Dual Luciferase Reporter Gene System Research Articles

Mechanism through which the hsa-circ_0000992– hsa- miR- 936–AKT3 regulatory network promotes the PM2.5-induced inflammatory response in human bronchial epithelial cells

BackgroundStudies have shown that fine particulate matter (PM2.5) remains a significant problem in developing countries and plays a critical role in the onset and progression of respiratory illnesses. Circular RNAs (circRNAs) are involved in many pathophysiological processes,but their relationship to PM2.5 pollution is largely unexplored. ObjectivesTo elucidate the functional role of hsa_circ_0000992 in PM2.5-induced inflammation in a human bronchial epithelial cell line (16HBE) and to clarify whether the competing endogenous RNA (ceRNA) mechanism is involved in the interrelationships between hsa_circ_0000992 and hsa-miR-936 and the inflammatory signaling pathways. MethodsDetection of inflammatory factors in 16HBE cells exposed to PM2.5 by RT-qPCR and ELISA.High throughput sequencing and bioinformatics analysis methods were used to screen circRNA.The bioinformatics analysis method western blotting and dual-luciferase reporter gene system were used to verify mechanisms associated with circRNA. ResultsPM2.5 cause inflammation in the 16HBE cells. High throughput sequencing and RT-qPCR result revealed that the expression of hsa_circ_0000992 was markedly up-regulated in 16HBE exposed to PM2.5. The binding sites between hsa_circ_0000992 and hsa-miR-936 was confirmed by dual-luciferase reporter gene system.Western blotting and RT-qPCR showed that hsa_circ_0000992 can interact with hsa-miR-936 to regulate AKT serine/threonine kinase 3(AKT3),thereby activating the PI3K/AKT pathway and ultimately promoting the expression of interleukin (IL)− 1β and IL-8. ConclusionPM2.5 can induce the inflammatory response in 16HBE cells by activating the PI3K/AKT pathway. The expression of hsa_circ_0000992 increased when PM2.5 stimulated 16HBE cells,and the circRNA could then regulate the inflammatory response.Hsa_circ_0000992 regulates the hsa-miR-936/AKT3 axis through the ceRNA mechanism,thereby activating the PI3K/AKT signaling pathway,increasing the expression of cellular inflammatory factors,and promoting PM2.5-induced respiratory inflammation.

MiR-26b-5p promotes osteogenesis of bone mesenchymal stem cells via suppressing FGF21.

miR-26b-5p actively participates in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs). The database showed that fibroblast growth factor (FGF)-21 is a potential binding site of miR-26b-5p. This study aimed to investigate the molecular osteogenic mechanisms of miR-26b-5p targeting FGF21. Bone marrow was aspirated from the anterior superior iliac spine during bone marrow puncture. BMSCs were used to establish an in vitro cell model, and BMSCs markers were analyzed by flow cytometry. miR-26b-5p were overexpressed for 48 hours, and then placed in an osteogenic induction medium for osteogenic induction culture, the expression of RNA was detected using RT-qPCR. On day 7 of induction, RT-qPCR was used to measure Runx2, Osterix (Osx), and target gene FGF21 expression levels in each group. RT-qPCR, the dual-luciferase reporter gene system and western blot were used to verify that FGF21 was a direct target of miR-26b-5p. BMSCs were identified according to the antigenic characteristics. miR-26b-5p expression was significantly upregulated after the expression of miR-26b-5p mimics, and FGF21 expression was downregulated; in miR-26b-5p inhibitor, the opposite results were revealed. After overexpression of miR-26b-5p, the alkaline phosphatase activity and nodules of Alizarin red S in the culture medium was increased; the opposite results were revealed in miR-26b-5p inhibitor. The expressions of Runx2 and Osx in the miR-26b-5p group were also significantly higher; in the miR-26b-5p inhibitor group, the opposite results were revealed. Luciferase reporter assays demonstrated that FGF21 was a direct target of miR-26b-5p. The western blotting analysis showed that FGF21 expression was significantly downregulated in the miR-26b-5p overexpressed group. Finally, the expressions of the characteristic osteogenic factors in the miR-26b-5p control + FGF21 group was significantly lower, but then increased significantly in the miR-26b-5p mimics + FGF21 group; the expressions of the characteristic osteogenic factors in the miR-26b-5p control + si-FGF21 group was significantly higher. miR-26b-5p can regulate the osteogenic differentiation of BMSCs and participate in PMOP pathogenesis via suppressing FGF21.

Targeting the LINC00324/miR-16-5p/SEPT2 Signaling Cascade is Effective to Reverse Malignant Phenotypes in Glioblastoma.

Long non-coding RNAs (LncRNAs) are identified as pivotal regulators and biomarkers for glioblastoma (GBM). However, the role of a novel LncRNA LINC00324 in regulating GBM progression has not been fully studied in the existing publications. In this study, we evidenced LINC00324 to act as an oncogene to facilitate GBM development, and the underlying mechanisms have also been uncovered. Clinicopathology and follow-up data of GBM patients were retrospectively studied, LINC00324 expression in clinical tissue or cell lines of GBM was measured by Real-time qPCR, and the role of LINC00324 in cell proliferation and migration was investigated by loss-of-function experiments in vitro and in vivo. The targeting genes of LINC00324 were predicted and verified by bioinformatic analysis and dual luciferase reporter gene system, respectively. LINC00324 was found to be significantly upregulated in GBM tissues and cells in contrast to normal counterparts, and the GBM patients with high-expressed LINC00324 tended to have a worse prognosis. Further, loss-offunction experiments showed that the silencing of LINC00324 suppressed cell proliferation, colony formation and migration, and promoted cell apoptosis in GBM cells in vitro. Consistently, the in vivo experiments supported that LINC00324 ablation also restrained tumorigenesis in nude mice models. The following mechanism studies showed that LINC00324 sponged miR-16-5p to upregulate SEPT2 in a competing endogenous RNA-dependent manner, and the inhibitory effects of LINC00324 downregulation on the malignant characteristics of GBM cells were abrogated by both miR-16-5p ablation and SEPT2 overexpression. LINC00324 promotes the malignant phenotypes in GBM via targeting the miR-16-5p/SEPT2 axis, and the study provides novel biomarkers for GBM diagnosis and therapy.

New insights into triazole fungicide-caused hematopoietic abnormality in zebrafish based on GRα screening developmental toxicity

Triazole fungicides (TFs) are known to be common environmental contaminants that can be toxic to aquatic animals, but their developmental toxicity is not fully understood. To address this gap, we first used a glucocorticoid receptor α (GRα)-mediated dual luciferase reporter gene system to explore the possible development toxicity of ten TFs and found that flusilazole (FLU) exhibited stronger agonistic activity against GRα. Subsequent transcriptome sequencing showed that FLU exposure affected GRα activation and hematopoiesis associated with a variety of biological processes, including responses to corticosteroid release, embryonic hematopoiesis, erythroid differentiation, and the development of hematopoietic or lymphoid organs. Furthermore, based on in situ hybridization and staining techniques, we clarified that FLU decreased the expression of the primitive hematopoietic marker genes gata1 and pu.1. and caused the defects in the posterior blood island (PBI), thereby impacting intermediate hematopoietic processes. Also, FLU significantly reduced the expression of the crucial hematopoietic gene cmyb and disrupted the production of erythrocytes and bone marrow cells during definitive hematopoiesis. Consistently, we found that FLU induced lesions in the kidney, a hematopoietic organ, including the infiltration of inflammatory cells, tubular collapse, reduced tubular filtration area, and interstitial hydronephrosis. We also found that FLU increased aberrant red blood cells in the peripheral blood of zebrafish. These findings provide new insights into the developmental toxicity and ecotoxicological risk of TFs.

Effects of P70 S6 kinase on the ageing process and reproduction of Daphnia pulex (Branchiopoda, Anomopoda)

Abstract Daphnia pulex is a natural food widely used in aquaculture, and S6K plays an important role in protein translation and ageing. To explore the function of S6K in the ageing process of D. pulex, we cloned and analysed the full-length cDNA of S6K. With the growth of D. pulex, the expression of S6K mRNA first increased, then peaked at 10 days, and then decreased. Western blot analysis showed that the S6K protein expression increased from 1 to 5 days of age, but did not change significantly from 5 to 25 days of age. RNAi knockdown of S6K, reduced the expression of S6K mRNA and S6K protein, and it causes changes in other genes associated with ageing in both sexes. S6K may be involved in reproductive transformation, affecting reproductive capacity, thus significantly affecting the ageing of D. pulex. miRNA with potential regulation of the S6K gene were screened by the bioinformatics method and validated using a dual luciferase reporter gene system. This study deepens our understanding of the mechanisms of ageing in D. pulex and expands our understanding of the role of S6K in the ageing process.

LncRNA MBNL1-AS1 Suppresses Cell Proliferation and Metastasis of Pancreatic Adenocarcinoma through Targeting Carcinogenic miR-301b-3p.

Pancreatic adenocarcinoma (PAAD) has been a huge challenge to public health due to its increasing incidence, frequent early metastasis, and poor outcome. The molecular basis of tumorigenesis and metastasis in PAAD is largely unclear. Here, we identified a novel tumor-suppressor long noncoding RNA (lncRNA) MBNL1-AS1, in PAAD and revealed its downstream mechanism. Quantitative real-time PCR (qRT-PCR) data showed that MBNL1-AS1 expression was significantly downregulated in PAAD tissues and cells, which was closely associated with metastasis and poor prognosis. Cell counting kit-8 (CCK-8) assay, transwell assay, and western blot verified that overexpression of MBNL1-AS1 suppressed cell proliferation, migration, and epithelial mesenchymal transformation (EMT) behavior in PAAD cells. By using a dual luciferase reporter gene system, we confirmed that miR-301b-3p was a direct target of MBNL1-AS1. Further mechanismic study revealed that upregulation of miR-301b-3p abolished the inhibitory effect of MBNL1-AS1 overexpression on cell proliferation, tumorigenesis, migration and EMT. Our results demonstrate that MBNL1-AS1 plays a tumor-suppressive role in PAAD mainly by downregulating miR-301b-3p, providing a novel therapeutic target for PAAD.

Development and optimization of a cell screening system for farnesoid X receptor agonist

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

Donor and recipient polymorphisms of MAPK signaling pathway genes influence post-transplant liver function in Chinese liver transplant patients taking tacrolimus

Tacrolimus (TAC) is an immunosuppressive drug that is widely used for patients who underwent liver transplantation. In addition to inhibiting the action of calcineurin, TAC also exerts its immunosuppressive effects by interfering with mitogen activated protein kinase (MAPK) pathway. In this study, we investigated the impact of both recipient and donor genetic polymorphisms of MAPK kinase kinase (MAP3K) genes on clinical events in Han Chinese liver transplantation recipients taking TAC. Fifty-seven tag SNPs of 11 genes (MEKK1, MEKK2, MEKK4, MLK1, MLK3, ASK1, TAO1, TAO2, Tpl2, TAK1 and ZAK1) in the MAPK pathway were detected by MALDI-TOF MS assay in 175 TAC-treated liver transplant recipients. The associations of SNPs with incidence of acute rejection, TAC-induced acute nephrotoxicity, and post-transplantation liver and kidney function were explored using Kaplan-Meier survival analysis, Cox-proportional hazard model and linear mixed model, respectively. For the sites significantly associated with clinical events, the dual-luciferase reporter gene system was used to perform preliminary function verification. The results showed that (1) Donor-recipient combinational (D-R) MEKK1 rs62355944 and D-R MLK1 rs8006424 genotypes were significant influence factors of post-transplantation γ-glutamyl transpeptidase (GGT) level (P < 0.0001); (2) D-R MLK1 rs8006424 genotypes were found to significantly affect the alkaline phosphatase (ALP) level after transplantation (P < 0.0001). The results of the dual luciferase reporter gene system demonstrated that the luciferase activity of the pGL3-rs8006424A was significantly higher than that of pGL3-rs8006424G (3.47 ± 0.10 vs 2.97 ± 0.08, P = 0.002). Therefore, MEKK1 rs62355944 and MLK1 rs8006424 might serve as biomarkers to predict post-transplant liver function in liver transplant patients.

MiRNA-181a-5p inhibits proliferation and migration of osteosarcoma cell line HOS by targeting HOXB4

To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.

TGF-β1 contributes to the hepatic inflammation in animal models with nonalcoholic steatohepatitis by Smad3/TLR2 signaling pathway

Non-alcoholic fatty liver disease (NAFLD) is increasingly affecting human health and the economy worldwide due to various factors. Here, we found that the expression of TGF-β1 and TLR2 was significantly up-regulated in liver samples from both rats and mice nonalcoholic steatohepatitis (NASH) models. By constructing corresponding cell model, we found that TGF-β1 challenge can positively regulate the expression of TLR2 and p-Smad2/3, and the dual luciferase reporter gene system and EMSA assay confirmed the existence of Smad3 binding site (−916 ∼ −906) in the promoter region of TLR2. The overexpression and interference changes of Smad2/3 further verified the above experimental results. Taken together, these findings suggest that TGF-β1 promotes TLR2 transcription and its target gene expression via Smad3, leading to malignant exacerbation of liver inflammation in NASH, which provides new insights into the treatment of NASH.

MiR-33a targets FOSL1 and EN2 as a clinical prognostic marker for sarcopenia by glioma.

To determine the relationship between glioma and muscle aging and to predict prognosis by screening for co-expressed genes, this study examined the relationship between glioma and sarcopenia. The study identified eight co-downregulated miRNAs, three co-upregulated miRNAs, and seven genes associated with overall glioma survival, namely, KRAS, IFNB1, ALCAM, ERBB2, STAT3, FOSL1, and EN2. With a multi-factor Cox regression model incorporating FOSL1 and EN2, we obtained ROC curves of 0.702 and 0.709, respectively, suggesting that glioma prognosis can be predicted by FOSL1 and EN2, which are differentially expressed in both cancer and aged muscle. FOSL1 and EN2 were analyzed using Gene Set Enrichment Analysis to identify possible functional pathways. RT-qPCR and a dual-luciferase reporter gene system verified that hsa-miR-33a targets FOSL1 and EN2. We found that hsa-mir-33a co-targeting FOSL1 and EN2 has a good predictive value for glioblastoma and skeletal muscle reduction.

Long intergenic noncoding RNA LINC01287 drives the progression of cervical cancer via regulating miR-513a-5p/SERP1.

Cervical cancer is one of the most frequent types of cancer in women, which is characterized by high invasion and metastatic tendency in its advanced stage. Emerging evidence indicated that long non-coding RNAs (LncRNAs) are involved in the pathogenesis of cervical cancer. LINC01287 has been reported to play crucial regulatory roles in the pathogenesis and progression of multiple cancers. However, up until now, whether LINC01287 is associated with the initiation and development of cervical cancer remains largely unknown. In the present study, expression levels of LINC01287, miR-513a-5p and stress-associated endoplasmic reticulum protein 1 (SERP1) mRNA were quantified utilizing qRT-PCR. A series of functional experiments including CCK-8 assay, colony formation assay, transwell assay, flow cytometry, and tumor xenograft growth of cervical cancer cells were performed for studying the effects of LINC01287. The luciferase reporter assay, pull-down assay, and western blot were used to confirm the downstream targets of LINC01287 and miR-513a-5p. The results demonstrate that LINC01287 was highly expressed in cervical cancer tissue samples and cell lines. High LINC01287 predicts a poor prognosis for cervical cancer patients. Additional gain- and loss-of-function experiments demonstrated that silencing LINC01287 inhibited cervical cancer cells proliferation, colony formation, migration, apoptosis in vitro and retarded tumor growth and metastasis in vivo. Furthermore, the dual-luciferase reporter gene system and RNA pulldown assay validated that LINC01287 positively regulated SERP1 expressions by sponging miR-513a-5p, and LINC01287 inhibited cervical cancer progression by regulating miR-513a-5p/SERP1 axis. In conclusion, the current study first identified that LINC01287/miR-513a-5p/SERP1 axis played an important role in cervical cancer progression. LINC01287 might be a prognostic biomarker and a target for new therapies in patients with cervical cancer.

The role of miR-135b-5p in inhibiting mice acute lung injury (ALI) induced by sepsis and its mechanism

Objective: To investigate the expression status of miR-135b-5p in the sepsis induced acute lung injury (ALI) mice and its effects on inflammatory responses and cell pyroptosis in mice pulmonary tissues. Methods: The cecal ligation puncture (CLP) method was employed to construct sepsis-induced ALI mice models. The C57BL/6 mice were randomly divided into 6 groups with 8 mice in each group. The sepsis mouse models were constructed by performing CLP surgery: mice were anesthetized by intraperitoneal injection of 0.1 mg/kg barbital, the abdomen was cut longitudinally to expose the cecum, the cecum was ligated and perforated with syringe needle, the wound was sutured after extruding part of the intestinal contents. The sham operation group (Sham group) did not undergo any treatment and suture wounds after laparotomy, and no CLP operation was performed. The treatment groups were divided into CLP+NC mimic group, CLP+ miR-135b-5p mimic group, CLP+NC mimic+ Empty vector group, CLP+GSDMD group, and CLP+ miR-135b-5p mimic+GSDMD group. One week before CLP surgery, mice in the treatment group were injected subcutaneously with 200 μ L NC mimic (200 nmol/L), miR-135b-5p mimic (200 nmol/L), Empty vector (100 nmol/L), GSDMD Vector (100 nmol/L), and miR-135b-5p mimic (200 nmol/L), once a day for a week. The euthanasia was performed 24 h after operation by carbon dioxide asphyxiation. The qRT-PCR was utilized to determine miR-135b-5p and GSDMD expressions;HE staing assay was performed to observe the pathological changes of pulmonary tissues. The mice right lung tissues were flushed with 5 ml saline for 3 times, and each flush lasted for 3~5 min to collect the BALF, and the levels of GSDMD, IL-1β and IL-18 in BALF were determined by ELISA. The protein levels of NLRP3, caspase 1 and cleaved GSDMD in mice lung tissues were examined by immunoblotting analysis; Dual-luciferase reporter gene system assay was employed to validate the targeting relationship of miR-135b-5p and GSDMD. Results: Compared with the control group mice, there were a large number of inflammatory cell infiltration, alveolar damage, interstitial edema and alveolar collapse in the lung tissues of the CLP group mice (P<0.01), and the expression levels of the pyroptosis-associated proteins (NLRP3, caspase-1 and GSDMD) were all increased, while miR-135b-5p was down-regulated in the CLP group (P<0.01). Further experiments confirmed that overexpression of miR-135b-5p significantly reduced CLP-induced pyroptotic cell death in mice lung tissues (P<0.01), and dual-luciferase reporter gene system assay confirmed that miR-135b-5p targeted GSDMD for its degradation. Moreover, the rescuing experiments validated that up-regulation of GSDMD abrogated the inhibition effects of miR-135b-5p overexpression on cell pyroptosis in CLP mice lung tissues (P<0.01). Consistently, we verified that miR-135b-5p also suppressed the expression levels of IL-1β and IL-18 in mice BALF via degrading GSDMD (P<0.05). Conclusion: Overexpression of miR-135b-5p attenuated sepsis-induced ALI by inhibiting GSDMD-mediated pyroptotic cell death, and this study provided potential therapeutic target and theoretical foundation for sepsis-induced ALI treatment.

PLAC8 Overexpression Promotes Lung Cancer Cell Growth via Wnt/β-Catenin Signaling.

The PLAC8 expression in lung cancer tissues and in vitro grown lung cancer cells, as well as the involvement of the Wnt/β-Catenin signaling pathway, was investigated in this process. PLAC8 protein expression in human lung cancer tissues and lung tumor cells of different strains was discovered using immunohistochemistry staining and Western blot, respectively. Animal models of PLAC8 overexpression and knockdown were created using lentivirus. The development in tumor tissue was seen both in vitro and vivo. The Wnt/β-Catenin signaling pathway played an important part in this process, as shown by the dual luciferase reporter gene system. PLAC8 expression was elevated in lung cancer tissues and plasma and decreased in plasma after lung tumor resection. PLAC8 upregulation promotes cell proliferation in vivo and in vitro, while PLAC8 downregulation inhibits cell viability and proliferation. The results of the dual luciferase reporter gene system suggest that PLAC8 can significantly activate the Wnt/β-Catenin signaling pathway in cells and can conduct signaling through it. A potential treatment targeting the prognosis of lung cancer patients may be PLAC8 overexpression, which promotes the lung cancer cell proliferation through controlling the Wnt/β-Catenin signaling pathway.

A novel miR-338-3p/SLC1A5 axis reprograms retinal pigment epithelium to increases its resistance to high glucose-induced cell ferroptosis.

Oxidative stress-induced cell ferroptosis occurs during the pathogenesis of diabetic retinopathy (DR), but the detailed molecular mechanisms are still unclear. The present study aimed to investigate this issue. The retinal pigment epithelium (RPE) was treated with high glucose (30 mM) in vitro to mimic the realistic conditions of DR progression in vivo. Cell viability was determined by MTT assay and trypan blue staining assay. Gene expressions were examined by Real-Time qPCR and Western Blot analysis. FCM was used to detect cell apoptosis and ROS generation. Dual-luciferase reporter gene system assay was used to verify the targeting sites. High glucose increased reactive oxygen species (ROS) levels, promoted cell ferroptosis, and suppressed cell proliferation and viability in RPE, which were reversed by co-treating cells with both a ferroptosis inhibitor ferrostatin-1 and an ROS scavenger, N-acetyl-L-Cysteine (NAC). In addition, we screened out a miR-338-3p/ASCT2 (SLC1A5) axis that played an important role in this process. Mechanistically, miR-338-3p targeted the 3' untranslated regions (3'UTR) of SLC1A5 for its inhibition and degradation, and high glucose downregulated SLC1A5 by upregulating miR-338-3p in RPE cells. Next, the miR-338-3p inhibitor and SLC1A5 overexpression vectors were delivered into the RPE cells, and the following gain- and loss-of-function experiments validated that both miR-338-3p ablation and SLC1A5 upregulation abrogated the regulating effects of high glucose on cell proliferation, viability, ferroptosis and ROS production in RPE cells. Collectively, data in the present study indicated that targeting the miR-338-3p/SLC1A5 axis could block high glucose-induced ferroptosis in RPE cells.

MicroRNA-140–5p exacerbates vascular cognitive impairment by inhibiting neurogenesis in the adult mouse hippocampus after global cerebral ischemia

Vascular cognitive impairment (VCI) is the most common type of dementia after Alzheimer’s disease (AD). Effective treatments for VCI are currently lacking. MicroRNA (miR)− 140–5p is associated with cerebral ischemia and poststroke depression, but its relationship with VCI remains unknown. A VCI model was established by bilateral common carotid artery occlusion (BCCAO) for 17 min in mice. Neurogenesis was evaluated by immunostaining for Nestin/bromodeoxyuridine (BrdU), NeuN/BrdU, and doublecortin (DCX)/BrdU. Neuroplasticity was assessed by quantifying synapsin-I and postsynaptic density protein 95 (PSD-95) protein levels. Predicted target genes were screened and verified using the dual luciferase reporter gene system. MiR-140–5p was upregulated in the hippocampus of the BCCAO mice 2 weeks following ischemia. Compared with control groups, the AAV-miR-140–5p group exhibited poorer cognitive performance alongside lower numbers of DCX/BrdU and NeuN/BrdU and less synapsin-I and PSD-95 in the dentate gyrus (P < 0.05). MiR-140–5p overexpression decreased the predicted target gene Prox1. Dual luciferase reporter system confirmed that Prox1 was a direct target site for miR-140–5p. In conclusion, our results suggest that miR-140–5p inhibits neurogenesis and neuroplasticity via downregulation of Prox1 and aggravates VCI. Our findings highlight that miR-140–5p is involved in the pathological process of VCI and provides information for the development of new treatments, which may need further inhibition tests to verify.

Cellular Lnc_209997 suppresses Bombyx mori nucleopolyhedrovirus replication by targeting miR-275-5p in B. mori.

Long non-coding RNA (lncRNA) is a type of non-coding RNA molecule, which exceeds 200 nucleotides in length and participates in the regulation of a variety of life activities. Recent studies showed that lncRNAs play important roles in viral infection and host immunity. At present, the researches on insect lncRNAs are relatively few. In this study, we found the expression of Lnc_209997 was significantly down-regulated in silkworm fat body infected with Bombyx mori nucleopolyhedrosis virus (BmNPV). Inhibition of Lnc_209997 promoted BmNPV replication. Enhancing the expression of Lnc_209997 inhibited the proliferation of BmNPV. miR-275-5p was up-regulated in silkworm fat body infected with BmNPV. Dual luciferase reporter gene system confirmed the interaction between Lnc_209997 and miR-275-5p. Over-expression of Lnc_209997 inhibited the expression of miR-275-5p, while inhibition of Lnc_209997 enhanced the expression of miR-275-5p. Further, over-expression of miR-275-5p can facilitate the replication of BmNPV. These results suggested that BmNPV could increase the expression of miR-275-5p by inhibiting cellular Lnc_209997 expression to promote their own proliferation. Our results are helpful for better understanding the role of lncRNAs in BmNPV infection, and provide insights into elucidating the molecular mechanism of interaction between Bombyx mori and virus.

Zhilong Huoxue Tongyu Capsule Alleviated the Pyroptosis of Vascular Endothelial Cells Induced by ox-LDL through miR-30b-5p/NLRP3.

Background Our previous studies have demonstrated a protective role of Zhilong Huoxue Tongyu capsule in atherosclerosis (AS); however, the molecular mechanisms are unclear. Methods Human coronary artery endothelial cells (HCAECs) were induced with oxidized low-density lipoprotein (ox-LDL) to obtain cellular AS models. Then, the medicated serum of Zhilong Huoxue Tongyu capsule was obtained and used for treatment with ox-LDL-induced HCAECs. The cell viability was detected by CCK-8 assay. Besides, the binding between miR-30b-5p and NLRP3 was determined by the dual-luciferase reporter gene system assay. Furthermore, ox-LDL-induced HCAECs were transfected with miR-30b-5p mimic or miR-30b-5p inhibitor. The pyroptosis of HCAECs was assessed by flow cytometry, LDH content detection, and qRT-PCR assays. Results 10% medicated serum of Zhilong Huoxue Tongyu capsule was the maximum nontoxic concentration and it was used in subsequent assays. The rate of pyroptosis, LDH content, and the mRNA expression level of pyroptosis-related genes including NLRP3, ASC, Caspase 1, IL-1β, and IL-18 were prominently enhanced after HCAECs were induced by ox-LDL, which were markedly rescued with medicated serum of Zhilong Huoxue Tongyu capsule. In addition, the medicated serum of Zhilong Huoxue Tongyu capsule significantly enhanced the ox-LDL-induced reduction of miR-30b-5p level. NLRP3 could bind to miR-30b-5p and was negatively corrected with miR-30b-5p. Moreover, all the rates of pyroptosis, LDH content, and the mRNA expression levels of pyroptosis-related genes including NLRP3, ASC, Caspase 1, IL-1β, and IL-18 were further observably decreased after ox-LDL-induced HCAECs treated with medicated serum were transfected with miR-30b-5p mimic, while these were significantly rescued with transfection of miR-30b-5p inhibitor. Conclusion Zhilong Huoxue Tongyu capsule alleviated the pyroptosis of vascular endothelial cells induced by ox-LDL through miR-30b-5p/NLRP3.

MiR-98 Protects Nucleus Pulposus Cells against Apoptosis by Targeting TRAIL in Cervical Intervertebral Disc Degeneration.

The excessive apoptosis of nucleus pulposus (NP) cells is a major risk factor in the progress of cervical intervertebral disc degeneration (IVDD). In this study, we investigated the impact of miR-98 on apoptosis of NP cells and the potential molecular mechanisms. Lipopolysaccharide (LPS) was used to establish an NP cell IVDD model. The sponging effect of miR-98 on TRAIL 3′UTR was predicted by ENCORI and assessed by the dual-luciferase reporter gene system. The expression levels of miR-98, TRAIL, and TRAIL pathway-related genes were tested by qRT-PCR, Western blot, and immunofluorescence analysis. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell viability was analyzed by MTT assay. It was found that the expression level of miR-98 was downregulated, while the level of TRAIL was upregulated in IVDD tissues, and their levels were negatively and positively associated with the clinical MRI grade, respectively. The LPS treatment resulted in a significant decrease of the miR-98 expression level and an increase of the TRAIL expression level in NP cells. miR-98 reduced NP cell apoptosis under LPS treatment in vitro. miR-98 directly targeted TRAIL. Moreover, the mRNA and protein levels of DR5, FADD, cleaved caspase8, cleaved caspase3, and cleaved PARP were downregulated by miR-98 overexpression. Overexpression of TRAIL partially reversed the suppressive roles of miR-98 on cell apoptosis and activation of the TRAIL pathway. We concluded that miR-98 inhibited apoptosis of NP cells by inactivating the TRAIL pathway via targeting TRAIL in IVDD NP cells. These results indicated that miR-98 might be a therapeutic target for IVDD.

Hsa_circ_0001020 accelerates the lower extremity deep vein thrombosis via sponging miR-29c-3p to promote MDM2 expression

BackgroundDeep vein thrombosis (DVT) is a serious venous thromboembolism and leads the morbidity and mortality worldwide. Circular RNAs (circRNAs) sever as the important function biomarkers in various diseases, including DVT. However, the regulatory mechanism of circRNAs in DVT remains unclear. Here, we aimed to explore the function and potential mechanism of circRNAs in lower extremity deep vein thrombosis formation in DVT. MethodsQRT-PCR and western blot were performed to detect the expression of hsa_circ_0001020, miR-29c-3p, and MDM2 expression in human peripheral blood of DVT and endothelial progenitor cells (EPCs), respectively. Flow cytometry, RNA FISH and immunofluorescence detected the expression of distribution of circ_0001020 and CD31+ and CD34+ cells. RIP, RNA-pull down, and dual-luciferase reporter gene system were used to determine the binding relationship between hsa_circ_0001020, miR-29c-3p, and MDM2. Wound healing, transwell, and tube formation assays detected cell migration, invasion, and angiogenesis in vitro. DVT mice model was constructed to validate the function of hsa_circ_0001020, and H&E and Carstairs staining were performed to evaluate the pathology of inferior vena cava (IVC). ResultsHsa_circ_0001020 and MDM2 upregulated, whereas miR-29c-3p downregulated in DVT patients and mouse model. Hsa_circ_0001020 sponged miR-29c-3p to promote MDM2 expression thus inhibited EPC migration, invasion and tube formation. And the function of hsa_circ_0001020 and regulatory mechanism was demonstrated by the lose-function of hsa_circ_0001020 and rescue experiment. In DVT mice, hsa_circ_0001020 knockdown suppressed thrombosis and promoted homing ability of EPCs into thrombi. ConclusionOur finding demonstrated a novel signaling pathway involving hsa_circ_0001020, miR-29c-3p, MDM2, which might be a potential therapeutic target for DVT.