siRNA Transfection Research Articles

Simvastatin Enhances the Radiosensitivity of Androgen-independent Prostate Cancer Cells via Inhibition of RAD51 Expression.

Statins exert antitumor effects via various mechanisms. Additionally, the recurrence rate of prostate cancer after radiation therapy is lower in patients taking statins. This study investigated the efficacy of combination therapy with statins and irradiation in androgen-independent prostate cancer cells. PC-3 and LNCaP human prostate cancer cell lines were used in this study. We developed androgen-independent LNCaP cells (LNCaP-LA) by gradually replacing fetal bovine serum (FBS) with charcoal-stripped FBS. Microarray analysis was performed, followed by Ingenuity Pathway Analysis. Cell viability was determined using the MTS assay. Simvastatin alters gene expressions in PC-3 cells. Microarray data showed that the number of differentially expressed genes was the highest in the pathway of "Role of BRCA1 in DNA Damage Response". In the validation, the expression of RAD51, listed in "Role of BRCA1 in DNA Damage Response", decreased significantly by simvastatin in PC-3 cells. Reduction in RAD51 expression following siRNA transfection increased the cytocidal effects of X-ray therapy in PC-3 and LNCaP-LA cells. The combination of simvastatin and irradiation further inhibited cell proliferation compared with monotherapy with either therapy in PC-3 or LNCaP-LA cells. In addition, compared with X-ray monotherapy, the combination of simvastatin and irradiation further enhanced the expression of γH2AX, which is reported to be one of the accurate markers of DNA damage in PC-3 cells. Simvastatin decreased the expression of RAD51 in androgen-independent prostate cancer cells. The combination of irradiation and drugs that reduce RAD51 expression can potentially affect androgen-independent prostate cancer growth.

Downregulation of TET2 Contributes to Nasal Polypogenesis Through Hypoxia-Inducible Factor 1α-Mediated Epithelial-to-Mesenchymal Transition.

Hypoxia-inducible factor 1α (HIF1α) and Tet methylcytosine dioxygenase 2 (TET2) have been reported to mediate nasal polypogenesis through the epithelial-to-mesenchymal transition (EMT). Additionally, HIF1α can regulate the expression and function of TET2. However, the precise mechanism of how TET2 regulates the EMT through HIF1α mediation in nasal epithelial cells is still poorly understood. Nasal tissue samples were collected from patients with chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps (CRSsNP), and controls. The expression of HIF1α and TET2 was detected using Western blotting and immunohistochemistry. EMT markers (E-cadherin and vimentin) were also evaluated by immunohistochemistry. Primary human nasal epithelial cells (hNECs) were stimulated with CoCl2 to mimic hypoxia. Vitamin C (VC), a TET2 non-specific activator, and small interfering RNA (siRNA) transfection of TET2 were used to further determine the role of TET2 in hypoxia-induced EMT. Finally, reactive oxygen species (ROS) and Nrf2 were measured to explore the downstream consequences of TET2 in hypoxic hNECs. TET2 levels were lower in the nasal epithelium of CRSwNP patients and were positively correlated with E-cadherin but negatively correlated with vimentin in CRS. However, HIF1α exhibited the opposite pattern and was negatively correlated with TET2 expression. CoCl2-simulated hypoxia led to EMT and increased HIF1α in hNECs in vitro, with simultaneous downregulation of TET2 expression. Addition of VC activated TET2 expression in hNECs, but inhibited EMT and HIF1α expression. Furthermore, siRNA knockdown of TET2 contributed to the EMT in CoCl2-simulated hNECs despite the addition of VC. Finally, TET2 regulated the EMT in hypoxic hNECs through Nrf2 expression and ROS generation. TET2 was negatively correlated with HIF1α and EMT in vivo. TET2 was downregulated by HIF1α, resulting in the EMT in CoCl2-hypoxic hNECs via regulation of oxidative stress in vitro. Hence, TET2 might provide a new therapeutic approach for CRSwNP.

Effect of inhibiting PDHα1 gene expression on the metabolism of fatty liver cells

PDHα1 gene encodes catalytic subunit PDHE1α of pyruvate dehydrogenase (PDH) complex. Based on previous studies, it is hypothesized that inhibition of PDH activity prevents the entry of glycolytic pyruvate into TCA cycle, while promotes fatty acid oxidation and reduces liver triglyceride (TG) level, thereby alleviating nonalcoholic fatty liver disease (NAFLD). In this study, an in vitro model for NAFLD was established with medical fat emulsion; the most effective siRNA for PDHα1 gene was screened out by qPCR technology; the alterations in metabolism of glucose and lipid, and structure & function of mitochondria in the NAFLD cells were primarily evaluated after transfecting PDHα1 siRNA. As the results showed, after inhibiting the expression of PDHα1 gene, glucose level in culture medium was time-dependently increased, and LDH activity in the cells was moderately elevated after 24 h of transfection and then returned to the normal level after 48 h; intracellular TG level was decreased while LPS activity was increased in a time-dependent manner; no significant change in mitochondrial structure was observed with or without siRNA transfection, and ATP content was obviously reduced after 24 h of transfection, followed by restoration after 48 h. It can be concluded that inhibiting PDHα1 gene in fatty liver cells enhances lipid degradation, and represses the utilization of glucose to an extent, thus reducing TG level without impacting energy generation required for cell survival.

Downregulation of JAM3 occurs in cholangiocarcinoma by hypermethylation: A potential molecular marker for diagnosis and prognosis.

Junctional adhesion molecular 3 (JAM3) is downregulated by hypermethylation in cancers but is unclear in cholangiocarcinoma. The JAM3 expression level was checked in cholangiocarcinoma cell lines and tissues. Methylated JAM3 was detected in cell lines, tissues and plasma cell-free DNAs (cfDNA). The roles of JAM3 in cholangiocarcinoma were studied by transfection of siRNA and pCMV3-JAM3. The survival analysis was based on the Gene Set Cancer Analysis (GSCA) database. JAM3 was downregulated in HCCC-9810 and HuCCT1 cell lines and tissues by hypermethylation. Methylated JAM3 was detected in cfDNAs with 53.3% sensitivity and 96.6% specificity. Transfection of pCMV3-JAM3 into HCCC-9810 and HuCCT1 induced apoptosis and suppressed cell proliferation, migration and invasion. The depletion of JAM3 in RBE cells using siRNA decreased apoptosis and increased cell proliferation, migration and invasion. Hypermethylation of JAM3 was associated with tumour differentiation, metastasis and TNM stage. Downregulation and hypermethylation of JAM3 were related to poor progression-free survival. Junctional adhesion molecular 3 may function as a tumour suppressor in cholangiocarcinoma. Methylated JAM3 DNA may represent a non-invasive molecular marker for the early detection of cholangiocarcinoma and prognosis.

Inhibition of Drp1 orchestrates the responsiveness of breast cancer cells to paclitaxel but insignificantly relieves paclitaxel-related ovarian damage in mice

Chemoresistance and chemotherapy-related ovarian damage are well-reported in breast cancer (BC) young patients. Herein, the inhibition of the mitochondrial fission was invested to explore its chemosensitizing role in Paclitaxel (PTX)-resistant cells, and its ability to restore the ovarian integrity in mice receiving PTX or cisplatin chemotherapy. To establish these aims, PTX-resistance was generated in BC cells, which were treated with PTX in combination with Drp1 deficiency, via mdivi-1, or Drp1-specific siRNA transfection. Furthermore, the alterations in the ovarian structure and the endocrine-related hormones were explored in mice receiving repetitive doses of PTX or cisplatin. We found that combining PTX with mdivi-1 improved cell responsiveness to PTX, induced apoptosis- and autophagy-mediated cell death, and relieved cellular oxidative stress. Additionally, the expression of PCNA1 and cyclin B1 genes were downregulated, meanwhile, p53, p21, and mitochondrial fusion proteins (Mfu1&Mfu2) were increased. The in vivo investigations in mice demonstrated that PTX induced gonadotoxic damage similar to cisplatin, whereas dual treatment of mice with PTX+ mdivi-1 failed to restore their normal follicular count and the circulating levels of E2 and AMH hormones. These results suggested that combining Drp1 inhibition with PTX resensitized breast cancer cells to PTX but failed to offer enough protection against chemotherapy-related gonadotoxicity.

TRPM2 exacerbates airway inflammation by regulating oxidized-CaMKⅡ in allergic asthma

BackgroundAirway epithelial cells play important roles in allergic asthma. Transient receptor potential melastatin-related 2 (TRPM2) and oxidized Ca2+/calmodulin-dependent protein kinase Ⅱ (ox-CaMKⅡ) participate in the airway inflammation. This study aimed to analyze the effects of TRPM2 on ox-CaMKⅡ in the airway epithelial cells during allergic asthma. MethodsBEAS-2B cells were treated with different dose of IL-13 (0, 5, 10, 20 ng/mL) for 24 h to analyze the changes of TRPM2 and ox-CaMKⅡ protein. Cells expressing different level of TRPM2 were obtained by transfection of TRPM2 siRNA or TRPM2-short cDNA. The transfected cells were treated with 10 ng/mL of IL-13 to analyze the effects of TRPM2 on the ox-CaMKⅡ. A CaMKⅡ inhibitor KN-93 was used to confirm the effects of TRPM2 on levels of ox-CaMKⅡ, p-MEK and p-ERK in the IL-13-treated BEAS-2B cells. Wild-type (WT) mice and TRPM2-knockout (TRPM2−/−) mice were induced by ovalbumin (OVA) to compare the differences of inflammation, levels of ox-CaMKII, p-MEK and p-ERK in airways. ResultsCell viability was clearly decreased by the 20 ng/mL of IL-13. The levels of TRPM2 and ox-CaMKII protein in cells were increased with increasing doses of IL-13. Transfection of TRPM2 siRNA or TRPM2-short cDNA respectively decreased or increased the levels of ox-CaMKⅡ in the IL-13-stimulated cells. The results of KN-93 treatment were similar to the results of TRPM2 siRNA transfection, that the levels of ox-CaMKⅡ, p-MEK and p-ERK were significantly decreased in the IL-13-treated cells. Compared with the OVA-induced WT mice, levels of inflammation, ox-CaMKⅡ, p-MEK and p-ERK in the airways were significantly weakened in the OVA-induced TRPM2−/− mice. ConclusionsTRPM2 plays a vital role in regulating ox-CaMKⅡ in airway epithelial cells during allergic asthma.

Identification of arnicolide C as a novel chemosensitizer to suppress mTOR/E2F1/FANCD2 axis in non-small cell lung cancer.

The mammalian target of rapamycin (mTOR) pathway plays critical roles in intrinsic chemoresistance by regulating Fanconi anaemia complementation group D2 (FANCD2) expression. However, the mechanisms by which mTOR regulates FANCD2 expression and related inhibitors are not clearly elucidated. Extracts of Centipeda minima (C.minima) showed promising chemosensitizing effects by inhibiting FANCD2 activity. Here, we have aimed to identify the bioactive chemosensitizer in C.minima extracts and elucidate its underlying mechanism. The chemosensitizing effects of arnicolide C (ArC), a bioactive compound in C.minima, on non-small cell lung cancer (NSCLC) were investigated using immunoblotting, immunofluorescence, flow cytometry, the comet assay, small interfering RNA (siRNA) transfection and animal models. The online SynergyFinder software was used to determine the synergistic effects of ArC and chemotherapeutic drugs on NSCLC cells. ArC had synergistic cytotoxic effects with DNA cross-linking drugs such as cisplatin and mitomycin C in NSCLC cells. ArC treatment markedly decreased FANCD2 expression in NSCLC cells, thus attenuating cisplatin-induced FANCD2 nuclear foci formation, leading to DNA damage and apoptosis. ArC inhibited the mTOR pathway and attenuated mTOR-mediated expression of E2F1, a critical transcription factor of FANCD2. Co-administration of ArC and cisplatin exerted synergistic anticancer effects in the A549 xenograft mouse model by suppressing mTOR/FANCD2 signalling in tumour tissues. ArC suppressed DNA cross-linking drug-induced DNA damage response by inhibiting the mTOR/E2F1/FANCD2 signalling axis, serving as a chemosensitizing agent. This provides insight into the anticancer mechanisms of ArC and offers a potential combinatorial anticancer therapeutic strategy.

Plasticizer acetyl triethyl citrate (ATEC) induces lipogenesis and obesity

Environmental chemicals, such as plasticizers, have been linked to increased rates of obesity, according to epidemiological studies. Acetyl triethyl citrate (ATEC) is a plasticizer that is commonly utilized in pharmaceutical products and food packaging as a non-phthalate alternative. Due to its direct contact with the human body and high leakage rate from the polymers, assessment of the potential risk of ATEC exposure at environmentally relevant low doses to human health is needed. Male C57BL/6 J mice were fed diets containing ATEC at doses of either 0.1 or 10 μg/kg per day in a period of 12 weeks to mimic the real exposure environment. The findings suggest that in C57BL/6 J mice, ATEC exposure resulted in increased body weight gain, body fat percentage, and benign hepatocytes, as well as adipocyte size. Consistent with in vivo models, ATEC treatment obviously stimulated the increase of intracellular lipid load in both mouse and human hepatocytes. Mechanically, ATEC induced the transcriptional expression of genes involved in de novo lipogenesis and lipid uptake. Using both enzyme inhibitor and small interfering RNA (siRNA) transfection, we found that stearoyl-coenzyme A desaturase 1 (SCD1) played a significant role in ATEC-induced intracellular lipid accumulation. This study for the first time provided initial evidence suggesting the obesogenic and fatty liver-inducing effect of ATEC at low doses near human exposure levels, and ATEC might be a potential environmental obesogen and its effect on human health need to be further evaluated.

A Synthetic Steroid 5α-Androst-3β, 5, 6β-triol Alleviates Radiation-Induced Brain Injury in Mice via Inhibiting GBP5/NF-κB/NLRP3 Signal Axis.

Radiotherapy for head and neck tumors can lead to a severe complication known as radiation-induced brain injury (RIBI). However, the underlying mechanism of RIBI development remains unclear, and limited prevention and treatment options are available. Neuroactive steroids have shown potential in treating neurological disorders. 5α-Androst-3β, 5, 6β-triol (TRIOL), a synthetic neuroprotective steroid, holds promise as a treatment candidate for RIBI patients. However, the neuroprotective effects and underlying mechanism of TRIOL on RIBI treatment are yet to be elucidated. In the present study, our findings demonstrate TRIOL's potential as a neuroprotective agent against RIBI. In gamma knife irradiation mouse model, TRIOL treatment significantly reduced brain necrosis volume, microglial activation, and neuronal loss. RNA-sequencing, immunofluorescence, real-time quantitative polymerase chain reaction, siRNA transfection, and western blotting techniques revealed that TRIOL effectively decreased microglial activation, proinflammatory cytokine release, neuron loss, and guanylate-binding protein 5 (GBP5) expression, along with its downstream signaling pathways NF-κB and NLRP3 activation in vitro. In summary, TRIOL effectively alleviate RIBI by inhibiting the GBP5/NF-κB/NLRP3 signal axis, reducing microglia activation and pro-inflammation cytokines release, rescuing neuron loss. This study highlights the potential of TRIOL as a novel and promising therapy drug for RIBI treatment.

MiR-99b-3p/Mmp13 axis regulates NLRP3 inflammasome-dependent microglial pyroptosis and alleviates neuropathic pain via the promotion of autophagy

BackgroundNeuropathic pain significantly impairs quality of life, and effective interventions are limited. NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated microglial pyroptosis and the subsequent proinflammatory cytokine production are critical in exacerbating pain. Considering microglial pyroptosis as a potential target for developing specific analgesic interventions for neuropathic pain, our study investigated the pathogenesis and therapeutic targets in this condition. MethodsIn vitro experiments involved the co-culture of the immortalized BV-2 microglia cell line with lipopolysaccharide (LPS) to induce microglial pyroptosis. Differentially expressed microRNAs (miRNAs) were identified using high-throughput sequencing analysis. The downstream target genes of these miRNAs were determined through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the downstream target genes, combined with miRNAs, were predicted and verified through dual luciferase reporter gene assays. In vivo experiments were conducted to construct a chronic constriction injury (CCI) neuropathic pain model in rats and evaluate the analgesic effects of intrathecal injection of an adeno-associated virus vector (AAV) carrying miR-99b-3p. Gene expression was modulated through mimic or siRNA transfection. Western blot analysis assessed the expression of microglial pyroptosis and autophagy-related proteins, whereas RT-qPCR measured changes in proinflammatory cytokines expression. ResultsLPS-stimulated up-regulation of proinflammatory cytokines in microglia, accompanied by NLRP3-dependent pyroptosis, including increased NLRP3, GSDMD-N, Caspase1-p20, and mature-IL-1β expression. High-throughput sequencing analysis revealed 16 upregulated and 10 downregulated miRNAs in LPS-stimulated microglia, with miR-99b-3p being the most downregulated. KEGG analysis revealed that the target genes of these miRNAs are primarily enriched in calcium, FoxO, and mitogen-activated protein kinase (MAPK) signal pathways. Furthermore, overexpression of miR-99b-3p through mimic transfection significantly inhibited the inflammatory response and NLRP3-mediated pyroptosis by promoting autophagy levels in activated microglia. In addition, we predicted that the 3′ untranslated region (UTR) of matrix metalloproteinase-13 (Mmp13) could bind to miR-99b-3p, and knockdown of Mmp13 expression through siRNA transfection similarly ameliorated enhanced proinflammatory cytokines expression and microglial pyroptosis by enhancing autophagy. In vivo, Mmp13 was co-localized with spinal dorsal horn microglia and was suppressed by intrathecal injection of the AAV-miR-99b-3p vector. Moreover, overpressed miR-99b-3p alleviated CCI-induced mechanical allodynia and neuroinflammation while suppressing pyroptosis by enhancing autophagy in the spinal cord of CCI rats. ConclusionmiR-99b-3p exerts analgesic effects on neuropathic pain by targeting Mmp13. These antinociceptive effects are, at least in part, attributed to the promotion of autophagy, thereby inhibiting neuroinflammation and NLRP3-mediated pyroptosis in activated microglia.

SiRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-β, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-β, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-β levels compared to mDCs that were not transfected. The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

Aerobic exercise improves BKCa channel-mediated vasodilation in diabetic vascular smooth muscle via AMPK/Nrf2/HO-1 pathway.

This study aims to investigate the effect of aerobic exercise training on BKCa channel in diabetic vascular smooth muscle and explore the underlying mechanism. Control m/m mice and diabetic db/db mice were randomly assigned to sedentary groups (W and D) and exercise training groups (WE and DE). Mice in exercise groups underwent training sessions lasting for 12weeks, with a speed of 12m/min for 60min, five times per week. The thoracic aorta was extracted isolated and examined for measurement of vascular structure, global levels of reactive oxygen species (ROS), vasodilation, and protein expression. Rat thoracic aorta vascular smooth muscle cells (USMCs) were cultured, and siRNA transfection was conducted to detect whether AMPK contributed to the regulation. ROS level and protein expression were measured. Compared with control mice, diabetic mice had a larger vascular medium thickness, impaired BKCa-mediated vasodilation, a higher level of ROS, and a lower expression of BKCa α, BKCa β1, Nrf2, p-Nrf2, p-Nrf2/Nrf2, HO-1, and p-AMPK/AMPK. Exercise training increased the expression of BKCa α, Nrf2, p-Nrf2, p-Nrf2/Nrf2, HO-1, and p-AMPK/AMPK. AMPK deletion led to lower ROS levels and expression of BKCa α, β1, Nrf2, and HO-1 in USMCs cultured in high glucose conditions. BKCa channel protein expression reduction in VSMCs contributes to vasodilation and vascular remodeling dysfunction in diabetes mellitus. Aerobic exercise can promote the expression of BKCa channel and improve BKCa-mediated vascular dysfunction in diabetic VSMCs through AMPK/Nrf2/HO-1 signaling pathway.

An AMPK agonist suppresses the progress of colorectal cancer by regulating the polarization of TAM to M1 through inhibition of HIF-1α and mTOR signal pathway.

This study aimed to evaluate the impact of an adenosine monophosphate-activated protein kinase (AMPK) agonist, metformin (MET), on the antitumor effects of macrophages and to determine the underlying mechanism involved in the process. M0 macrophages were derived from phorbol-12-myristate-13-acetate-stimulated THP-1 cells. The levels of tumor necrosis factor-alpha (TNF-α) and human leukocyte antigen-DR (HLA-DR) were decreased in macrophages incubated with HCT116 cells, whereas those of arginase-1 (Arg-1), CD163, and CD206 were elevated; these effects were reversed by MET. The transfection of small interfering (si) RNA abrogated the influence of MET on the expression of the M1/M2 macrophage biomarkers. MET significantly suppressed the proliferation and migration abilities of HCT116 cells incubated with M0 macrophages; these actions were reversed by siRNA transfection against AMPK. The hypoxia-inducible factor 1-alpha (HIF-1α), phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target of rapamycin (p-mTOR) levels were reduced by the introduction of MET and promoted by siRNA transfection against AMPK. In addition, the levels of HIF-1α, p-AKT, and p-mTOR suppressed by MET were markedly increased following the transfection of siRNA against AMPK. These findings indicate that MET can repress the progression of colorectal cancer by transforming tumor-associated macrophages to the M1phenotype via inhibition of the HIF-1α and mTOR signaling pathways.

Lnc-ANRIL modulates the immune response associated with NF-κB pathway in LPS-stimulated bovine mammary epithelial cells.

The antisense noncoding RNA in the INK4 locus (ANRIL) has been confirmed related to multiple disease progression, but the role and exact mechanisms of lnc-ANRIL in lipopolysaccharide (LPS)-induced inflammation of bovine mammary epithelial cells (MAC-T) remain unclear. This manuscript focused on expounding the functional role of lnc-ANRIL through experiments performed in MAC-T. At the in vitro level, we established a Bovine mammary epithelial cell (BMEC) cell model of mastitis by LPS treatment. Transfection of siRNA was examined by immunofluorescence localization and RT-qPCR. CCK8, clonogenic assay and EdU were used to detect the proliferation ability of the cells. Cell cycle and apoptosis were detected by flow cytometry and Western blot. The levels of inflammatory factors and oxidative stress markers were detected by ELISA kits. Cell Counting Kit-8, colony formation, and 5-ethynyl-20-deoxyuridine were adopted and the data illustrated that LPS could significantly suppress the cell proliferation, while knockdown of lnc-ANRIL expression obviously promoted MAC-T cell proliferation compared with LPS or LPS + si-NC group. Flow cytometry analysis demonstrated that lnc-ANRIL could induce MAC-T cell apoptosis. In addition, downregulation of lnc-ANRIL affected LPS-induced immune response by regulating inflammatory factor expressions and modulating the nuclear factor kappa B (NF-κB) axis in MAC-T cells. Our results suggest that lnc-ANRIL is involved in the regulation of cell proliferation, cell cycle, and cell apoptosis of MAC-T cells, and plays an important role in the inflammatory and immune response of MAC-T cells through the regulation of the NF-κB pathway, proposing new therapeutic strategies for the treatment of innate immune response-related disease such as bovine mastitis.

Intravenous Injection of Na Ions Aggravates Ang II-Induced Hypertension-Related Vascular Endothelial Injury by Increasing Transmembrane Osmotic Pressure.

Extracellular protein nanoparticles (PNs) and ions perform synergistical functions in the control of transmembrane osmotic pressure (OP) under isotonic conditions. Intravenous injection may disrupt the ion balance and alter PN levels in blood plasma, changing transmembrane OP and damaging vascular endothelial cells. Na ions were injected into AngII-induced HUVECs to simulate cell injury in vitro, and tail vein infusion of Na ions into hypertensive rats was performed to assess vascular damage. Optical measurements using an intermediate filament (IF) tension probe were conducted to detect indicators related to transmembrane OP. Immunofluorescence, Western blotting and small interfering RNA (siRNA) transfection were employed to investigate inflammasomes and the relationship between Abl2 and inflammation. Electrolyte injections with sodium ions (but not glucose and hydroxyethyl starch) induced the production of ASC and NLRP3 inflammasomes in Ang II-induced HUVECs; this in turn resulted in the disorder of calcium signals, and changes in transmembrane OP and cell permeability. Moreover, injection of Na ions into Ang II-induced HUVECs activated the mechanosensitive protein Abl2, involved in inflammation-induced transmembrane OP changes. A drug combination was identified that could induce OP recovery and block hyperpermeability induced by cytoplasmic inflammatory corpuscles in vivo and in vitro. Changes in extracellular PNs and ions following chemical stimuli (Ang II) participate in the regulation of transmembrane OP. Furthermore, injection of Na ions causes vascular endothelial injury in Ang II-induced cells in vitro and hypertension rats in vivo, suggesting it is not safe for hypertensive patients, and we propose a new drug combination as a solution.

Effect of NFATc2- and Sp1-mediated TNFalpha Regulation on the Proliferation and Migration Behavior of Pancreatic Cancer Cells.

One in two people will develop a tumor during their lifetime. Adenocarcinoma of the pancreas is one of the most aggressive types of cancer in humans with very poor long-term survival. A central role in the carcinogenesis of pancreatic cancer has been attributed to NFAT transcription factors. Previous studies have identified the transcription factor Sp1 as a binding partner of NFATc2 in pancreatic cancer. Using expression profile analysis, our group was able to identify the tumor necrosis factor TNFalpha as a target gene of the interaction between NFATc2 and Sp1. The present study investigated the effect of TNFalpha over-expression via the transcription factors NFATc2 and Sp1 on the pancreatic cancer cell lines PaTu 8988t and PANC-1. Transient transfection of NFATc2, Sp1, and TNFalpha siRNAs and their effects on the expression were investigated with immunoblot. Cell proliferation was measured with the ELISA BrdU assay. Cell migration was assayed with a Cell Migration Assay Kit using a Boyden chamber. Inhibition of the transfection factors NFATc2, Sp1, or TNFalpha by siRNA significantly inhibited proliferation, which was exacerbated when using the combination of NFATc2 and Sp1. TNFalpha was able to counterbalance this effect. In contrast to proliferation, migration of pancreatic cancer cells was increased by inhibiting these transfection factors. Tumor progression is strongly influenced by transcriptional changes in signaling cascades and oncogene mutations as well as by changes in tumor suppressor genes. Further studies are needed to understand the underlying mechanisms of these processes.

Zebrafish spop promotes ubiquitination and degradation of mavs to suppress antiviral response via the lysosomal pathway

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathways are required to be tightly controlled to initiate host innate immune responses. Fish mitochondrial antiviral signaling (mavs) is a key determinant in the RLR pathway, and its ubiquitination is associated with mavs activation. Here, we identified the zebrafish E3 ubiquitin ligase Speckle-type BTB–POZ protein (spop) negatively regulates mavs-mediated the type I interferon (IFN) responses. Consistently, overexpression of zebrafish spop repressed the activity of IFN promoter and reduced host ifn transcription, whereas knockdown spop by small interfering RNA (siRNA) transfection had the opposite effects. Accordingly, overexpression of spop dampened the cellular antiviral responses triggered by spring viremia of carp virus (SVCV). A functional domain assay revealed that the N-terminal substrate-binding MATH domain regions of spop were necessary for IFN suppression. Further assays indicated that spop interacts with mavs through the C-terminal transmembrane (TM) domain of mavs. Moreover, zebrafish spop selectively promotes K48-linked polyubiquitination and degradation of mavs through the lysosomal pathway to suppress IFN expression. Our findings unearth a post-translational mechanism by which mavs is regulated and reveal a role for spop in inhibiting antiviral innate responses.

Primary cilium participates in radiation-induced bystander effects through TGF-β1 signaling.

Many studies have indicated that tumor growth factor-beta (TGF-β) signaling mediates radiation-induced bystander effects (RIBEs). The primary cilium (PC) coordinates several signaling pathways including TGF-β signaling to regulate diverse cellular processes. But whether the PC participates in TGF-β induced RIBEs remains unclear.The cellular levels of TGF-β1 were detected by western blot analysis and the secretion of TGF-β1 was measured by ELISA kit. The ciliogenesis was altered by CytoD treatment, STIL siRNA transfection, IFT88 siRNA transfection, or KIF3a siRNA transfection, separately, and was detected by western blot analysis and immunofluorescence staining. G0 /G1 phase cells were arrested by serum starvation and S phase cells were induced by double thymidine block. The TGF-β1 signaling was interfered by LY2109761, a TGF-β receptor 1 (TβR1) inhibitor, or TGF-β1 neutral antibody. The DNA damages were induced by TGF-β1 or radiated conditional medium (RCM) from irradiated cells and were reflected by p21 expression, 53BP1 foci, and γH2AX foci.Compared with unirradiated control, both A549 and Beas-2B cells expressed and secreted more TGF-β1 after carbon ion beam or X-ray irradiation. RCM collected from irradiated cells or TGF-β1 treatment caused an increase of DNA damage in cocultured unirradiated Beas-2B cells while blockage of TGF-β signaling by TβR1 inhibitor or TGF-β1 neutral antibody alleviates this phenomenon. IFT88 siRNA or KIF3a siRNA impaired PC formation resulted in an aggravated DNA damage in bystander cells, while elevated PC formation by CytoD or STIL siRNA resulted in a decrease of DNA damage. Furthermore, TGF-β1 induced more DNA damages in S phases cells which showed lower PC formation rate and less DNA damages in G0 /G1 phase cells which showed higher PC formation rate.This study demonstrates the particular role of primary cilia during RCM induced DNA damages through TGF-β1 signaling restriction and thereby provides a functional link between primary cilia and RIBEs.

Transient receptor potential ankyrin 1 channels in the bladder mediate low temperature elicited bladder overactivity in rats.

This study aimed to investigate whether pathways involving transient receptor potential ankyrin 1 (TRPA1) channels in the urinary bladder mediate the bladder overactivity elicited by exposure to a low temperature in rats. At postnatal week 10, female Sprague-Dawley (SD) rats were intraperitoneally injected with the TRPA1 channel antagonist, HC030031, at room temperature (RT) and subsequently exposed to low temperature (LT). Bladder specimens treated with HC030031 were evaluated for contractions through cumulative addition of the TRPA1 channel agonist trans-cinnamaldehyde. Two days before cystometric investigation, small interfering RNA (siRNA) targeting TRPA1 was transfected into urinary bladders. Then, cystometric investigations were performed on rats subjected to TRPA1 siRNA transfection at both RT and LT. Expression of TRPA1 channels in the urinary bladder was assessed through immunohistochemistry and real-time reverse transcription-polymerase chain reaction. At RT, micturition patterns were unaffected by HC030031 treatment. However, upon exposure to LT, rats treated with HC030031 exhibited a reduction of LT-elicited bladder overactivity, as evidenced by inhibited decreases in voiding interval, micturition volume, and bladder capacity. Additionally, HC030031 inhibited trans-cinnamaldehyde-induced contractions. Immunohistochemical analysis showed the presence of TRPA1 channels in the urinary bladder. Notably, rats with TRPA1 siRNA-transfected bladders could partially inhibit bladder overactivity during LT exposure. These findings indicate that pathways involving TRPA1 channels expressed in the urinary bladder could mediate the LT-elicited bladder overactivity.

Cationic amino-acid functionalized polymethacrylamide vectors for siRNA transfection based on modification of poly(2-isopropenyl-2-oxazoline)

Poly(2-isopropenyl-2-oxazoline) (PiPOx) is a functional polymer showing great potential for the development of smart biomaterials. The straightforward synthesis and post-polymerization functionalization of PiPOx offers many opportunities for tailoring the properties of the polymer towards biomaterials. In this study we report for the first time PiPOx-based cationic charged polymethacrylamides with amino acid side chains that can complex siRNA and promote transfection in vitro. Therefore, PiPOx was fully modified via ring opening addition reactions with the carboxylic acid groups of a series of N-Boc-L-amino acids and their reaction kinetics were investigated. Based on the determined kinetic constants, another series of PiPOx-based copolymers with balanced hydrophilic/hydrophobic content of N-Boc-L-amino acids were obtained via one-pot modification reaction with two different N-Boc-L-amino acids. The N-Boc protected homopolymers and related copolymers were deprotected to obtain (co)polymers with the targeted side chain cationic charged units. The (co)polymers' structures were fully investigated via FT-IR and 1H NMR spectroscopy, size exclusion chromatography (SEC), and TGA–DSC–MS analysis. The polarimetry measurements revealed that the homopolymers retain their chiroptical properties after post-modification, and a sign inversion is noticed from (L) N-Boc-protected analogues to (D) for the TFA cationic charged homopolymers. Generally, cationically charged homopolymers with hydrophilic amino acids on the side chain showed efficient complexation of siRNA, but poor transfection while cationic copolymers having both tryptophan and valine or proline side chains revealed moderate siRNA binding, high transfection efficiency (> 90% of the cells) and potent gene silencing with IC50 values down to 5.5 nM. Particularly, these cationic copolymers showed higher gene silencing potency as compared to the commercial JetPRIME® reference, without reducing cell viability in the concentration range used for transfection, making this a very interesting system for in vitro siRNA transfection.