Bone Marrow Mesenchymal Stem Cells (BMSCs) with a Disintegrin and Metalloprotease 17 (ADAM17) Overexpression Increase Cervical Cancer Cell Proliferation and Migration Through Epidermal Growth Factor Receptor (EGFR)/Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase b (AKT) Signaling

ADAM-17 is a membrane-bound protease and highly expressed in multiple tumors. BMSCs carrying target genes are delivered to damaged sites. This study aimed to investigate the mechanism underlying BMSCs with ADAM-17 in cervical cancer (CC). BMSCs were transfected with ADAM-17 mimics and co-cultured with CC cells followed by analysis of cell proliferation and migration by MTT assay and scratch assay, ADAM-17 and target genes (LAMB3, Robol) level by Western blot and RT-qPCR. As the effectiveness of ADAM-17 transfection was confirmed by its increased level, the presence of empty vector rarely affected ADAM-17 expression and biological activities of CC cells compared to control group (p > 0.05). BMSCs with ADAM-17 overexpression increased CC cell proliferation and enhanced scratch healing rate (p < 0.05), accompanied with upregulated LAMB3 and Robol. The difference in LAMB3 and Robol expression between empty vector group and control group did not reach a significance. In conclsuion, this study elucidates that BMSCs with ADAM-17 overexpression promotes CC cell progression through up-regulation of LAMB3 and Robol and activation EGFR/PI3K/Akt signaling, providing a novel BMSC-based targeted therapy.

R354X Mutation in PRPF31 Affects Its Capacity in Inflammation Response and Splicing

Pre-mRNA processing factor 31(PRPF31) is a key component of RNA splicing and also a disease-causing gene of Retinitis Pigmentosa (RP). Previously, we found that the nonsense mutation R354X in PRPF31induces RP in a Chinese RP family. In order to investigate the underlining molecular mechanisms of RP pathogenesis induced by this mutation, we generated cell lines stably expressing R354X mutant, wild type (WT) of PRPF31 and corresponding empty vector using HEK293T cells ,the resulting cell lines were used for Long non-coding RNA sequencing(LncRNA -sequencing). The results of LncRNA sequencing showed that, comparing to WT, R354X mutation changed the expression and splicing of coding and non-coding transcripts. Interestingly, in HEK293T and APRE-19 cells, inflammation-associated genes such as IFI6, OAS3and STAT3, enhanced their expression in response to the overexpression of WT PRPF31; however, in R354X mutation cells, those genes’ expression remained basal levels. Moreover, increased H2AFX expression and attenuated growth capacity were found in cells expressing R354X PRPF31. In contrast with WT, R354X mutant showed varied splicing model for dihydrofolate reductase (DHFR) in HEK293T , reduced CPSF1 and SORBS1 mRNAs binding in ARPE-19 ,and its binding with CTNNBL1 was also interfered in ARPE-19 cells. On the other hand, the R354X mutant also increased the level of transcripts read-through. Taken together, R354X mutation in PRPF31 affected cell survival, changed gene’s expression and splicing. Those findings indicate that inflammation and oxidation might contribute the pathogenesis of RP induced by the R354X mutation.

Cystic Fibrosis Transmembrane Conductance Regulator Folding Mutations Reveal Differences in Corrector Efficacy Linked to Increases in Immature Cystic Fibrosis Transmembrane Conductance Regulator Expression

Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin–proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B.Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio.Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level.Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Abstract MP10: Furin-Mediated Modification Is Required For Epithelial Sodium Channel-Activating Activity Of Soluble (Pro)Renin Receptor In Cultured Collecting Duct Cells

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in cultured M1 cells. M1 cells were transfected with pcDNA3.4 containing full-length PRR with (Furin Mut) or without (WT) mutagenesis of furin cleavage site; empty vector (EM) served a control. As compared with EM, overexpression of WT and Furin Mut vectors induced a more than 16-fold comparable increase in the release of sPRR. Amiloride-sensitive short circuit current as assessed by Ussing chamber technique was elevated by overexpression of WT PRR which was reduced by 37% by Furin Mut (ENaC activity: 1.00 + 0.06 μA/cm 2 in EM, 1.67 + 0.05 μA/cm 2 in WT, and 1.04 + 0.07 μA/cm 2 in Furin Mut, p < 0.05). In parallel, the expression of α-ENaC but not β or γ subunit as assessed by immunoblotting and qRT-PCR analysis was elevated by WT PRR and this increase was blunted by Furin Mut. In a separate experiment, M1 cells were transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P) or with furin cleavage (AA 1-279) (sPRR-furin); empty vector was used as a control. Overexpression of cDNA for the two types of sPRR induced a significant and comparable increase in the release of sPRR. By Ussing chamber technique, ENaC activity was 1.00 + 0.09 μA/cm 2 in EM, 1.03 + 0.10 μA/cm 2 in sPRR-S1P, and 1.39 + 0.14 μA/cm 2 in sPRR-furin, p < 0.05. Lastly, 293 cells were pretreated for 1 h with furin inhibitor α1-antitrypsin Portland followed by transfection with empty vector, WT PRR, or Furin Mut vectors. sPRR in the condition medium was enriched by using protein centrifugal filter devices and applied to M1 cells for 10 min followed by measurement of ENaC activity. Pretreatment with furin inhibition attenuated ENaC-acting activity induced by overexpression of WT PRR. Overall, the three independent approaches consistently demonstrated that furin-mediated modification is required for the activity of sPRR to increase ENaC-mediated Na + transport in the CD cells.

Cytokeratin 8 Inhibits Hepatic Glycogen Synthesis in Type 2 Diabetes Mellitus by Modulating Insulin-dependent IRS1/PI3K/Akt-GSK3β Pathway

Purpose: Cytokeratin 8(CK8) is a cytoskeletal protein mainly expressed in the liver. Recent studies have found that CK8 was closely related to glycogen synthesis. However, the role and the underlying mechanisms of CK8 in hepatic glycogen synthesis in type 2 diabetes mellitus (T2DM) have remained to be fully elucidated. Therefore, this study aimed to investigate the effects and the underlying mechanisms of CK8 on hepatic glycogen synthesis in T2DM.Methods The T2D mouse model was constructed by high energy feed of 8-10 weeks old C57BL/6J male mice. The model was validated by OGTT test and ITT test. The liver samples of T2DM patients were collected and the expression levels of CK8, IRS1, PI3K, Akt, GSK3β, p-PI3K p-Akt and p-GSK3β were determined by Western blotting. Then, at the cellular level the murine NCTC 1469 cells were used, and to up-regulate and down-regulate the CK8 gene the overexpression plasmid was constructed and transfected and RNA interference technology was applied, and CK8, IRS1, PI3K, Akt, GSK3β, p-IRS1, p-PI3K p-Akt and p-GSK3β were detected by Western blotting. Immunohistochemistry was used to detect the level of glycogen synthase (GS) and glycogen staining experiments was performed by PAS. At the animal level, the T2D mouse model was used to up-regulate and down-regulate the CK8 gene using an adenovirus vector. The levels of CK8, PI3K, Akt, GSK3β, p-PI3K, p-Akt and p-GSK3β in rat liver were detected by Western blotting, immunohistochemistry was used to detect the level of GS and glycogen staining experiments was performed by PAS.Results The expression levels of IRS1, p-PI3K, p-Akt and p-GSK3β were significantly higher, while the expression levels of CK8 was significantly lower in control group than in the liver of T2D mice or T2DM patients. Upregulation of CK8 in murine NCTC 1469 cells treated with high-glucose medium, we found IRS1, p-IRS1, p-PI3K, p-Akt and p-GSK3β were significantly decreased, the level of GS was significantly decreased, and glycogen synthesis was inhibited compared with NCTC1469 cells transfected with empty vector. Downregulation of CK8 in murine NCTC 1469 cells murine treated with high-glucose medium, we found IRS1, p-IRS1, p-PI3K, p-Akt and p-GSK3β were significantly higher, the level of GS was significantly increased, and glycogen synthesis was promoted compared with NCTC1469 cells transfected with sh-NC. Upregulation of CK8 in the T2D mouse model, we found p-PI3K, p-Akt and p-GSK3β were significantly lower compared with T2D mouse model transfected with empty vector. The level of GS was significantly decreased, and glycogen synthesis was inhibited. Downregulation of CK8 in the T2D mouse model, we found p-PI3K, p-Akt and p-GSK3β were significantly increased, and the level of GS was significantly increased, and glycogen synthesis was promoted compared with T2D mouse model transfected with sh-NC.Conclusions Overall, this experiment provides a new molecular target for the treatment of T2DM by revealing the role of CK8 inhibiting hepatic glycogen synthesis in T2DM via regulating insulin-dependent IRS1/PI3K-Akt-GSK3β pathway. CK8 may play an important role in the pathogenesis of glycogen synthesis in T2DM.

In situ treatment of H. pylori infection in mice stomach with bioengineered probiotic bacteria releasing guided Antimicrobial peptides

Objectives: Targeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome. Results: Three AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. The probiotics were then fed to mice infected with H. pylori, both as a therapeutic and prophylactic measure, and the samples were collected using a novel gavage method and analyzed using qPCR and Illumina sequencing of the extracted stomach samples over a 10-day period. Microbiome analysis with Next-Gen sequencing also revealed a dysbiosis created by H. pylori, determined by creating a Correlation network model with the relative abundances of taxa across the samples, and this dysbiosis was palliated by the bioengineered probiotics which preserved and boosted key microbiome species and reducing the load of deleterious ones. The bioengineered probiotic also significantly improved the OTU diversity compared to antibiotics and L. lactis cloned with empty vector, with gAMP-L. lactis faring the best. Conclusions: Probiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.

Overexpression of a novel microRNA IamiR-4-3p from water spinach (Ipomoea aquatica Forsk.) increased Cd toxicity and accumulation in Arabidopsis thaliana

The function of IamiR-4-3p was investigated by using wild type (WT), transfected with empty vector pCambia1302 (CK) and IamiR-4-3p transgenic Arabidopsis in this study. The expression level of GST3 was reduced by 20% in the transgenic Arabidopsis (p35S::miR-4-3p Arabidopsis) when compared to WT, and both of its shoot and root were shorter than WT and CK. After 3 d Cd treatment, root Cd concentrations of p35S::miR-4-3p Arabidopsis was significantly higher than WT and CK, while no significant difference was found in shoot Cd concentrations. MDA and H2O2 concentrations were positively correlated with the Cd concentrations in Arabidopsis. Interestingly, even though there was no significant difference among the shoot Cd concentrations, shoot MDA and H2O2 of p35S::miR-4-3p Arabidopsis were higher than those of WT and CK, and shoot T-AOC exhibited a opposite trend. These results are clearly related to the lowered expression of GST3 by the overexpression of miR-4-3p in p35S::miR-4-3p Arabidopsis. It is suggested that the function of IamiR-4-3p is able to diminish the expression level of GST3, and is responsible to the growth dwarf, higher Cd uptake and oxidative damage but not the Cd translocation from root to shoot in Arabidopsis.

NGFR Increases the Chemosensitivity of Colorectal Cancer Cells by Enhancing the Apoptotic and Autophagic Effects of 5-fluorouracil via the Activation of S100A9

Colorectal cancer (CRC) is currently the third leading cause of cancer-related deaths worldwide, and 5-fluorouracil (5-FU)-based chemotherapies serve as important adjuvant therapies before and after surgery for CRC. However, the efficacy of CRC chemotherapy is limited by chemoresistance, and therefore the discovery of novel markers to indicate chemosensitivity is essential. Nerve growth factor receptor (NGFR), a cell surface receptor, is involved in cell death and survival. Our previous study indicated that NGFR acts as a tumor suppressor, and high expression is associated with better outcomes in patients receiving 5-FU-based adjuvant chemotherapy after surgery. The aim of this study was to investigate the effect of NGFR on the chemotherapeutic response in CRC. Chemosensitivity was investigated using DLD1 and HCT8 cells after NGFR transfection. Apoptosis was investigated by flow cytometry. Autophagy was assessed using GFP-LC3B transient transfection. Gene expression was measured using an mRNA microarray. Beclin-1 and Bcl-2 protein expressions were assessed by western blot. NGFR and S100 calcium-binding protein A9 (S100A9) expressions in CRC patients were investigated by immunohistochemistry. The results showed that the half maximal inhibitory concentration of NGFR-transfected cells was lower than that of controls in DLD1 and HCT8 cells after 5-FU treatment, and cell viability was lower than in empty-vector cells. Tumor sizes were also smaller than in empty-vector cells in vivo. The percentages of apoptotic and autophagic cells were higher in NGFR-transfected cells. NGFR elevated the expression of S100A9 after 5-FU treatment. The combination of Bcl-2 and Beclin-1 was significantly suppressed by overexpressed NGFR. Five-year overall and disease-free survival in NGFR+/S100A9+ patients was better than in NGFR−/S100A9− patients. This study’s findings suggest that NGFR may serve as a marker predicting CRC patients’ chemosensitivity.

Sympathetic Nervous System Mediates Cardiac Remodeling After Myocardial Infarction in a Circadian Disruption Model

Background: Circadian rhythms have a considerable impact on the daily physiology of the heart, and their disruption causes pathology. Several studies have revealed that circadian disruption impaired cardiac remodeling after myocardial infarction (MI); however, the underlying brain-heart mechanisms remain unknown. We aim to discuss whether circadian disruption facilitates cardiac remodeling after MI by activating sympathetic nervous system.Methods: Rats were randomly divided into three groups: Sham group (Sham), MI group (MI), and MI+ circadian disruption group (MI+Dis); rats were treated with pseudorabies virus (PRV) injections for trans-synaptic retrograde tracing; rats were randomly divided into two groups: MI+ circadian disruption + Empty Vector+ clozapine N-oxide (CNO) (Empty Vector), and MI+ circadian disruption + hM4D(Gi)+ CNO [hM4D(Gi)].Results: Circadian disruption significantly facilitated cardiac remodeling after MI with lower systolic function, larger left ventricular volume, and aggravated cardiac fibrosis. Cardiac sympathetic remodeling makers and serum norepinephrine levels were also significantly increased by circadian disruption. PRV virus-labeled neurons were identified in the superior cervical ganglion (SCG), paraventricular nucleus (PVN), and suprachiasmatic nucleus (SCN) regions. Ganglionic blockade via designer receptors exclusively activated by designer drugs (DREADD) technique suppressed the activity of sympathetic nervous system and significantly alleviated the disruption-related cardiac dysfunction.Conclusion: Circadian disruption adversely affected cardiac remodeling after MI possibly by activating sympathetic nervous system, and suppressing sympathetic activity can attenuate this disruption-related cardiac dysfunction.

The Prolactin Inducible Protein Modulates Antitumor Immune Responses and Metastasis in a Mouse Model of Triple Negative Breast Cancer

The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs in vitro compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis in vivo, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.