while in COPD LAMP-1 negatively correlated to lung function. The active presence of ACE2 protein along with endocytic vacuoles such as early/late endosomes and lysosomes in the small airways of smokers and COPD patients provides evidence that these patient groups could be more susceptible to COVID-19.

ACE2 expression is elevated in airway epithelial cells from older and male healthy individuals but reduced in asthma.

BACKGROUND AND OBJECTIVE: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter AEC. We determined what factors are associated with ACE2 expression particularly in patients with asthma and COPD. METHODS: We obtained lower AEC from 145 people from two independent cohorts, aged 2-89 years, Newcastle (n = 115) and Perth (n = 30), Australia. The Newcastle cohort was enriched with people with asthma (n = 37) and COPD (n = 38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry was assessed by qPCR, and protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AEC. RESULTS: Increased gene expression of ACE2 was associated with older age (P = 0.03) and male sex (P = 0.03), but not with pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma patients (P = 0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in patients with asthma (P = 0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface, was increased (P = 0.02). ACE2 protein expression was also reduced in endobronchial biopsies from asthma patients. CONCLUSION: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

The rise of electronic nicotine delivery systems and the emergence of electronic-cigarette-driven disease.

In 2019, the United States experienced the emergence of the vaping-associated lung injury (VALI) epidemic. Vaping is now known to result in the development and progression of severe lung disease in the young and healthy. Lack of regulation on electronic cigarettes in the United States has resulted in over 2,000 patients and 68 deaths. We examine the clinical representation of VALI and the delve into the scientific evidence of how deadly exposure to electronic cigarettes can be. E-cigarette vapor is shown to affect numerous cellular processes, cellular metabolism, and cause DNA damage (which has implications for cancer). E-cigarette use is associated with a higher risk of developing crippling lung conditions such as chronic obstructive pulmonary disease (COPD), which would develop several years from now, increasing the already existent smoking-related burden. The role of vaping and virus susceptibility is yet to be determined; however, vaping can increase the virulence and inflammatory potential of several lung pathogens and is also linked to an increased risk of pneumonia. As it has emerged for cigarette smoking, great caution should also be given to vaping in relation to SARS-CoV-2 infection and the COVID-19 pandemic. Sadly, e-cigarettes are continually promoted and perceived as a safer alternative to cigarette smoking. E-cigarettes and their modifiable nature are harmful, as the lungs are not designed for the chronic inhalation of e-cigarette vapor. It is of interest that e-cigarettes have been shown to be of no help with smoking cessation. A true danger lies in vaping, which, if ignored, will lead to disastrous future costs.

Exosomal KRAS mutation promotes the formation of tumor-associated neutrophil extracellular traps and causes deterioration of colorectal cancer by inducing IL-8 expression.

BACKGROUND: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. The current study aimed to elucidate the mechanism by which exosomes carrying KRAS mutant contribute to neutrophil recruitment as well as the formation of the neutrophil extracellular trap (NET) in CRC. METHODS: APC-WT and APC-KRASG12D mouse models were initially developed. Peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) were isolated to detect neutrophil content. Then, APC-WT and APC-KRASG12D mice were injected with exosomes isolated from APC-WT and APC-KRASG12D mice. The ratio of neutrophils, NETs formation and IL-8 protein content were subsequently quantified in colon tissues. DKs-8 (wild type) and DKO-1 (KRAS mutant) cells were employed for in vitro experimentation. Then, DKs-8 cells were cultured with exosome-treated PMA stimulated neutrophil-forming NETs culture medium, with cell viability, invasion, migration, and adhesion evaluated. RESULTS: Compared with APC-WT mice, the numbers of polyps and neutrophils in the peripheral blood, spleen and mLNs were increased in APC-KRASG12D mice, accompanied with increased NET formation, IL-8 expression and exosomes. Meanwhile, IL-8 upregulation, neutrophil recruitment and NET formation were observed in the mice injected with exosomes derived from APC-KRASG12D. The in vitro investigation results revealed that more NETs were formed in the presence of DKO-1-Exos, which were inhibited by DNAse. In addition, DKs-8- and DKO-1 cells-derived exosomes could adhere to NETs under static conditions in vitro. Exosomal KRAS mutants were noted to exert stimulatory effects on the IL-8 production and NET formation to promote the growth of CRC cells. CONCLUSION: The results provide evidence suggesting that exosomes may transfer mutant KRAS to recipient cells and trigger increases in IL-8 production, neutrophil recruitment and formation of NETs, eventually leading to the deterioration of CRC.

Dynamic changes of Chest CT follow-up in Coronavirus Disease-19 (COVID-19) pneumonia: relationship to clinical typing.

BACKGROUND: To investigate the CT changes of different clinical types of COVID-19 pneumonia. METHODS: This retrospective study included 50 patients with COVID-19 from 16 January 2020 to 25 February 2020. We analyzed the clinical characteristics, CT characteristics and the pneumonia involvement of the patients between the moderate group and the severe and critical group, and the dynamic changes of severity with the CT follow-up time. RESULTS: There were differences in the CT severity score of the right lung in the initial CT, and total CT severity score in the initial and follow-up CT between the moderate group and the severe and critical group (all p < 0.05). There was a quadratic relationship between total CT severity score and CT follow-up time in the severe and critical group (r2 = 0.137, p = 0.008), the total CT severity score peaked at the second follow-up CT. There was no correlation between total CT severity score and CT follow-up time in the moderate group (p > 0.05). There were no differences in the occurrence rate of CT characteristics in the initial CT between the two groups (all p > 0.05). There were differences in the occurrence rate of ground-glass opacity and crazy-paving pattern in the second follow-up CT, and pleural thickening or adhesion in the third follow-up CT between the two groups (all p < 0.05). CONCLUSIONS: The CT changes of COVID-19 pneumonia with different severity were different, and the extent of pneumonia involvement by CT can help to assess the severity of COVID-19 pneumonia rather than the initial CT characteristics.

miR-381-3p restrains cervical cancer progression by downregulating FGF7.

This study aimed at elucidating the molecular mechanism of miR-381-3p in cervical cancer progression, which may provide a novel therapeutic target for patients with cervical cancer. The expression of miR-381-3p was confirmed by quantitative reverse transcription polymerase chain reaction. Microarray analysis was conducted to screen out differentially expressed genes, and the target gene of microRNA (miRNA) was predicted on TargetScan. Dual-luciferase reporter assay then verified the targeting relationship between miR-381-3p and FGF7. The protein expression of FGF7 was examined via Western blot assay. Colony formation assay was used to detect the cell proliferation, while flow cytometry was used to analyze cell cycle and apoptosis. The influence of miR-381-3p and FGF7 on cell migration and invasion was confirmed by transwell migration/invasion assay. Finally, we demonstrated that miR-381-3p was lowly expressed, while FGF7 was highly expressed in cervical cancer cells. There was a direct target relationship and a negative correlation between miR-381-3p and FGF7. miR-381-3p could downregulate FGF7 expression, inhibiting cell proliferation and metastasis, and inducing cell cycle arrest and apoptosis in cervical cancer.

Mechanism of miR-137 regulating migration and invasion of melanoma cells by targeting PIK3R3 gene.

OBJECTIVE: To investigate the effect of microRNA-137 (miR-137) on the migration and invasion of melanoma cells and its mechanism. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-137 in melanoma tissues and cells. miR-137 mimics, phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) small interfering RNA and corresponding controls were transfected into A375 and WM451 cells by lipofection. The expression of PIK3R3 was examined by qRT-PCR and Western blot analysis. The Trans-well assay was conducted to measure cell migration and invasion. Dual luciferase reporter assay was used to detect the interaction between miR-137 and PIK3R3. RESULTS: Compared with normal pigmented nevus tissue, miR-137 expression was significantly reduced in melanoma tissues. Compared with keratinous HaCaT cells, the level of miR-137 was significantly decreased in melanoma SK-MEL-1, A375, and WM451 cells. Knockdown of miR-137 significantly reduced the migrated and invasive abilities of melanoma A375 and WM451 cells. Moreover, inhibition of PIK3R3 obviously suppressed the migration and invasion abilities of melanoma A375 and WM451 cells. Luciferase activity assay showed that PIK3R3 was a direct target of miR-137. In addition, overexpression of miR-137-inhibited PIK3R3 expression, while knockdown of miR-137-enhanced PIK3R3 abundance. Restoration of PIK3R3 reversed the regulatory effect of miR-137 on cell migration and invasive in melanoma A375 and WM451 cells. CONCLUSION: miR-137 inhibited melanoma cell migration and invasion by targeting PIK3R3 gene.

Heparin-binding epidermal growth factor (HB-EGF) drives EMT in patients with COPD: implications for disease pathogenesis and novel therapies.

Chronic obstructive pulmonary disease (COPD) is a progressive and devastating chronic lung condition that has a significant global burden, both medically and financially. Currently there are no medications that can alter the course of disease. At best, the drugs in clinical practice provide symptomatic relief to suffering patients by alleviating acute exacerbations. Most of current clinical research activities are in late severe disease with lesser attention given to early disease manifestations. There is as yet, a lack of understanding of the underlying mechanisms of disease progression and the molecular switches that are involved in their manifestation. Small airway fibrosis and obliteration are known to cause fixed airflow obstruction in COPD, and the consequential damage to the lung has an early onset. So far, there is little evidence of the mechanisms that underlie this aspect of pathology. However, emerging research confirms that airway epithelial reprogramming or epithelial to mesenchymal transition (EMT) is a key mechanism that drives fibrotic remodelling changes in smokers and patients with COPD. A recent study by Lai et al. further highlights the importance of EMT in smoking-related COPD pathology. The authors identify HB-EGF, an EGFR ligand, as a key driver of EMT and a potential new therapeutic target for the amelioration of EMT and airway remodelling. There are also wider implications in lung cancer prophylaxis, which is another major comorbidity associated with COPD. We consider that improved molecular understanding of the intricate pathways associated with epithelial cell plasticity in smokers and patients with COPD will have major therapeutic implications.

Knockdown of long noncoding RNA PVT1 suppresses cell proliferation and invasion of colorectal cancer via upregulation of microRNA-214-3p.

Long noncoding RNAs (lncRNAs) have been reported to be involved in the occurrence and tumorigenesis of numerous malignant cancers. Microarray expression profiles were used to screen colorectal cancer (CRC)-related differentially expressed genes and lncRNAs, which revealed that insulin receptor substrate 1 (IRS1) and lncRNA plasmacytoma variant translocation 1 (PVT1) were highly expressed in CRC. This study aimed to investigate the regulatory role of lncRNA PVT1 in CRC. Subcellular localization detected by fluorescence in situ hybridization identified that lncRNA PVT1 was primarily located in the cytoplasm. The interaction between lncRNA PVT1 and microRNA-214-3p (miR-214-3p) and IRS1 was predicted using the RNA22 website. Next the dual luciferase reporter gene assay, RNA pull-down, and RNA immunoprecipitation assays verified lncRNA PVT1 to be a competitive endogenous RNA (ceRNA) against miR-214-3p, and IRS1 was found to be a target of miR-214-3p. The expression pattern of lncRNA PVT1, miR-214-3p, IRS1, phosphoinositide 3-kinase (PI3K), and Akt was characterized in response to lncRNA PVT1 silencing or miR-214-3p upregulation. Meanwhile, their regulatory effects on cell proliferation, invasion, and apoptosis were detected in CRC cells. With increased levels of miR-214-3p and decreased levels of lncRNA PVT1 in CRC cells, the expression of phosphatidylinositol 3-kinase, putative (PI3K) and Akt was reduced, and consequently, the cell apoptosis was stimulated and cell proliferation and invasion were suppressed. All in all, lncRNA PVT1 competitively binds to miR-214-3p to upregulate the expression of IRS1 thus activating the PI3K/Akt signaling pathway, thus accelerating CRC progression. This study suggests that lncRNA PVT1 might be a potential target of therapeutic strategies for CRC treatment.NEW & NOTEWORTHY This study mainly suggests that long noncoding (lnc)RNA plasmacytoma variant translocation 1 (PVT1) is a downregulated lncRNA in colorectal cancer (CRC), accelerating CRC progression. Strikingly, lncRNA PVT1 acts as a competitive endogenous RNA against microRNA (miR)-214-3p, whereas miR-214-3p targets insulin receptor substrate 1, which draws a comprehensive picture of the potential molecular mechanisms of lncRNA PVT1 in CRC.

Molecular mechanism of the extended oil accumulation phase contributing to the high seed oil content for the genotype of tung tree (Vernicia fordii).

BACKGROUND: Oil from seeds of the tung tree (Vernicia fordii) has unique drying properties that are industrially important. We found that the extended oil accumulation period was related to the high seed oil content at maturity among tung tree population. In order to understand the molecular mechanism underlying the high oil content in tung tree seed, Tree H and L were adopted for the further investigation, with seed oil content of about 70 and 45%, respectively. We compared the transcriptomic changes of seed at various times during oil accumulation between the two trees. RESULTS: Transcriptomes analysis revealed that many genes involved in glycolysis, fatty acid synthesis, and tri-acyl glyceride assembly still kept high expression in the late period of seed oil accumulation for Tree H only. Many genes in fatty acid degradation pathway were largely up regulated in the late period of seed oil accumulation for Tree L only. Four transcription factors related to fatty acid biosynthesis had different expression pattern in the seed oil accumulation period for the two trees. WRI1 was down regulated and kept the low expression in the late period of seed oil accumulation for the two trees. PII, LEC1 and LEC1-LIKE extended the high expression in the late period of seed oil accumulation in Tree H only. CONCLUSIONS: The continued accumulation of oil in the late period of seed oil accumulation for Tree H was associated with relatively high expression of the relevant genes in glycolysis, fatty acid synthesis and tri-acyl glyceride assembly. PII, LEC1, and LEC1-LIKE rather than WRI1 should play an important role in the oil continual accumulation in the late period of seed oil accumulation in Tree H. This study provides novel insight into the variation in seed oil content and informs plant breeding strategies to maximize oil yield.

A Conserved Glycine Is Identified to be Essential for Desaturase Activity of IpFAD2s by Analyzing Natural Variants from Idesia polycarpa.

High amounts of polyunsaturated fatty acids (PUFAs) in vegetable oil are not desirable for biodiesel or food oil due to their lower oxidative stability. The oil from Idesia polycarpa fruit contains 65⁻80% (mol%) linoleic acid (C18:2). Therefore, development of Idesia polycarpa cultivars with low PUFAs is highly desirable for Idesia polycarpa oil quality. Fatty acid desaturase 2 (FAD2) is the key enzyme converting oleic acid (C18:1) to C18:2. We isolated four FAD2 homologs from the fruit of Idesia polycarpa. Yeast transformed with IpFAD2-1, IpFAD2-2 and IpFAD2-3 can generate appreciable amounts of hexadecadienoic acid (C16:2) and C18:2, which are not present in wild-type yeast cells, revealing that the proteins encoded by these genes have Δ12 desaturase activity. Only trace amounts of C18:2 and little C16:2 were detected in yeast cells transformed with IpFAD2-4, suggesting IpFAD2-4 displays low activity. We also analyzed the activity of several FAD2 natural variants of Idesia polycarpa in yeast and found that a highly conserved Gly376 substitution caused the markedly reduced products catalyzed by IpFAD2-3. This glycine is also essential for the activity of IpFAD2-1 and IpFAD2-2, but its replacement in other plant FAD2 proteins displays different effects on the desaturase activity, suggesting its distinct roles across plant FAD2s proteins.

MiR-1-3p Suppresses the Proliferation, Invasion and Migration of Bladder Cancer Cells by Up-Regulating SFRP1 Expression.

BACKGROUND: Bladder cancer is of compelling morbidity and mortality due to its high recurrence rate. Little development has been made in the last decades in the therapy methods. Thus, the mechanism of its growth and invasiveness involving novel molecular targets are needed. OBJECTIVE: Our research objective is to confirm the hypothesis that miR-1-3p suppresses the proliferation, invasion and migration of bladder cancer cells. METHODS: The expression levels of miR-1-3p and SFRP1 were evaluated using RT-qPCR in bladder cancer tissues and cells as well as in normal tissues and cells. J82 cell lines were selected as experiment subjects due to their low expression levels of miR-1-3p. Plasmids carrying miR-1-3p mimics, miR-1-3p inhibitors and SFRP1 were transfected into the J82 cell lines. Subsequently, the protein expression of SFRP1 was detected using Western Blot analysis, and cell proliferation, apoptosis, invasion and migration ability was measured using MTT, the flow cytometry, the Transwell test and wound healing assays, respectively Results: Bladder cancer tissues and cells exhibited significant decrease in the expression of miR-1-3p and SFRP1 compared to normal tissues and cells, and human bladder cancer cell line J82 exhibited the most significant decrease in these expressions (P < 0.05). MiR-1-3p up-regulates SFRP1 expression in bladder cancer cells, and the over-expression of miR-1-3p can suppress the proliferation, invasion and migration ability of bladder cancer cells. This mechanism is similar to the effect of SFRP1 over-expression on bladder cancer cells. CONCLUSION: MiR-1-3p suppresses the proliferation, invasion and migration of bladder cancer cells by up-regulating SFRP1 expression.

MiR-1-3p inhibits the proliferation and invasion of bladder cancer cells by suppressing CCL2 expression.

We attempted to analyze the effects of miR-1-3p and CCL2 on the proliferation, migration, and invasion of bladder cancer cells. A total of 18 pairs of bladder cancer tissues with corresponding adjacent tissues and the 6 cases of normal tissues were collected. The expressions of miR-1-3p and CCL2 in the cancer tissues were evaluated using quantitative real-time polymerase chain reaction and western blot. The relationship between miR-1-3p and CCL2 was assessed using luciferase reporter assay. The UM-UC-3 bladder cancer cells were transfected with CCL2 small interfering RNA and miR-1-3p mimics. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, wound healing assay, Transwell assay, and the flow cytometry test were used to detect the proliferation, migration, invasion, and apoptosis of bladder cancer cells. Bladder cancer tissues had lower levels of miR-1-3p but higher levels of CCL2 than normal tissues ( p < 0.05). The transfection of miR-1-3p mimics and CCL2 small interfering RNA remarkably suppressed cell proliferation and invasion and promoted apoptosis of cells ( p < 0.05). Results of the luciferase reporter gene assay demonstrated that miR-1-3p targeted CCL2. MiR-1-3p suppresses the proliferation and invasion of urinary bladder cancer cells by targeting CCL2.