Short Hairpin RNA Transfection Research Articles

The mGluR5-mediated Arc activation protects against experimental traumatic brain injury in rats.

Traumatic brain injury (TBI) is a complex pathophysiological process, and increasing attention has been paid to the important role of post-synaptic density (PSD) proteins, such as glutamate receptors. Our previous study showed that a PSD protein Arc/Arg3.1 (Arc) regulates endoplasmic reticulum (ER) stress and neuronal necroptosis in traumatic injury invitro. In this study, we investigated the expression, regulation and biological function of Arc in both invivo and invitro experimental TBI models. Traumatic neuronal injury (TNI) induced a temporal upregulation of Arc in cortical neurons, while TBI resulted in sustained increase in Arc expression up to 24 h in rats. The increased expression of Arc was mediated by the activity of metabotropic glutamate receptor 5 (mGluR5), but not dependent on the intracellular calcium (Ca2+) release. By using inhibitors and antagonists, we found that TNI regulates Arc expression via Gq protein and protein turnover. In addition, overexpression of Arc protects against TBI-induced neuronal injury and motor dysfunction both invivo and invitro, whereas the long-term cognitive function was not altered. To determine the role of Arc in mGluR5-induced protection, lentivirus-mediated short hairpin RNA (shRNA) transfection was performed to knockdown Arc expression. The mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG)-induced protection against TBI was partially prevented by Arc knockdown. Furthermore, the CHPG-induced attenuation of Ca2+ influx after TNI was dependent on Arc activation and followed regulation of AMPAR subunits. The results of Co-IP and Ca2+ imaging showed that the Arc-Homer1 interaction contributes to the CHPG-induced regulation of intracellular Ca2+ release. In summary, the present data indicate that the mGluR5-mediated Arc activation is a protective mechanism that attenuates neurotoxicity following TBI through the regulation of intracellular Ca2+ hemostasis. The AMPAR-associated Ca2+ influx and ER Ca2+ release induced by Homer1-IP3R pathway might be involved in this protection.

Transcription factor DDIT3 is a potential driver in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and aggressive tumor that affects the digestive tract, leading to high mortality and poor survival rates. The purpose of the present study was to evaluate the expression levels of DNA damage-inducible transcript 3 (DDIT3) in pancreatic cancer and to investigate its effects in in vitro and in vivo experiments. Bioinformatics analysis indicated that DDIT3 expression was higher in pancreatic cancer tumor tissues and associated with a poor prognosis. Positive or strong positive DDIT3 expression was observed in PDAC, and no or weak expression was observed in normal pancreatic tissues. It was also highly expressed in PDAC cells, while being expressed at lower levels in normal pancreatic ductal epithelial cells. Transfection of short hairpin RNA targeting the DDIT3 gene reduced the proliferation, migration and invasion of PANC-1 cells. In vivo, in an in situ implantation tumor model with Pan02 cells, the size and weight of the tumors were reduced in the DDIT3 knockdown Pan02 cell-implanted group. These data suggested that DDIT3 represents a novel predictive biomarker for the potential treatment of patients presenting with PDAC.

The activation of adenosine monophosphate-activated protein kinase inhibits the migration of tongue squamous cell carcinoma cells by targeting Claudin-1 via epithelial-mesenchymal transition.

The role of Claudin-1 in tongue squamous cell carcinoma (TSCC) metastasis needs further clarification, particularly its impact on cell migration. Herein, our study aims to investigate the role of Claudin-1 in TSCC cell migration and its underlying mechanisms. 36 TSCC tissue samples underwent immunohistochemical staining for Claudin-1. Western blotting and immunofluorescence analyses were conducted to evaluate Claudin-1 expression and distribution in TSCC cells. Claudin-1 knockdown cell lines were established using short hairpin RNA transfection. Migration effects were assessed through wound healing assays. Furthermore, the expression of EMT-associated molecules was measured via western blotting. Claudin-1 expression decreased as TSCC malignancy increased. Adenosine monophosphate-activated protein kinase (AMPK) activation led to increased Claudin-1 expression and membrane translocation, inhibiting TSCC cell migration and epithelial-mesenchymal transition (EMT). Conversely, Claudin-1 knockdown reversed these inhibitory effects on migration and EMT caused by AMPK activation. Our results indicated that AMPK activation suppresses TSCC cell migration by targeting Claudin-1 and EMT pathways.

A novel role of AIM2 inflammasome-mediated pyroptosis in radiofrequency ablation of hepatocellular carcinoma

Introduction and objectivesThe absence of melanoma 2 (AIM2) protein triggers the activation of the inflammasome cascade. It is unclear whether AIM2 plays a role in hepatocellular carcinoma (HCC) and radiofrequency ablation (RFA), which uses radiofrequency waves to treat tumors. In this study, we investigated if RFA could induce pyroptosis, also called cell inflammatory necrosis, in HCC through AIM2-inflammasome signaling in vivo and in vitro. Materials and methodsBALB/c nude mice were used to generate HepG2 or SMMC-7721 cell-derived tumor xenografts. HCC cells with knockdown or overexpression of AIM2 were created using short hairpin RNA (shRNA) and expression vector transfection, respectively, for functional and mechanistic studies. Downstream effects were examined using flow cytometry, qRT-PCR, ELISAs, and other molecular assays. ResultsRFA significantly suppressed tumor growth in HCC cell xenografts. Flow cytometry analysis revealed that RFA could induce pyroptosis. Furthermore, AIM2, NLRP3, caspase-1, γ-H2AX, and DNA-PKc had significantly greater expression levels in liver tissues from mice treated with RFA compared with those of the controls. Additionally, interleukin (IL)-1β and IL-18 expression levels were significantly higher in the HCC cell-derived xenograft mice treated with RFA compared with those without RFA. Notably, a significantly greater effect was achieved in the RFA complete ablation group versus the partial ablation group. Knockdown or overexpression of AIM2 in HCC cells demonstrated that AIM2 exerted a role in RFA-induced pyroptosis. ConclusionsRFA can suppress HCC tumor growth by inducing pyroptosis via AIM2. Therefore, therapeutically intervening with AIM2-mediated inflammasome signaling may help improve RFA treatment outcomes for HCC patients.

TXNIP knockdown protects rats against bupivacaine-induced spinal neurotoxicity via the inhibition of oxidative stress and apoptosis

Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting protein (TXNIP) is a member of the α-arrestin protein superfamily that binds covalently to thioredoxin (TRX) to inhibit its function, leading to increased oxidative stress and activation of apoptosis. The role of TXNIP in BUP-induced oxidative stress and apoptosis remains to be elucidated. In this context, the present study aimed to explore the effects of TXNIP knockdown on BUP-induced oxidative stress and apoptosis in the spinal cord of rats and in PC12 cells through the transfection of adeno-associated virus-TXNIP short hairpin RNA (AAV-TXNIP shRNA) and siRNA-TXNIP, respectively. In vivo, a rat model of spinal neurotoxicity was established by intrathecally injecting rats with BUP. The BUP + TXNIP shRNA and the BUP + Control shRNA groups of rats were injected with an AAV carrying the TXNIP shRNA and the Control shRNA, respectively, into the subarachnoid space four weeks prior to BUP treatment. The Basso, Beattie & Bresnahan (BBB) locomotor rating score, % MPE of TFL, H&E staining, and Nissl staining analyses were conducted. In vitro, 0.8 mM BUP was determined by CCK-8 assay to establish a cytotoxicity model in PC12 cells. Transfection with siRNA-TXNIP was carried out to suppress TXNIP expression prior to exposing PC12 cells to BUP. The results revealed that BUP effectively induced neurological behavioral dysfunction and neuronal damage and death in the spinal cord of the rats. Similarly, BUP triggered cytotoxicity and apoptosis in PC12 cells. In addition, treated with BUP both in vitro and in vivo exhibited upregulated TXNIP expression and increased oxidative stress and apoptosis. Interestingly, TXNIP knockdown in the spinal cord of rats through transfection of AAV-TXNIP shRNA exerted a protective effect against BUP-induced spinal neurotoxicity by ameliorating behavioral and histological outcomes and promoting the survival of spinal cord neurons. Similarly, transfection with siRNA-TXNIP mitigated BUP-induced cytotoxicity in PC12 cells. In addition, TXNIP knockdown mitigated the upregulation of ROS, MDA, Bax, and cleaved caspase-3 and restored the downregulation of GSH, SOD, CAT, GPX4, and Bcl2 induced upon BUP exposure. These findings suggested that TXNIP knockdown protected against BUP-induced spinal neurotoxicity by suppressing oxidative stress and apoptosis. In summary, TXNIP could be a central signaling hub that positively regulates oxidative stress and apoptosis during neuronal damage, which renders TXNIP a promising target for treatment strategies against BUP-induced spinal neurotoxicity.

Myricetin, a natural inhibitor of CD147, increases sensitivity of cisplatin in ovarian cancer

ABSTRACT Background Ovarian cancer (OC) is the most lethal gynecological tumor, but it currently lacks effective therapeutic targets. CD147, which is overexpressed in OC, plays a crucial role in promoting malignant progression and is associated with poor prognosis in patients. Therefore, CD147 has been identified as a potential therapeutic target. However, there is a limited amount of research on the development of CD147 inhibitors. Methods Surface plasmon resonance (SPR) assay and virtual molecular docking analysis were performed to identify potential natural compounds targeting CD147. The anti‑tumor effects of myricetin were evaluated using various assays, including CCK8, Alkaline comet, immunofluorescence and xenograft mouse models. The underlying mechanism was investigated through western blot analysis and lentivirus short hairpin RNA (LV-shRNA) transfection. Results Myricetin, a flavonoid commonly found in plants, was discovered to be a potent inhibitor of CD147. Our findings demonstrated that myricetin exhibited a strong affinity for CD147 and down-regulated the protein level of CD147 by facilitating its proteasome-dependent degradation. Additionally, we observed synergistic antitumor effects of myricetin and cisplatin both in vivo and in vitro. Mechanistically, myricetin suppressed the expression of FOXM1 and its downstream DNA damage response (DDR) genes E×O1and BRIP1, thereby enhancing the DDR induced by cisplatin. Conclusion Our data demonstrate that myricetin, a natural inhibitor of CD147, may have clinical utility in the treatment of OC due to its ability to increase genomic toxicity when combined with cisplatin.

Scinderin promotes glioma cell migration and invasion via remodeling actin cytoskeleton.

Glioma is one of the most common intracranial tumors, characterized by invasive growth and poor prognosis. Actin cytoskeletal rearrangement is an essential event of tumor cell migration. The actin dynamics-related protein scinderin (SCIN) has been reported to be closely related to tumor cell migration and invasion in several cancers. To investigate the role and mechanism of SCIN in glioma. The expression and clinical significance of SCIN in glioma were analyzed based on public databases. SCIN expression was examined using real-time quantitative polymerase chain reaction and Western blotting. Gene silencing was performed using short hairpin RNA transfection. Cell viability, migration, and invasion were assessed using cell counting kit 8 assay, wound healing, and Matrigel invasion assays, respectively. F-actin cytoskeleton organization was assessed using F-actin staining. SCIN expression was significantly elevated in glioma, and high levels of SCIN were associated with advanced tumor grade and wild-type isocitrate dehydrogenase. Furthermore, SCIN-deficient cells exhibited decreased proliferation, migration, and invasion in U87 and U251 cells. Moreover, knockdown of SCIN inhibited the RhoA/focal adhesion kinase (FAK) signaling to promote F-actin depolymerization in U87 and U251 cells. SCIN modulates the actin cytoskeleton via activating RhoA/FAK signaling, thereby promoting the migration and invasion of glioma cells. This study identified the cancer-promoting effect of SCIN and provided a potential therapeutic target for the treatment of glioma.

Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

Cisplatin (CDDP) plays a central role in chemotherapy for head and neck squamous cell carcinoma (HNSCC), but drug resistance in HNSCC chemotherapy remains a problem, and the mechanism of CDDP resistance is unclear. We investigated CDDP-resistance mechanisms mediated by extracellular vesicles (EVs) and ATPase copper transporting beta (ATP7B) in HNSCC. We established CDDP-resistant sublines of HNSCC cells and verified their ATP7B expression. We used an EV secretion inhibitor (GW4869) and ATP7B short hairpin (sh)RNA transfection to examine the correlation between EV secretion and ATP7B expression. The CDDP-resistant HNSCC sublines showed decreased CDDP sensitivity and increased ATP7B expression. GW4869 suppressed ATP7B expression, and ATP7B shRNA transfection suppressed EV secretion. The suppressions of EV secretion and ATP7B expression both enhanced CDDP's cell-killing effect. EVs were involved in the ATP7B-mediated mechanism underlying CDDP resistance. Further clarification of the EV-induced CDDP-resistance mechanism may lead to novel therapeutic strategies for HNSCC.

The HIF-1/ BNIP3 pathway mediates mitophagy to inhibit the pyroptosis of fibroblast-like synoviocytes in rheumatoid arthritis

BackgroundSynovial hypoxia, a critical pathological characteristic of rheumatoid arthritis (RA), significantly contributes to synovitis and synovial hyperplasia. In response to hypoxic conditions, fibroblast-like synoviocytes (FLS) undergo adaptive changes involving gene expression modulation, with hypoxia-inducible factors (HIF) playing a pivotal role. The regulation of BCL2/adenovirus e1B 19 kDa protein interacting protein 3 (BNIP3) and nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3) expression has been demonstrated to be regulated by HIF-1. The objective of this study was to examine the molecular mechanism that contributes to the aberrant activation of FLS in response to hypoxia. Specifically, the interaction between BNIP3-mediated mitophagy and NLRP3-mediated pyroptosis was conjointly highlighted. MethodsThe research methodology employed Western blot and immunohistochemistry techniques to identify the occurrence of mitophagy in synovial tissue affected by RA. Additionally, the levels of mitophagy under hypoxic conditions were assessed using Western blot, immunofluorescence, quantitative polymerase chain reaction (qPCR), and CUT&Tag assays. Pyroptosis was observed through electron microscopy, fluorescence microscopy, and Western blot analysis. Furthermore, the quantity of reactive oxygen species (ROS) was measured. The silencing of HIF-1α and BNIP3 was achieved through the transfection of short hairpin RNA (shRNA) into cells. ResultsIn the present study, a noteworthy increase in the expression of BNIP3 and LC3B was observed in the synovial tissue of patients with RA. Upon exposure to hypoxia, FLS of RA exhibited BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis. It appears that hypoxia regulates the expression of BNIP3 and NLRP3 through the transcription factor HIF-1. Additionally, the activation of mitophagy has been observed to effectively inhibit hypoxia-induced pyroptosis by reducing the intracellular levels of ROS. Conclusion: In summary, the activation of FLS in RA patients under hypoxic conditions involves both BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis. Additionally, mitophagy can suppress hypoxia-induced FLS pyroptosis by eliminating ROS and inhibiting the HIF-1α/NLRP3 pathway.

Ultrasonic Microbubble Cavitation Deliver Gal-3 shRNA to Inhibit Myocardial Fibrosis after Myocardial Infarction

Galectin-3 (Gal-3) participates in myocardial fibrosis (MF) in a variety of ways. Inhibiting the expression of Gal-3 can effectively interfere with MF. This study aimed to explore the value of Gal-3 short hairpin RNA (shRNA) transfection mediated by ultrasound-targeted microbubble destruction (UTMD) in anti-myocardial fibrosis and its mechanism. A rat model of myocardial infarction (MI) was established and randomly divided into control and Gal-3 shRNA/cationic microbubbles + ultrasound (Gal-3 shRNA/CMBs + US) groups. Echocardiography measured the left ventricular ejection fraction (LVEF) weekly, and the heart was harvested to analyze fibrosis, Gal-3, and collagen expression. LVEF in the Gal-3 shRNA/CMB + US group was improved compared with the control group. On day 21, the myocardial Gal-3 expression decreased in the Gal-3 shRNA/CMBs + US group. Furthermore, the proportion of the myocardial fibrosis area in the Gal-3 shRNA/CMBs + US group was 6.9 ± 0.41% lower than in the control group. After inhibition of Gal-3, there was a downregulation in collagen production (collagen I and III), and the ratio of Col I/Col III decreased. In conclusion, UTMD-mediated Gal-3 shRNA transfection can effectively silence the expression of Gal-3 in myocardial tissue, reduce myocardial fibrosis, and protect the cardiac ejection function.

GATA2 promotes oxidative stress to aggravate renal ischemia-reperfusion injury by up-regulating Redd1

Renal ischemia-reperfusion injury (RIRI) is a common pathophysiological process, and it is also an important cause of acute renal failure. Therefore, finding an effective therapeutic target for RIRI is extremely urgent. In our study, we constructed hypoxia-reoxygenation (HR) model in vitro and a renal ischemia-reperfusion (IR) model in vivo. Elevated levels of serum creatinine (Cr), blood urea nitrogen (BUN) tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and malondialdehyde (MDA) along with the decreased levels of superoxide dismutase (SOD) and glutathione (GSH) proved that kidney function was damaged after IR, and pathological changes of renal tissues were observed using HE staining and TUNEL staining. The protein of Redd1 expression level was detected to be upregulated after IR by western blot (WB). However, transfection of short hairpin RNA of Redd1 (sh-Redd1) alleviated the HR injury on LLC-PK1 cells, as evidenced by increased cell viability, proliferation and decreased cell apoptosis; additionally, the accumulation of ROS was inhibited. Sh-Redd1 also alleviated IR injury in the mouse model. Subsequently, GATA2 was proved to be upregulated in IR and HR models and was the transcription factor of Redd1. Knockdown of GATA2 efficiently mitigated the oxidative stress induced damages in vivo and in vitro, while these mitigations were reversed by transfection of Redd1 overexpression plasmid. In conclusion, our study clarified the possible underlying mechanism of protecting RIRI.

Low-dose decitabine modulates myeloid-derived suppressor cell fitness via LKB1 in immune thrombocytopenia

AbstractMyeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic fitness of MDSCs is fundamental for its suppressive activity toward effector T cells. Our previous studies showed that the number and inhibitory function of MDSCs were impaired in patients with immune thrombocytopenia (ITP) compared with healthy controls. In this study, we analyzed the effects of decitabine on MDSCs from patients with ITP, both in vitro and in vivo. We found that low-dose decitabine promoted the generation of MDSCs and enhanced their aerobic metabolism and immunosuppressive functions. Lower expression of liver kinase 1 (LKB1) was found in MDSCs from patients with ITP, which was corrected by decitabine therapy. LKB1 short hairpin RNA (shRNA) transfection effectively blocked the function of MDSCs and almost offset the enhanced effect of decitabine on impaired MDSCs. Subsequently, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP in murine models. Passive transfer of decitabine-modulated MDSCs significantly raised platelet counts compared with that of phosphate buffered saline–modulated MDSCs. However, when LKB1 shRNA-transfected MDSCs were transferred into SCID mice, the therapeutic effect of decitabine in alleviating thrombocytopenia was quenched. In conclusion, our study suggests that the impaired aerobic metabolism of MDSCs is involved in the pathogenesis of ITP, and the modulatory effect of decitabine on MDSC metabolism contributes to the improvement of its immunosuppressive function. This provides a possible mechanism for sustained remission elicited by low-dose decitabine in patients with ITP.

PI3K/Akt and Wnt/β-catenin Signaling Cross-regulate NF-κB Signaling in TNF-α-induced Human Lgr5+ Intestinal Stem Cells.

Intestinal stem cells (ISCs) are responsible for intestinal proliferation, differentiation, and neoplasia, and also play a crucial role in inflammation. Thus, it is important to investigate the effect of TNF-α on the activities of NF-κB, PI3K/Akt, and Wnt/β-catenin signaling pathways. The Lgr5+ intestinal cells were isolated using fluorescence-activated cell sorting from NCM460 spheroid cells, and the potential molecular mechanisms were investigated via short hairpin RNA (shRNA) transfection or the use of an inhibitor. The Lgr5+ cells were termed ISCs because of the higher expression of stem cell genes, including Sox2, Nanog, Oct4, Lgr5, and CD133. The Lgr5+ ISCs had a higher proliferation capacity, invasive ability, and drug resistance to 5-fluorouracil, as well as higher expression levels of anti-apoptotic proteins but lower expression levels of pro-apoptotic proteins, compared with Lgr5~ cells. The PI3K/Akt and Wnt/β-catenin pathways were triggered by the TNF-α-induced activation of NF-κB signaling. Notably, when p65 expression was knocked-down via shRNA transfection in Lgr5+ ISCs, the TNF-α-induced activation of the NF-κB, PI3K/Akt, and Wnt/β-catenin pathways were reversed. The same effect was observed with regards to β-catenin shRNA transfection. Moreover, the Akt inhibitor MK2206 inhibited the TNF-α-induced activation of the PI3K/Akt pathway, as well as the NF-κB and Wnt/β-catenin pathways. PI3K/Akt and Wnt/β-catenin signaling cross-regulate NF-κB signaling in TNF-α-induced human Lgr5+ ISCs.

Bone morphogenetic protein 4 inhibits pulmonary fibrosis by modulating cellular senescence and mitophagy in lung fibroblasts.

Accumulation of myofibroblasts is critical to fibrogenesis in idiopathic pulmonary fibrosis (IPF). Senescence and insufficient mitophagy in fibroblasts contribute to their differentiation into myofibroblasts, thereby promoting the development of lung fibrosis. Bone morphogenetic protein 4 (BMP4), a multifunctional growth factor, is essential for the early stage of lung development; however, the role of BMP4 in modulating lung fibrosis remains unknown. The aim of this study was to evaluate the role of BMP4 in lung fibrosis using BMP4-haplodeleted mice, BMP4-overexpressed mice, primary lung fibroblasts and lung samples from patients with IPF. BMP4 expression was downregulated in IPF lungs and fibroblasts compared to control individuals, negatively correlated with fibrotic genes, and BMP4 decreased with transforming growth factor (TGF)-β1 stimulation in lung fibroblasts in a time- and dose-dependent manner. In mice challenged with bleomycin, BMP4 haploinsufficiency perpetuated activation of lung myofibroblasts and caused accelerated lung function decline, severe fibrosis and mortality. BMP4 overexpression using adeno-associated virus 9 vectors showed preventative and therapeutic efficacy against lung fibrosis. In vitro, BMP4 attenuated TGF-β1-induced fibroblast-to-myofibroblast differentiation and extracellular matrix (ECM) production by reducing impaired mitophagy and cellular senescence in lung fibroblasts. Pink1 silencing by short-hairpin RNA transfection abolished the ability of BMP4 to reverse the TGF-β1-induced myofibroblast differentiation and ECM production, indicating dependence on Pink1-mediated mitophagy. Moreover, the inhibitory effect of BMP4 on fibroblast activation and differentiation was accompanied with an activation of Smad1/5/9 signalling and suppression of TGF-β1-mediated Smad2/3 signalling in vivo and in vitro. Strategies for enhancing BMP4 signalling may represent an effective treatment for pulmonary fibrosis.

Knockdown of CDCA5 suppresses malignant progression of breast cancer cells by regulating PDS5A.

Breast cancer is one of the most common malignant tumors in women. Cell division cycle-associated 5 (CDCA5) is closely associated with the behavior of various cancer types. The aim of the present study was to explore the effect of CDCA5 on breast cancer. Western blot analysis and reverse transcription-quantitative PCR were used to detect the expression level of CDCA5 in human normal mammary cells and human breast cancer cell lines. To determine its function in MDA-MB-231 cells, CDCA5 was silenced in MDA-MB-231 cells by transient short hairpin RNA transfection. Cell Counting Kit-8 and clonogenicity assays were used to evaluate cell proliferation. Wound healing and Transwell assays were used to detect cell invasion and migration. Western blot analysis was used to detect the protein expressions of Ki67 and PCNA associated with proliferation, MMP2 and MMP9 associated with migration. CDCA5 was found to be markedly increased in breast cancer cell lines. CDCA5 knockdown was able to suppress cell proliferation, invasion and migration. CDCA5 inhibition downregulated PDS5 cohesin-associated factor A (PDS5A) expression in breast cancer cells. PDS5A overexpression was found to reverse the effect of CDCA5 inhibition on breast cancer cell proliferation and migration. CDCA5 knockdown was shown to suppress the malignant progression of breast cancer cells by regulating PDS5A. The present findings may provide new potential targets for breast cancer therapy.

Vitamin D3 alleviates cigarette smoke extract-mediated epithelial-mesenchymal transition and fibrogenesis by upregulating CC16 expression in bronchial epithelial cells.

Vitamin D3 supplementation has been previously reported to inhibit the occurrence and development of chronic obstructive pulmonary disease (COPD). However, the underlying mechanism remains unclear. Epithelial-mesenchymal transition (EMT) and fibrogenesis have been associated with the development of COPD. The aim of the present study was to investigate the potential effects and mechanism of vitamin D3 in an in vitro model of cigarette smoke (CS)-induced EMT and fibrosis, with specific focus on the role of club cell protein 16 (CC16). CS extract (CSE) at different concentrations (5, 10 and 20%) was used to treat 16-HBE cells to induce EMT and fibrogenesis following which they were treated with vitamin D3. Subsequently, the 20% CSE group was selected for further experiments, where 16-HBE cells were divided into the following five groups: The control group; the CSE group; the low-dose vitamin D3 group (250 nM); the medium-dose vitamin D3 group (500 nM); and the high-dose vitamin D3 group (1,000 nM). Western blot analysis was used to detect the protein expression levels of the EMT-related proteins E-cadherin, N-cadherin, Slug and α-SMA, fibrogenesis-related proteins collagen Ⅳ and fibronectin 1, proteins involved in the TGF-β1/SMAD3 signaling pathway and CC16. Immunofluorescence was used to measure the protein expression levels of E-cadherin, N-cadherin and collagen Ⅳ. Specific CC16 knockdown was performed using short hairpin RNA transfection to investigate the role of CC16. The results of the present study found that vitamin D3 could increase the protein expression level of CC16 to inhibit the activation of the TGF-β1/SMAD3 signaling pathway; thereby reducing the 20% increase in CSE-induced EMT- and fibrogenesis-related protein expression levels. Following CC16 knockdown, the inhibitory effects of vitamin D3 on EMT- and fibrogenesis-related protein expression were partially reversed. To conclude, these results suggest that vitamin D3 can inhibit the protein expression levels of EMT- and fibrogenesis-related proteins induced by CSE, at least partially through the function of CC16. These findings are expected to provide novel theoretical foundations and ideas for the pathogenesis and treatment of COPD.

Transcriptional Expression Profiles of Olive Flounder (Paralichthys olivaceus) Toll-like Receptor 3 (TLR3) and TLR22 in Response to Extracellular Poly I:C Stimuli

Toll-like receptors (TLRs) recognize extracellular and intracellular double-stranded RNA (dsRNA) and play crucial roles in innate and adaptive immune systems. Here, we aimed to investigate the changes in the transcriptional profile of TLR3 and TLR22 genes of olive flounder (Paralichthys olivaceus) in response to polyinosinic:polycytidylic acid (poly I:C; a dsRNA analog) stimuli in vivo, using healthy olive flounders and in vitro, using Hirame natural embryonic (HINAE) cells. The role of JfTLR3 and JfTLR22 in inducing type I interferon response in HINAE cells was analyzed through construction and transfection of short-hairpin RNA (shRNA)-producing vector targeting the JfTLR22 and JfTLR3. Poly I:C stimuli upregulated the JfTLR3 and JfTLR22 genes both in vivo and in vitro, and JfTLR22 showed an earlier (at 6 and 12 h post-stimulation) response than JfTLR3, suggesting that JfTLR22 is a more sensitive receptor and recognizes the extracellular dsRNA more quickly than JfTLR3. After 12 h of poly I:C stimulation, the expression of ISG15 and Mx genes in HINAE cells harboring TLR22-targeting shRNA vector was markedly reduced, wherein the reduction in expression of these genes was weakly reduced in HINAE cells carrying TLR3-targeting shRNA vector. These results suggested that JfTLR22 plays an important role, wherein JfTLR3 partially induces type I interferon response to extracellular dsRNA at 12 h after poly I:C stimulation.

Inhibition of macrophage migration inhibitory factor alleviates LPS-induced inflammation response of HEI-OC1 cells via suppressing NF-κB signaling

Sudden sensorineural hearing loss (SSNHL) is acute and unexplained. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine in several inflammatory diseases. However, its role in SSNHL remains elusive. Lipopolysaccharide (LPS) was used to induce the inflammatory response of murine auditory cells, HEI-OC1. Silencing of MIF in HEI-OC1 cells was achieved by transfection of short hairpin RNA against MIF. 740Y-P and IMD0354 were used to stimulate the PI3K pathway and suppress the NF-κB pathway, respectively. RT-qPCR and western blotting were used to examine MIF and cyclooxygenase 2 (COX2) expression in LPS-treated HEI-OC1 cells. ELISA was employed to assess prostaglandin E2 (PGE2) concentrations. MIF was upregulated in LPS-treated HEI-OC1 cells. MIF knockdown reduced PGE2 synthesis and COX2 expression in LPS-treated HEI-OC1 cells. Moreover, MIF knockdown suppressed activation of the PI3K/AKT and NF-κB pathway in LPS-treated HEI-OC1 cells. Additionally, inhibition of MIF decreased PGE2 production and COX2 expression via inactivation of the NF-κB pathway. Inhibition of MIF alleviated LPS-induced inflammation in HEI-OC1 cells via inactivating the NF-κB signaling, which might provide a better understanding for SSNHL development.

Elevated expression of NFE2L3 promotes the development of gastric cancer through epithelial-mesenchymal transformation

ABSTRACT Gastric cancer (GC) is a malignant tumor with high mortality, but research on its molecular mechanisms remain limited. This study is the first to explore the biological role of nuclear factor NFE2L3 (nuclear factor, erythroid 2 like 3) in GC. We used Western blot and RT–qPCR to detect gene expression at the protein or mRNA level. Short hairpin RNA (shRNA) transfection was used to inhibit NFE2L3 expression. CCK-8 and colony formation assays were used to detect cell proliferation. Cell migration, invasion, cell cycle and apoptosis were detected by Transwell assays and flow cytometry. The results showed that NFE2L3 was highly expressed in gastric cancer tissues and promoted gastric cancer cell proliferation and metastasis. Inhibiting NFE2L3 expression blocks the cell cycle and increases the proportion of apoptotic cells, whereas NFE2L3 expression promotes the epithelial-mesenchymal transformation (EMT) process. In summary, NFE2L3 is highly expressed in gastric cancer and promotes gastric cancer cell proliferation and metastasis and the EMT process.

Notoginsenoside R1 protects hypoxia-reoxygenation deprivation-induced injury by upregulation of miR-132 in H9c2 cells.

Myocardial ischemia/reperfusion injury (IRI) is a common perioperative complication of heart and great vessels surgery, aggravating the original myocardial damage and seriously affecting the postoperative recovery of cardiac function. The aim of this study was to reveal the functional effects and potential mechanisms of notoginsenoside R1 (NG-R1) in myocardial cells injured by hypoxia-reoxygenation (H/R). The rat cardiomyocyte line H9c2 was subjected to H/R with or without NG-R1 treatment. The levels of miR-132 and HBEGF in the cell were altered by microRNA or short-hairpin RNA transfection. Cell viability, apoptosis, lactate dehydrogenase (LDH) and malondialdehyde (MDA) were monitored. Dual luciferin was used to detect the relationship between miR-132 and HBEGF. NG-R1 (20 μM) had no impact on H9c2 cells, but cell viability was significantly reduced at 80 μM. NG-R1 (20 μM) protected H9c2 cells against H/R-induced cell damage, accompanied by increased cell viability, reduced cell apoptosis, and downregulation of LDH and MDA. Furthermore, the level of miR-132 was decreased in response to H/R exposure but then increased after NG-R1 treatment. When miR-132 was overexpressed, H/R-induced cell damage could be recovered. Downregulation of miR-132 limited the protective effect of NG-R1 on H/R damage. We also found that HBEGF was a direct target of miR-132. The expression of HBEGF was increased upon H/R damage, and this increase was reversed after NG-R1 treatment. This study demonstrated that NG-R1 markedly protected H9c2 cells against H/R-induced damage via upregulation of miR-132 and downregulation of its target protein HBEGF.