NC siRNA Research Articles

A Comprehensive Analysis of HOXB13 Expression in Hepatocellular Carcinoma.

Background and objectives: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and is caused by multiple factors. To explore novel targets for HCC treatment, we comprehensively analyzed the expression of HomeoboxB13 (HOXB13) and its role in HCC. Materials and Methods: The clinical significance of HCC was investigated using open gene expression databases, such as TIMER, UALCAN, KM, OSlihc, and LinkedOmics, and immunohistochemistry analysis. We also analyzed cell invasion and migration in HCC cell lines transfected with HOXB13-siRNA and their association with MMP9, E2F1, and MEIS1. Results: HOXB13 expression was higher in fibrolamellar carcinoma than in other histological subtypes. Its expression was associated with lymph node metastasis, histological stage, and tumor grade. It was positively correlated with immune cell infiltration of B cells (R = 0.246), macrophages (R = 0.182), myeloid dendritic cells (R = 0.247), neutrophils (R = 0.117), and CD4+ T cells (R = 0.258) and negatively correlated with immune cell infiltration of CD8+ T cells (R = -0.107). A positive correlation was observed between HOXB13, MMP9 (R = 0.176), E2F1 (R = 0.241), and MEIS1 (R = 0.189) expression (p < 0.001). The expression level of HOXB13 was significantly downregulated in both HepG2 and PLC/PFR/5 cell lines transfected with HOXB13-siRNA compared to that in cells transfected with NC siRNA (p < 0.05). Additionally, HOXB13 significantly affected cell viability and wound healing. Conclusions: HOXB13 overexpression may lead to poor prognosis in patients with HCC. Additional in vivo studies are required to improve our understanding of the biological role and the exact mechanism of action of HOXB13 in HCC.

The Molecular Mechanism of LncRNA HOTAIR Regulating the Proliferation, Invasion and Migration of Lymphoma Cells through Target Gene miR-20a-5p

To investigate the effects of lncRNA HOTAIR on the proliferation, invasion and migration of lymphoma cells through target gene miR-20a-5p and its molecular mechanism. After synthesizing HOTAIR siRNA and siRNA NC plasmids, they were transfected into lymphoma Raji cells, respectively. The expression of HOTAIR mRNA was detected by RT-qPCR. The proliferation, invasion and migration of lymphoma Raji cells were detected by CCK-8 assay, Transwell assay and cell scratch healing assay, respectively. The target gene of lncRNA HOTAIR was predicted by miRcode software, and the relationship between HOTAIR and target gene was analyzed by dual luciferase assay. After synthesis of miR-20a-5p inhibitor and inhibitor NC, Raji cells were transiently transfected. The expression of miR-20a-5p was detected by RT-qPCR, and the effects of down-regulation of miR-20a-5p on the proliferation, invasion and migration of Raji cells were analyzed. The overexpression plasmid of lncRNA HOTAIR and miR-20a-5p mimics were transfected into Raji cells simultaneously to analyze the proliferation, invasion and migration ability of Raji cells. After overexpression or down-regulation of miR-20a-5p, the expression of JAK/STAT3 signaling pathway related proteins was analyzed. HOTAIR expression in Raji cells was decreased after transfection of HOTAIR siRNA (P<0.01), and miR-20a-5p expression was also decreased after transfection of miR-20a-5p inhibitor (P<0.01). HOTAIR had a targeting and negative regulation relationship with miR-20a-5p (r=-0.826). Silencing HOTAIR promoted the expression of miR-20a-5p and inhibited the proliferation, invasion and migration of Raji cells. Down-regulation of miR-20a-5p expression promoted the proliferation, invasion and migration of Raji cells. Effect of HOTAIR overexpression on the proliferation, invasion and migration of Raji cells could be reversed by up-regulation of miR-20a-5p. Down-regulation of miR-20a-5p expression activated the intracellular JAK/STAT3 signaling pathway. HOTAIR affects the proliferation, invasion and migration of lymphoma cells by targeting miR-20a-5p, and its mechanism may be related to the activation of JAK/STAT3 signaling pathway.

Effects of MFG-E8 expression on the biological characteristics of ovarian cancer cells via the AKT/mTOR/S6K signalling pathway

In this study, we assessed the effects of MFG-E8 on the biological characteristics of ovarian cancer cells and explored the underlying mechanisms. Human ovarian cancer SKOV3 cells were transfected with MFG-E8 siRNA or NC siRNA. CCK-8, cell adhesion, scratch-wound, and Transwell assays were used to detect changes in cell metastatic processes. Effects of MFG-E8 silencing on the proteins involved in AKT/mTOR/S6K signalling pathway were assessed using qRT-PCR and Western blotting. Transient silencing of MFG-E8 in SKOV3 cells decreased cell proliferation and downregulated the expression of CDK4, cyclin D1, and caspase-3 proteins. Cell adhesion, migration, and invasion were also suppressed. p-AKT, p-mTORC1, and p-p70S6K levels decreased following MFG-E8 knockdown. Hence, MFG-E8 enhances carcinogenesis and affects the AKT/mTOR/S6K signalling pathway in ovarian cancer cells. In conclusion, our results suggested that MFG-E8 could promote ovarian cancer via AKT/mTOR/S6K signalling pathway which improved our understanding of the molecular mechanisms involved in ovarian cancer. IMPACT STATEMENT What is already known on this subject? Milk fat globule-epidermal growth factor 8 (MFG-E8) is expressed in several types of cancers such as oesophageal, breast, and liver. However, the mechanism of MFG-E8 involving in EOC remains unknown. We previously found that MFG-E8 expression was related to pathological staging, tissue differentiation, platinum sensitivity, ascites state, and other clinicopathological characteristics. What the results of this study add? Due to a series of in vitro studies, we confirmed that MFG-E8 is involved in the process of proliferation, invasion and metastasis. Our results show that silencing MFG-E8 can significantly inhibit the expression of cyclin D1 and CDK4 in EOC SKOV3 cells. MFG-E8 enhances carcinogenesis and affects the AKT/mTOR/S6K signaling pathway in ovarian cancer. What the implications are of these findings for clinical practice and/or further research? Taken together, our findings suggest that MFG-E8 may be an oncogene in EOC and provide new insights into the mechanism of MFG-E8 in the progression of EOC.

Intervention effects of estradiol on myocardial ischemia- reperfusion injury of rat and its mechanisms

Objective: To study the effects of estradiol (E2) on alleviating myocardial ischemia/reperfusion(I/R) injury through estrogen receptorβ(ERβ) mediated extracellular regulated protein kinases(ERK) pathway activation. Methods: Eighty-four adult female SD rats were ovariectomized and randomly divided into control group, NC siRNA adeno-associated virus (AAV) group received sham operation, the myocardial I/R injury model was prepared by ligation of the left anterior descending coronary artery in I/R group, E2+I/R group, NC siRNA AAV+I/R group, NC siRNA AAV+E2+I/R group and ERβ-siRNA AAV+E2+I/R group. E2+I/R group, NC siRNA AAV+E2+I/R group and ERβ-siRNA AAV+E2+I/R group were treated with E2 0.8 mg/kg by gavage for 60 days before modeling. NC siRNA AAV+I/R group, NC siRNA AAV+E2+I/R group, and ERβ-siRNA AAV+E2+I/R group were treated with AAV by caudal vein injection 24 h before modeling. After 120 min of reperfusion, the contents of serum lactate dehydrogenase (LDH), phosphocreatine kinase (CK), phosphocreatine kinase isoenzyme (CK-MB), myocardial infarction area and the expressions of ERβ, p-ERK, the contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1 β), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in myocardium were measured. Results: The contents of serum LDH, CK, CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β, MDA in myocardium of I/R group were higher than those of the control group, the expression levels of ERβ and p-ERK and the content of T-AOC were lower than those in the control group (P＜0.05). The contents of serum LDH, CK and CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β and MDA in myocardium of E2+I/R group were lower than those of the I/R group, the expression levels of ERβ and p-ERK and the content of T-AOC were higher than those of the I/R group(P＜0.05). After knockdown ERβ by caudal vein injection of ERβ-siRNA AAV, the contents of serum LDH, CK and CK-MB, myocardial infarction area and the contents of TNF-α, IL-1 β and MDA in myocardium of ERβ-siRNA AAV+E2+I/R group were higher than those of NC-siRNA AAV+E2+I/R, the expression levels of ERβ and p-ERK and the content of T-AOC were lower than those of NC-siRNA AAV+E2+I/R(P＜0.05). Conclusion: E2 has protective effects on myocardial I / R injury in ovariectomized rats, which are related to the promotion of ERβ mediating the activation of ERK pathway, reducing inflammatory and oxidative stress responses.

OAT3A1/NKD2 mediates increased calcification of aortic valvular interstitial cells under in vitro conditions of uremia

Abstract Background Chronic kidney disease is closely associated with cardiovascular disease which contributes to a high mortality and morbidity in these patients. Calcific aortic valve stenosis is the most common valve disease among adults. CKD leads to a higher incidence of AS and is associated with impaired clinical outcomes after valve replacement. CKD leads to retention of uremic toxins, such as indoxyl sulfate (IS), which is known to induce inflammatory and pro-calcific processes. The influence of IS on AS pathophysiology is currently not known. Methods and results Human aortic valvular interstitial cells (VICs) were stimulated by 4 different conditions: Control medium (CM), CM and IS in a concentration of 50 mmol/l, pro-calcifying medium (PCM) which was produced by adding NaH2PO4 and L-ascorbic acid to CM and PCM+IS. After 7 days of incubation, VICs were fixed in formalin and calcification was evaluated through staining with alizarin red solution (B). Staining was quantified by photometric measurements at 540 nm. We observed a significantly higher degree of VIC calcification under the influence PCM+IS compared to PCM alone (C). VICs were stimulated for 7 days and next-generation RNA Sequencing (RNA Seq) was performed. Analysis of RNA Seq dataset identified naked cuticle homologue 2 (NKD2) to be strongly regulated by IS. This effect was confirmed by qPCR using VICs from a total of three different human donors and immunoblot analysis (D, E). We observed a more pronounced upregulation of NKD2 gene expression under PCM+IS vs. PCM alone (p&amp;lt;0.0001) (D). To investigate the influence of NKD2 on VIC calcification genetic knockdown of NKD2 was performed by transfecting VICs with NKD2 siRNA compared and scrambled siRNA as negative control (nc siRNA) (F). After 21 days of stimulation and subsequent alizarin staining, we observed a significant decrease in VIC calcification under NKD2 knockdown compared to negative control under PCM+IS conditions (G, H). To identify upstream mechanisms that lead to NKD2 upregulation by IS we investigated the influence of organic anion transporters (OAT) which are responsible for cellular uptake of IS. Probenecid, known as a potent inhibitor of OATs showed a dose dependent suppression of NKD2 upregulation under PCM+IS (I). Systematic analysis of RNA Seq data regarding SLCO genes coding for OAT and OAT-peptides revealed that only SLCO3A1, coding for OAT3A1 is highly abundant in VICs (J). SLCO3A1 knockdown showed a significant decrease of NKD2 expression after stimulation with PCM+IS for 7 days compared to negative control (K). Conclusion Our findings show that in vitro conditions of uremia increase calcification of aortic VICs. NKD2 expression is regulated by PCM and IS, and PCM+IS treated cells show the strongest upregulation of NKD2. Genetic knockdown of NKD2 attenuates VIC calcification suggesting a role of NKD2 in this process. Further, our findings suggest that OAT3A1 is involved in IS mediated regulation of NKD2 expression. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Medical faculty of the University of Bonn, German Cardiac Society (DGK), German Research Foundation (DFG), TRR259

Inhibition Mechanism of PinX1 Gene on Cancer Stem Cells of Nasopharyngeal Carcinoma.

The recurrence and long-term metastasis of these tumors are important causes of treatment failure and death. On the other hand, PinX1 is a nucleolar protein found in recent years that can interact with telomere/telomerase simultaneously, and it is highly conserved in human and yeast. Some studies have shown that the PinX1 gene can inhibit the tumor stem cells of NPC. Therefore, the mechanism of inhibition of the PinX1 gene on the tumor stem cells of NPC has been studied in this paper. In this paper, CNE2 cells of NPC were used as experimental materials, CD133 as a marker, PinX1 overexpression plasmids and their corresponding empty plasmids were respectively transfected in CD133+ cells, PinX1 siRNA and their corresponding NC siRNA were respectively transfected in CD133- cells for control experiments. In this study, we found that the telomerase activity of the CD133 - + NC group was 1.001 ± 0.086, the CD133 - + pinx1sirna group was 0.974 ± 0.046, CD133+ + vector group was 0.928 ± 0.102, CD133+ + over PinX1 group was 0.703 ± 0.086. Therefore, the PinX1 gene can inhibit NPC stem cells by inhibiting telomerase activity.

CpG Oligodeoxynucleotides Inhibit RANKL-Induced Osteoclast Formation by Upregulating A20 Deubiquitinase in RAW 264.7 Cells.

Objective Activation of toll-like receptor 9 (TLR9) has been proposed to play an inhibitory role in RANKL-induced osteoclastogenesis. A20 deubiquitinase has been found to be related to bone loss. This study investigated the role of CpG oligodeoxynucleotides (CpG-ODNs) through regulation of A20 deubiquitinase in RANKL-induced osteoclast formation. Methods RAW 264.7 cells, a murine monocyte-macrophage cell line, were incubated with or without CpG-ODN in the presence of RANKL. Osteoclast-specific genes and their related signaling molecules were measured by real-time quantitative polymerase chain reaction and Western blot assay. Morphological assessment for osteoclast formation was performed using tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring formation staining. Results RANKL-induced osteoclast-related genes and proteins, c-Fos, NFATc1, TRAP, cathepsin K, and carbonic anhydrase II were significantly inhibited in RAW 264.7 cells stimulated with CpG-ODN. CpG-ODN attenuated TNF receptor-associated factor 6 (TRAF6), p-IκBα, and p-NF-κB expression in RAW 264 cells mediated by RANKL. CpG-ODN increased A20 gene and proteins in time-dependent manner. A20 expression under costimulation with CpG-ODN and RANKL was more decreased than under stimulation with RANKL alone. Cells transfected with A20 siRNA augmented expression of osteoclast-related genes and proteins. Number of TRAP-positive cells transfected with A20 siRNA was higher than those transfected with NC siRNA. A20 expression in cells transfected with IL-1β siRNA in the presence of both RANKL and CpG-ODN was more decreased than those with NC siRNA. Conclusion This study showed that CpG-ODN suppressed RANKL-induced osteoclast formation through regulation of the A20-TRAF6 axis in RAW 264.7 cells.

Effects of Long Noncoding RNA HOTAIR Targeting miR-138 on Inflammatory Response and Oxidative Stress in Rat Cardiomyocytes Induced by Hypoxia and Reoxygenation.

Objective To investigate the effects of HOX transcript antisense RNA (HOTAIR) and miR-138 on inflammatory response and oxidative stress (OS) induced by IRI in rat cardiomyocytes. Methods H9C2 cells were divided into the control group, H/R group, H/R+siRNA NC group, H/R+si-HOTAIR group, and H/R+si-HOTAIR+inhibitor group. Expression levels of HOTAIR, miR-138, and inflammatory factors were detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The double luciferase reporter gene assay was used to detect the targeting relationship between HOTAIR and miR-138. Results Compared with the control group, the level of miR-138 and SOD in the H/R group was obviously reduced, while the expression levels of the HOTAIR, MDA, and NF-κB pathway were obviously increased. Compared with the H/R group, the level of miR-138 and SOD in the H/R+si-HOTAIR group was obviously increased, and the expression levels of the HOTAIR, MDA, and NF-κB pathway were obviously decreased. Compared with the H/R+si-HOTAIR group, the level of SOD in the H/R+si-HOTAIR+inhibitor group decreased; MDA content and the NF-κB pathway expression level increased. In the double luciferase reporter gene assay, compared with the HOTAIR wt+NC group, the luciferase activity of the HOTAIR wt+miR-138 mimic group was obviously decreased. Conclusions Silent HOTAIR can promote the expression of miR-138 and inhibit H/R-induced inflammatory response and OS by regulating the NF-κB pathway, thus protecting cardiomyocytes.

Effects of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition of type II alveolar epithelial cell and its mechanism

Objective: To study the role of Notch-1/Twist-1 axis in the process of epithelial-mesenchymal transition (EMT) of type II alveolar epithelial cells in pulmonary fibrosis (PF) and hope to provide a new theoretical basis for the pathogenesis of PF. Methods: Thirty rats were randomly divided into control group and bleomycin (BLM) group, 15 rats in each group. The PF rat model was induced by intratracheal injection of BLM (7 500 U/kg). Excised inferior lobe of left lung was fixed in 10% formalin for HE staining, Masson staining and transforming growth factor-beta 1 (TGF-β1) immunohistochemistry staining after BLM injection for 28 days. The cultured type II alveolar epithelial cells (RLE-6TN) were divided into 4 groups (Control group, transforming growth factor-beta 1 (TGF-β1) group, Notch-1 negative control siRNA (NC siRNA, 100 pmol/L) group and Notch-1 siRNA (100 pmol/L) group), each group was established nine holes. The cells were treated with TGF-β1 (5.0 ng/ml) for 24 h following NC siRNA or Notch-1 siRNA for 48 h. The mRNA and (or) proteins levels of TGF-β1, collagen I, collagen III, E-Cadherin, zonula occludens-1 (ZO-1), Vimentin, E-Cadherin, Notch-1, Notch-1 intracellular domain (NICD), Hes-1 and Twist-1 were detected in lung tissue and type II alveolar epithelial cells. Results: In vivo, compared with the control group, the alveolar atrophy, collapse and fusion occurred, alveolar septum widened significantly, and a large number of inflammatory cells infiltrated in the pulmonary interstitial of the rats in the BLM group. And compared with control group, BLM obviously increased collagen deposition and collagen I and collagen III expressions, while the expressions of ZO-1 and E-cadherin were decreased, and the expressions of Vimentin and N-cadherin were increased, and concomitantly with increasing Notch-1, NICD, Hes-1 and Twist-1 expression in lung tissues of rats (P＜0.01). In vitro, compared with control group, TGF-β1 treatment obviously induced collagen I, collagen III, Notch-1, NICD, Hes-1 and Twist-1 expressions, and the expressions of E-cadherin and ZO-1 were decreased and the expressions of Vimentin and N-cadherin were increased(P＜0.01). Compared with TGF-β1 group, Notch-1 siRNA treatment significantly inhibited the expressions of Notch-1, NICD, Hes-1 and Twist-1, and the expressions of E-cadherin and ZO-1 were increased and the expressions of Vimentin and N-cadherin were decreased, and also obviously reduced the expressions of collagen I and collagen III induced by TGF-β1 (P＜0.05 or P＜0.01). Conclusion: Notch-1/Twist-1 axis is involved in the EMT process of type II alveolar epithelial cells, suggesting that Notch-1/Twist-1 signaling may be involved in the development of pulmonary fibrosis.

The effects of miR-26b-5p on fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) via targeting EZH2

ObjectiveTo study the possible effects of miR-26b-5p on fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) through targeting enhancer of zeste homolog 2 (EZH2). MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to detect miR-26b-5p and EZH2 expressions in synovial tissues of RA patients and healthy controls. Dual luciferase reporter assay was adopted to verify the targeting relationship between miR-26b-5p and EZH2. RA-FLS was divided into Blank, mimics NC, mimics, NC siRNA, EZH2 siRNA and inhibitors + EZH2 siRNA groups, followed by the assessment of proliferation, apoptosis, migration and invasion. The expression of genes and proteins in RA-FLS was tested by qRT-PCR and western blotting, respectively. ResultsMiR-26b-5p expression was lower, while EZH2 expression was higher in synovial tissue of RA patients than healthy controls; and miR-26b-5p was negatively correlated with the EZH2 in synovial tissue of RA patients, which were both related with disease activities. MiR-26b-5p can target EZH2 in RA-FLS. In vitro, miR-26b-5p mimics down-regulated EZH2 expression in RA-FLS. Compared with EZH2 siRNA group, the miR-26b-5p expression in inhibitors + EZH2 siRNA group was reduced, but EZH2 expression was increased. EZH2 siRNA inhibited the proliferation, invasion and migration of RA-FLS, promoted cell apoptosis, and inhibited the expression of TNF-α, IL-1β, IL-6, IL-17, MMP-2, MMP-9, which were reversed by miR-26b-5p inhibitor. ConclusionMiR-26b-5p may affect the biological characteristics of RA-FLS via targeting EZH2, including proliferation, apoptosis, invasion and migration, as well as the secretion of cytokines, thus playing a potential therapeutic role in RA.

Hsa_circ_0006948 regulates the proliferation, migration and invasion in osteosarcoma by regulation of the expression of miR-490-3p target ATG7

Objective: To investigate the effect of hsa_circ_0006948 (circ_0006948) on the proliferation, migration and invasion of osteosarcoma cells and the underlying mechanism. Methods: A total of 120 osteosarcoma tissues and 40 adjacent normal tissue samples were collected from patients admitted to the First People's Hospital of Shangqiu City from 2009 to 2015. Microarray analysis was performed to detect the differential expressions of circRNA in Saos-2 cell. The mRNA expressions of circ_0006948, microRNA (miR)-490-3p and autophagy-related protein 7 (ATG7) in osteosarcoma cells, NHOst cells, osteosarcoma tissues and adjacent tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cell clone formation assay was used to detect cell proliferation ability, Transwell assay was used to detect cell invasion ability, and cell scratch assay was used to detect cell migration ability. The interactions between circ_0006948 and miR-490-3p, miR-490-3p and ATG7 were detected by dual luciferase reporter gene assay. The correlation between miR-490-3p and ATG7 was analyzed by TargetScan database, and the expression levels of Bcl-2 and Bax proteins in cells were detected by western blot. Results: The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in SAOS-2 cells were significantly different from NHOst cells (P<0.01). The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in osteosarcoma tissues were significantly different from adjacent tissues (P<0.01). The numbers of cell clone, migration and mobility in circ_0006948-siRNA group were (32.78±1.76), (37.58±1.82) and (36.93±1.45)%, respectively, lower than (65.72±1.45), (78.63±1.93) and (65.32±1.74)% in the siRNA NC group (all P<0.01). The numbers of cell clone, migration and mobility in the miR-490-3p mimics group were (20.08±1.54), (30.24±1.78) and (21.15±1.68)%, respectively, lower than (60.36±1.83), (76.93±1.64) and (40.56±1.27)% in the mimics NC group (all P<0.01). The numbers of cell clone, migration and mobility in the miR-490-3p inhibitor+ siRNA NC group were (90.34±1.72), (120.89±2.34) and (70.83±1.93)%, respectively, higher than (61.27±1.73), (75.82±1.82) and (42.38±1.74)% in the inhibitor NC+ siRNA NC group (P<0.01). The numbers of cell clone, migration and mobility in the circ_0006948 siRNA+ miR-490-3p inhibitor group were (58.74±1.98), (73.46±1.04) and (40.35±1.72)%, respectively, lower than (90.34±1.72), (120.89±2.34) and (70.83±1.93)% in the miR-490-3p inhibitor+ siRNA NC group (P<0.01). The numbers of cell clone, migration and mobility in the ATG7 siRNA group were (20.56±1.87), (40.36±1.76) and (20.96±1.73)%, lower than (65.46±1.74), (90.87±2.32) and (40.87±2.03)% in the siRNA NC group (P<0.01). The absorbance of miR-490-3p mimics+ pcDNA-ATG7 group was 0.54±0.11, higher than (0.36±0.08) of miR-490-3p mimics group (P<0.05). The expression levels of Bax and Bcl-2 protein in Saos-2 cells of miR-490-3p mimics group were significantly different from mimics NC group (P<0.01). The protein expression levels of Bax and Bcl-2 in Saos-2 cells of miR-490-3p mimics + pcDNA-ATG7 group were significantly different from miR-490-3p mimics group (P<0.01). Conclusion: Circ_0006948 regulates ATG7 expression through miR-490-3p, therefore regulates the proliferation, migration and invasion of osteosarcoma cells.

Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway

Increasing lines of evidence identified that dexmedetomidine (DEX) exerted protective effects against sepsis-stimulated acute lung injury via anti-inflammation, anti-oxidation and anti-apoptosis. However, the mechanisms remain unclear. Herein, we investigated whether DEX afforded lung protection by regulating the process of mitochondrial dynamics through the HIF-1a/HO-1 pathway in vivo and in vitro. Using C57BL/6J mice exposed to lipopolysaccharide, it was initially observed that preemptive administration of DEX (50μg/kg) alleviated lung pathologic injury, reduced oxidative stress indices (OSI), improved mitochondrial dysfunction, upregulated the expression of HIF-1α and HO-1, accompanied by shifting the dynamic course of mitochondria into fusion. Moreover, HO-1-knockout mice or HO-1 siRNA transfected NR8383 cells were pretreated with HIF-1α stabilizer DMOG and DEX to validate the effect of HIF-1a/HO-1 pathway on DEX-mediated mitochondrial dynamics in a model of endotoxin-induced lung injury. We found that pretreatment with DEX and DMOG distinctly relieved lung injury, decreased the levels of mitochondrial ROS and mtDNA, reduced OSI, increased nuclear accumulation of HIF-1a and HO-1 protein in wild type mice but not HO-1 KO mice. Similar observations were recapitulated in NC siRNA transfected NR8383 cells after LPS stimulation but not HO-1 siRNA transfected cells. Concertedly, DEX reversed the impaired mitochondrial morphology in LPS stimulated-wild type mice or NC siRNA transfected NR8383 cells, upregulated the expression of mitochondrial fusion protein, while downregulated the expression of fission protein in HIF-1a/HO-1 dependent pathway. Altogether, our data both in vivo and in vitro certified that DEX treatment ameliorated endotoxin-induced acute lung injury by preserving the dynamic equilibrium of mitochondrial fusion/fission through the regulation of HIF-1a/HO-1 signaling pathway.

Linc‑ROR facilitates hepatocellular carcinoma resistance to doxorubicin by regulating TWIST1‑mediated epithelial‑mesenchymal transition.

Long non-coding RNAs are associated with cancer progression. Long intergenic non-protein coding RNA (linc)-regulator of reprogramming (ROR) enhances tumor development in hepatocellular carcinoma (HCC). However, the effect of chemoresistance and its underlying mechanisms in HCC are not completely understood. The present study aimed to identify the effect of ROR on sensitivity to doxorubicin (DOX) in HCC cells. In the present study, Cell Counting Kit-8 and EdU assays were performed to assess cell viability and proliferation, respectively. In addition, E-cadherin and vimentin protein expression levels were assessed via western blotting and immunofluorescence. The results of the present study demonstrated that HCC cells with high linc-ROR expression levels were more resistant to DOX, and linc-ROR knockdown increased HCC cell DOX sensitivity compared with the control group. The results indicated that compared with the NC siRNA group, linc-ROR knockdown notably suppressed epithelial-mesenchymal transition by downregulating twist family bHLH transcription factor 1 (TWIST1) expression. TWIST1 knockdown displayed a similar effect on HCC cell DOX sensitivity to linc-ROR knockdown. Moreover, linc-ROR knockdown-induced HCC cell DOX sensitivity was inhibited by TWIST1 overexpression. The present study provided evidence that linc-ROR promoted HCC resistance to DOX by inducing EMT via interacting with TWIST1. Therefore, linc-ROR might serve as a therapeutic target for reducing DOX resistance in HCC.

Transcription factor EB promotes rheumatoid arthritis of Sprague-Dawley rats via regulating autophagy.

This study investigated the effect of autophagy-related gene transcription factor EB (TFEB) on the rheumatoid arthritis (RA) and explored whether TFEB regulated RA by autophagy. The Sprague-Dawley rats were divided into two groups (n = 6). The rats were stimulated with the mixture of the type II collagen and Freund's adjuvant or PBS at the root of the tail. Results showed that swollen and deformed joints were discovered, the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were elevated, and hematoxylin and eosin staining showed the inflammatory cells infiltrate the synovial tissue in the RA rats, compared to the control group. Immunohistochemistry displayed that the expressions of TFEB and LC3B increased in the synovial tissues of RA rats, whereas p62 decreased. The silence of TFEB in the RA-fibroblast-like synoviocytes (RA-FLS) decreased the protein expressions of LC3B, compared to the siRNA NC group. Meanwhile, the activity of FLS was raised, whereas the levels of TNF-α and IL-6 decreased in RA-FLS with TFEB knockdown. In conclusion, our study revealed that TFEB plays a crucial role in the progress of RA by regulating autophagy, which might provide novel targets for the therapy of RA.

Roles of TNF receptor-associated and Fas-associated death domain proteins in the apoptosis of Eimeria tenella host cells

The present study aimed to investigate the effects of death receptor adapter proteins, namely, TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD) proteins, on Eimeria tenella-induced host cell apoptosis. Gene silencing, culture technique for primary chick embryo cecal epithelial cells, enzyme-linked immunosorbent assay, Hoechst–Annexin V/PI apoptosis staining, fluorescence quantitative PCR, and flow cytometry were used to detect the E. tenella host cell apoptotic rate, RIP1 and FADD protein expression levels, and caspase-8 activity of the TRADD siRNA-treated and FADD siRNA-treated groups. Results showed that the apoptotic rate in the TRADD siRNA group was significantly higher than that in the NC siRNA group at 4 h post-infection with E. tenella (P < 0.05). The RIP1 protein expression level in the TRADD siRNA group was significantly lower than that in the NC siRNA group at 4–24 h (P < 0.05). The FADD expression and apoptotic rates in the TRADD siRNA group were significantly lower than those in the NC siRNA group at 24–120 h (P < 0.05). The caspase-8 activity and apoptotic rates in the FADD siRNA group were significantly lower than those in the NC siRNA group (P < 0.05) at 24–120 h. These findings indicated that E. tenella inhibited the host cell apoptosis through the TRADD-RIP1 pathway at the early developmental stage and promoted host cell apoptosis via the TRADD-FADD-caspase-8 apoptotic pathway at the middle and late developmental stages.

Long Non-Coding RNA SNHG1 Regulates the Wnt/β-Catenin and PI3K/AKT/mTOR Signaling Pathways via EZH2 to Affect the Proliferation, Apoptosis, and Autophagy of Prostate Cancer Cell.

BackgroundProstate cancer (PCa) is the most common malignant cancer in western developed countries, which has seriously threatened the life style and life quality of men. Its pathogenesis and causes remain indistinct. Currently, it is found that lncRNA-SNHG1 (SNHG1) is highly expressed in multiple tumors with proto-oncogene effect, but its function and mechanism in PCa need to be further studied.MethodsThe expression of SNHG1 and EZH2 was detected by RT-qPCR in the 20 pairs of PCa tissue, adjacent tissue and PCa cell lines. They were transfected with siRNA NC, SNHG1 siRNA, EZH2 siRNA, SNHG1 siRNA+empty, and SNHG1 siRNA+EZH2 overexpression. Then, MTT and colony formation assay were used to detect the proliferation and cloning ability of PCa cells LNCaP and PC3. Transwell and flow cytometry were used to measure cell migration and invasion ability and apoptosis level respectively. Immunofluorescence was used to detect the LC3 spot formation. Western blot was used to detect the expression of the autophagy-related proteins, and PI3K/AKT/mTOR and Wnt/β-catenin signaling pathway related proteins. Finally, in vivo nude mice tumorigenesis experiment to explore the effect of SNHG1 expression on PCa.ResultsWe found that SNHG1 and EZH2 were up-regulated in PCa tissue and cells. The expression of SNHG1 and EZH2 was positively correlated. RNA pull down and RNA IP assay further confirmed that SNHG1 bound to EZH2. The proliferation, colony formation, migration and invasion of LNCaP and PC3 cells were significantly reduced with the interference with SNHG1or EZH2 compared with the control group. The related proteins of Wnt/β-catenin and PI3K/AKT/mTOR signaling pathway were significantly reduced after the interference with SNHG1 or EZH2; after simultaneous interference with SNHG1 and overexpression of EZH2, the functional effects on LNCaP and PC3 cells interfered with SNHG1 were reversed. These results were also confirmed in vivo nude mice tumor formation experiments.ConclusionsThis study reveals that lncRNA-SNHG1 regulates Wnt/β-catenin and PI3K/AKT/mTOR signaling pathways via EZH2 gene to affect proliferation, apoptosis and autophagy of PCa cells. This experiment provides ideas and experimental basis for the improvement and treatment of PCa.

Expression of fibroblast growth factor receptor like 1 protein in oral squamous cell carcinoma and its influence on tumor cell proliferation and migration

This study aims to investigate the expression of fibroblast growth factor receptor like 1 (FGFRL1) in oral squamous cell carcinoma (OSCC) and reveals its association with tumor cell proliferation and migration. Western blot was performed to detect the expression of FGFRL1 protein in OSCC tissues, adjacent normal tissues, OSCC cell lines and normal epithelial cells. After knocking down of FGFRL1 in HN4 cells, CCK-8 and Ki67 assays were performed to detect cell proliferation, wounding healing assay and transwell were performed to detect cell-migration. Western blot was used to detect the expression of protein related to epithelial-mesenchymal transition (EMT). The expression of FGFRL1 in OSCC tissues was higher than that in adjacent nontumor tissues, respectively (t=2.820, P=0.047 8). Moreover, the expression of FGFRL1 in OSCC cells was higher than that in HOK cells. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that FGFRL1 expression of FGFRL1 RNA in HOK cells was lower than that in OSCC cells. HN4 cells transfected with FGFRL1 siRNA were included in the experimental group, whereas HN4 cells treated with NC siRNA were included in the control group. CCK-8 experiment showed no significant difference between the experimental and control groups with regard to proliferation ability at 48 h (P=0.478 1) and 72 h (P=0.334 2). Migration experiment showed that the wound healing areas in the experimental group after 12 h (P=0.022 8), 24 h (P=0.005 1), and 36 h (P=0.009 5）were smaller than that in the control group. Transwell invasion assay showed that the number of invaded cells in the experimental group after 16 h (P=0.008 7) and 24 h (P=0.008 6) were lower than that in the control group. Knocking-down FGFRL1 up-regulated the expression of E-cadherin and down-regulated the expression of N-cadherin and Vimentin in HN4 cells. FGFRL1 expression in the OSCC tissues was significantly higher than that in the adjacent nontumor tissues. FGFRL1 expression in the OSCC cells was significantly higher than that in the HOK cells, and FGFRL1 had no effect on cell proliferation but promoted tumor cell migration and EMT.

Inhibition of miR-96 enhances the sensitivity of colorectal cancer cells to oxaliplatin by targeting TPM1.

Colorectal cancer (CRC) is one of the major threats to human health worldwide. In the treatment of CRC, chemoresistance affects the efficacy of platinum-based therapies. Oxaliplatin is one of the most commonly used first-line medications for the treatment of CRC; however, chemoresistance is common among patients receiving oxaliplatin treatment, which significantly decreases its therapeutic efficacy. The present study focused on the roles of microRNA (miR)-96 in the oxaliplatin resistance of CRC cells and the underlying mechanisms. First, the expression of miR-96 was compared between CRC and adjacent tissues. Furthermore, target genes of miR-96 were predicted, and a dual-luciferase reporter assay was employed to confirm whether the candidate tropomyosin 1 (TPM1) is a direct target of miR-96. In addition, CRC cells were transfected with miR-96 inhibitor, miR-negative control, small interfering RNA (siRNA) targeting TPM1 or siRNA NC, and then treated with oxaliplatin. CCK-8 assay and flow cytometry were performed to examine the proliferation and apoptosis of the CRC cell line SW480. Next, reverse transcription-quantitative PCR and western blot analysis were performed to determine the mRNA and/or protein levels of miR-96, Bcl-2, BAX and TPM1. The results indicated that miR-96 was upregulated in CRC compared with normal adjacent tissues, while TPM1 was downregulated. The luciferase activity was reduced following transfection with miR-96 mimics and luciferase reporter plasmid containing the wild-type sequence of the 3'-untranslated region of TPM1. Furthermore, knockdown of miR-96 combined with oxaliplatin reduced the viability and induced apoptosis of CRC cells, which was further verified by decreased expression of Bcl-2 and the increased expression of TPM1 and BAX. Taken together, the downregulation of miR-96 enhanced the sensitivity of CRC cells to oxaliplatin.

Correlation of Fibulin-2 expression with proliferation, migration and invasion of breast cancer cells.

Expression level of Fibulin-2 gene in breast cancer cells was evaluated to explore the impact of Fibulin-2 gene on the proliferation, migration and invasion of breast cancer cells. MDA-MB-231, BT483, MCF-7 and SK-BR-3 breast cancer cells were cultured in vitro. Then, expression of Fibulin-2 in cells was upregulated and downregulated using ribonucleic acid interference (RNAi) and lentiviral transfection techniques, respectively. Thereafter, expression levels of Fibulin-2 messenger RNA (mRNA) and protein were measured via quantitative real-time reverse transcription-polymerase chain reaction and western blotting, respectively. Cell Counting Kit-8 assay was applied to detect the proliferation ability, and wound healing assay was performed to determine the effect of transfection on the metastatic capacity of cells. The influence of transfection on the invasive ability of breast cancer cells was detected through Transwell chamber assay. MDA-MB-231 and MCF-7 cells did not express Fibulin-2, while BT483 and SK-BR-3 cells expressed Fibulin-2. Expression of Fibulin-2 mRNA and protein in SK-BR-3 Fibulin-2 siRNA group was significantly lower than that in SK-BR-3 NC siRNA group 48 h after transfection (P<0.01), while the expression of Fibulin-2 mRNA and protein in MDA-MB-231 Fibulin-2 lentiviral transfection (LAP) group was significantly higher than that in MDA-MB-231 NC LAP group. Compared with the MDA-MB-231 NC LAP group, the cell proliferation, migration and invasion ability of MDA-MB-231 Fibulin-2 LAP group were weakened. The tumor volume and weight of the MDA-MB-231 Fibulin-2 LAP group were significantly lower than those of the MDA-MB-231 NC LAP group. Low expression of Fibulin-2 is able to promote proliferation, migration and invasion of breast cancer cells, and can reduce the rate of tumor growth in nude mice. Therefore, Fibulin-2 may be a potential therapeutic target and an indicator of prognosis for breast cancer.

Nimodipine Improves Cognitive Impairment After Subarachnoid Hemorrhage in Rats Through IncRNA NEAT1/miR-27a/MAPT Axis.

BackgroundSubarachnoid hemorrhage (SAH) is a cerebral hemorrhage disease that severely damages the brain and causes cognitive impairment (CI). Therefore, accurate and appropriate treatment strategies are urgently needed. The application of nimodipine can not only improve blood circulation in patients with SAH but also repair ischemic neuron injury.PurposeTo investigate the effects of nimodipine and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1)/miR-27a/microtubule-associated protein tau (MAPT) axis on CI after SAH.MethodsOne hundred and twenty healthy male rats were selected and equally divided into control group, sham operation group, model group, PBS group, nimodipine group (drug group), NC siRNA group, NC mimics group, NEAT1 siRNA, miR-27a mimics, MAPT siRNA, drug + NEAT1-ad, and drug + NC-ad groups by random number table. Rats in the model group were constructed by double-hemorrhage model, and expression vectors were injected into the tail to regulate the expression of lncRNA NEAT1, miR-27a and MAPT. In addition, Western blot was employed to detect brain tissue protein, flow cytometry was applied to measure brain tissue apoptosis, and MTT was utilized to determine cell activity, so as to evaluate brain damage and cognitive function in each group.ResultsNimodipine, down-regulated lncRNA NEAT1, up-regulated miR-27a and down-regulated MAPT all improved brain damage and CI, inhibited brain tissue cell apoptosis, and enhanced brain cell activity. The common binding sites of lncRNA NEAT1 and MAPT were found on the miR-27a sequence fragment, and miR-27a could be paired with the former two. Nimodipine was found to cause the down-regulation of lncRNA NEAT1 and MAPT, as well as the up-regulation of miR-27a.ConclusionNimodipine can improve CI after SAH in rats through the lncRNA NEAT1/miR-27a/MAPT axis.