Dual-luciferase Reporter Gene Detection Research Articles

Feasibility study of lncRNA DHRS4-AS1 sponge miR-222-3p in the diagnosis of thyroid cancer.

Thyroid cancer is a commonly occurring malignant tumour within the endocrine system, the incidence of which has been increasing steadily in our country. It has been the focus and direction of research in recent decades to continuously explore the diagnostic markers and molecular mechanisms of thyroid cancer and provide new possibilities for the healing of patients. In this study, lncRNA DHRS4-AS1 was identified as the research target, and the regulatory function of abnormal expression of DHRS4-AS1 on thyroid cancer was discussed. Thyroid cancer (116) and non-cancer normal (82) tissue samples were collected in this paper, and the expression of DHRS4-AS1 and miR-222-3p in tissues and cells were evaluated by RT-qPCR. CCK-8 and flow cytometry were used to detect cell survival status. The mechanism of DHRS4-AS1 sponge miR-222-3p was analysed by dual-luciferase reporter gene detection. In the present study, DHRS4-AS1 was down-regulated in both thyroid tissue and cell samples, while miR-222-3p expression was elevated. The ROC curve reflected the diagnostic value of DHRS4-AS1 in thyroid cancer [area under the curve (AUC) = 0.887, sensitivity = 76.7%, specificity = 95.1%]. DHRS4-AS1 regulates the development of thyroid cancer by targeting miR-222-3p. In addition, in vitro experiments demonstrated that overexpression of DHRS4-AS1 (pcDNA3.1-DHRS4-AS1) inhibited the proliferation of thyroid cancer cells and promoted cell apoptosis, while down-regulating the level of miR-222-3p. DHRS4-AS1 acts as a miR-222-3p sponge in thyroid cancer, and overexpression of DHRS4 AS1 down-regulates cell proliferation and promotes cell apoptosis. These findings demonstrate the potential of DHRS4-AS1 as a diagnostic factor for thyroid cancer.

Treatment of Intracerebral Hemorrhage with Traditional Chinese Medicine Monomer Wogonin by Modifying NLRP3 with METTL14 to Inhibit Neuronal Cell Pyroptosis.

The aim of this study was to investigate the alleviating effect of wogonin on intracerebral hemorrhage (ICH) and its mechanism. The hemin-treated PC-12 cells were constructed to mimic ICH in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis was used for cell viability measurement and flow cytometry was for pyroptosis detection. Enzyme-linked immunosorbent assay (ELISA) assay and western blot were used to detect the protein levels of pyroptosis-related proteins. The modification level of N6-methyladenosine (m6A) methylation was detected by quantitative real-time polymerase chain reaction (qRT-PCR) combined with m6A dot blot assays. Molecular docking experiments analyzed the binding of wogonin and METTL14 protein. The correlation between METTL14 and NLRP3 was confirmed by bioinformatics analysis and dual luciferase reporter gene detection. ICH was induced in mice injected with collagenase into the basal ganglia, and the neurobehavioral damage was evaluated. Triphenyltetrazolium chloride monohydrate (TTC) staining and neurological scores were used to assess brain damage in mice. The results demonstrated that wogonin alleviated neuronal cell pyroptosis, and was molecularly docked with METTL14. Overexpression of METTL14 partly reversed the protecting effects of wogonin on brain in vitro and in vivo. Furthermore, NLRP3 was methylated by METTL14. Taken together, wogonin inhibits neuronal pyroptosis and thus treats IHC by inhibiting METTL14 and its methylated NLRP3.

The intervention effect of Aitongxiao prescription on primary liver cancer rats was evaluated based on high-throughput miRNA sequencing and bioinformatics analysis.

Liver cancer is a common malignant tumor known for its difficult treatment and poor prognosis. As a traditional Chinese medicine prescription, Aitongxiao prescription (ATXP) has been used in clinical treatment of primary liver cancer (PLC) for more than ten years, and its therapeutic effect is obvious and has been verified over time. However, the mechanism of ATXP in treating PLC has not been fully elucidated. This study aimed to detect the liver-protective effect of ATXP on a PLC rat model and explore its potential mechanism from the perspective of plasma extracellular vesicle miRNAs. Fifty SPF male SD rats were randomly selected, with six rats as the control group, and the remaining rats were injected with DEN to establish a primary liver cancer model. The model rats were randomly divided into the model group and the ATXP group. After 4 weeks of intervention, the liver-protective effect of ATXP was evaluated using plasma biochemical indicators and histopathological methods. Plasma extracellular vesicles were isolated and extracted, and identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Significant differentially expressed miRNAs in extracellular vesicles were screened by Illumina sequencing to explore the therapeutic targets of ATXP and conduct functional analysis. The results showed that ATXP significantly reduced plasma liver function in PLC rats and alleviated liver pathological damage. In addition, plasma extracellular vesicles were isolated and identified. According to the results of GO and KEGG analysis, they were related to multiple biological processes and covered multiple signaling pathways (PI3K-Akt and MAPK signaling pathways, etc.). The interaction between miR-199a-3p and MAP3K4 was determined by bioinformatics methods and dual-luciferase reporter gene detection, confirming that MAP3K4 is the target gene of miR-199a-3p. In conclusion, ATXP protects the liver from DEN-induced PLC, which may be related to the regulation of plasma extracellular vesicle miR-199a-3p. This study further reveals the mechanism of ATXP in treating liver cancer and provides a theoretical basis for subsequent research.

Up-regulation of oxidized low-density lipoprotein receptor 1 correlates with decreased miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465, higher recurrence rate, and poor prognosis in ovarian cancer.

MicroRNAs (miRNAs) are widely involved in cell metabolism, and their abnormal expression is involved in the regulation of ovarian cancer development, metastasis, and recurrence. The current study aimed to explore the potential mechanism and prognostic value of miRNAs related to the targeted regulation of oxidized low-density lipoprotein receptor 1 (OLR1) expression in ovarian cancer. A prospective study was conducted including 132 ovarian cancer patients. Patients were followed up for 36 months. The dual-luciferase reporter gene detection was used to verify the targeting relationship between miRNA and OLR1. Cell Counting Kit-8 assay, flow cytometric analysis, transwell migration, and invasion assays were performed to address the malignant biological behaviors of ovarian cancer cells. OLR1 protein and gene expression levels were significantly higher in ovarian cancer tissues and cell lines than in adjacent tissues and normal ovarian epithelial cells. OLR1 depletion facilitated apoptosis and impeded cell proliferation, migration, and invasion in ovarian cancer. Predictive software and dual-luciferase reporter assays showed that miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465 targeted OLR1 expression. Functionally, the introduction of miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465 mimics abrogated the aggressive phenotype in ovarian cancer cells. Lastly, compared with adjacent tissues, the levels of miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465 in cancer tissues were significantly lower (P<0.001). Compared with high miR-106b-5p, high miR-93-5p, high miR-3129-5p, high miR-199b-3p, high miR-4465 group and low OLR1 group, the low miR-106b-5p, low miR-93-5p, low miR-3129-5p, low miR-199b-3p, low miR-4465 group and high OLR1 group have significantly higher recurrence (all P<0.05) and higher mortality (all P<0.05). The identification value of recurrence assessment model [Y = 4.267+0.336*(miR-106b-5p)+0.168*(miR-93-5p)+1.847*(miR-3129-5p)+2.119*(miR-199b-3p)+0.872*(miR-4465)-3.408*(OLR1)] was high with an AUC of 0.918. The prognosis assessment model [Y = 3.914+0.143*(miR-106b-5p)+0.102*(miR-93-5p)+0.115*(miR-3129-5p)+1.369*(miR-199b-3p)+0.186*(miR-4465)-0.334*(OLR1)] also had high identification value with an AUC of 0.934. The combined detection of miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, miR-4465, and OLR1 is expected to become a molecular biomarker for the long-term prognostic assessment of ovarian cancer.

Circular RNA LPAR3 targets JPT1 via microRNA-513b-5p to facilitate glycolytic activation but repress prostate cancer radiosensitivity

A great many circular RNAs (circRNAs) are considered key modulators of human malignancies. However, the function of circRNA lysophosphatidic acid receptor 3 (LPAR3) in the radioresistance of prostate cancer (PCa) cells is still uncertain. circLPAR3, microRNA (miR)-329-3p, and JPT1 expression in PCa tissues and cells were detected by real-time quantitative PCR or western blot. Cell proliferation was detected by CCK-8 (cell proliferation assay) and colony formation assay, apoptosis was by flow cytometry, and migration and invasion ability were by Transwell assay. Cell glycolysis was analyzed by glucose uptake, lactate production, and ATP metabolism. Under different doses of radiation, the radiosensitivity of PCa cells was detected by colony formation assay. The relationship between circLPAR3, miR-513b-5p, and JPT1 was confirmed by dual luciferase reporter gene detection and RIP assay. The data presented that circLPAR3 and JPT1 expression was elevated in PCa, while miR-513b-5p expression was reduced. Repression of circLPAR3 depressed cell advancement, and restrained glycolysis, but enhanced the radiosensitivity of PCa cells. CircLPAR3's target miRNA was miR-513b-5p which targeted JPT1. Elevated JPT1 reversed the repressive effects of circLPAR3 knockdown or miR-513b-5p overexpression on PCa advancement, glycolysis, and radiosensitivity. In summary, the knockdown of circLPAR3 reduces glycolysis, but promotes PCa radiosensitivity via the miR-513b-5p/JPT1 axis, discovering a novel mechanism in PCa progression.

Peptide RL-QN15 promotes wound healing of diabetic foot ulcers through p38 mitogen-activated protein kinase and smad3/miR-4482-3p/vascular endothelial growth factor B axis.

Wound management of diabetic foot ulcers (DFUs) is a complex and challenging task, and existing strategies fail to meet clinical needs. Therefore, it is important to develop novel drug candidates and discover new therapeutic targets. However, reports on peptides as molecular probes for resolving issues related to DFUs remain rare. This study utilized peptide RL-QN15 as an exogenous molecular probe to investigate the underlying mechanism of endogenous non-coding RNA in DFU wound healing. The aim was to generate novel insights for the clinical management of DFUs and identify potential drug targets. We investigated the wound-healing efficiency of peptide RL-QN15 under diabetic conditions using in vitro and in vivo experimental models. RNA sequencing, in vitro transfection, quantitative real-time polymerase chain reaction, western blotting, dual luciferase reporter gene detection, in vitro cell scratches, and cell proliferation and migration assays were performed to explore the potential mechanism underlying the promoting effects of RL-QN15 on DFU repair. Peptide RL-QN15 enhanced the migration and proliferation of human immortalized keratinocytes (HaCaT cells) in a high-glucose environment and accelerated wound healing in a DFU rat model. Based on results from RNA sequencing, we defined a new microRNA (miR-4482-3p) related to the promotion of wound healing. The bioactivity of miR-4482-3p was verified by inhibiting and overexpressing miR-4482-3p. Inhibition of miR-4482-3p enhanced the migration and proliferation ability of HaCaT cells as well as the expression of vascular endothelial growth factor B (VEGFB). RL-QN15 also promoted the migration and proliferation ability of HaCaT cells, and VEGFB expression was mediated via inhibition of miR-4482-3p expression by the p38 mitogen-activated protein kinase (p38MAPK) and smad3 signaling pathways. RL-QN15 is an effective molecule for the treatment of DFUs, with the underlying mechanism related to the inhibition of miR-4482-3p expression via the p38MAPK and smad3 signaling pathways, ultimately promoting re-epithelialization, angiogenesis and wound healing. This study provides a theoretical basis for the clinical application of RL-QN15 as a molecular probe in promoting DFU wound healing.

MiRNA-139-3p inhibits malignant progression in urothelial carcinoma of the bladder via targeting KIF18B and inactivating Wnt/beta-catenin pathway.

Bladder cancer is a highly prevalent disease worldwide. We aimed to investigate the effect of miRNA/mRNA signaling on bladder urothelial carcinoma (BUC). MiRNA-139-3p wasselected from The Cancer Genome Atlas database, and its downstream target gene was predicted. The correlation between miRNA-139-3p and intersected mRNAs was analyzed. The mRNA expression levels of miRNA-139-3p and KIF18B in BUC were assayed via quantitative real-time polymerase chain reaction. Effects of miRNA-139-3p on cell proliferation, invasion, migration and cell cycle were detected via Cell Counting Kit-8, colony formation, transwell, wound healing and flow cytometry assays, respectively. Binding relationship between miRNA-139-3p and KIF18B was verified by dual-luciferase reporter gene detection. The protein expression levels of KIF18B, β-catenin and Cyclin D1 were detected by Western blot. Rescue assays were performed for verifying the interaction among miRNA-139-3p, KIF18B and Wnt/β-catenin signaling pathway, which revealed effects of miRNA-139-3p/KIF18B on BUC cells. MiRNA-139-3p was remarkably underexpressed, and KIF18B was dramatically overexpressed in BUC cells, respectively. It was also demonstrated that overexpressing miRNA-139-3p could prominently inhibit proliferation, invasion and migration of BUC, and block BUC cells at G0-G1 phase. Afterwards, we found that miRNA-139-3p could bind to KIF18B mRNA 3'UTR, and miRNA-139-3p had a negative regulatory effect with KIF18B. Subsequent experimental results presented that overexpressing KIF18B could reverse inhibitory effect of overexpressing miRNA-139-3p on BUC. Finally, this study also ascertained that miRNA-139-3p/KIF18B could repress oncogenic effects of BUC via modulating Wnt/β-catenin signaling pathway. MiRNA-139-3p/KIF18B/Wnt/β-catenin could significantly inhibit the malignant progression of BUC, and its targeting mechanism might provide an effective therapeutic target for BUC patients.

MiR-29c-3p represses gastric cancer development via modulating MEST.

Gastric cancer (GC) triggers a great number of deaths worldwide. Although great efforts have been made in treating this cancer, GC patients' survival rate remains unsatisfactory. An increasing amount of evidence indicates that miR-29c-3p inhibits cancer progression. However, the mechanism of miR-29c-3p in GC remains to be fully defined. Hence, this work aimed to analyze the underlying mechanism of miR-29c-3p in GC. Outcomes showed marked downregulation of miR-29c-3p in GC tissue and cell lines. Functional experiments exhibited that miR-29c-3p repressed GC cell malignant behaviors. Moreover, bioinformatics analysis and dual-luciferase reporter gene detection indicated that MEST was targeted by miR-29c-3p. Rescue assay further proved that MEST participated in functions of miR-29c-3p in GC. To sum up, miR-29c-3p/MEST signaling pathway suppressed formation of malignant phenotypes of GC, and targeting the signaling pathway may be a new method for treating GC.

Effects of salinity stress on methylation of the liver genome and complement gene in large yellow croaker (Larimichthys crocea)

Salinity is an important environmental factor that affects the yield and quality of large yellow croaker (Larimichthys crocea) during aquaculture. Here, whole-genome bisulfite sequencing (WGBS), RNA-seq, bisulfite sequencing PCR (BSP), quantitative real-time PCR (qPCR), and dual luciferase reporter gene detection technologies were used to analyze the DNA methylation characteristics and patterns of the liver genome, the expression and methylation levels of important immune genes in large yellow croaker in response to salinity stress. The results of WGBS showed that the cytosine methylation of CG type was dominant, CpGIsland and repeat regions were important regions where DNA methylation occurred, and the DNA methylation in upstream 2k (2000bp upstream of the promoter) and repeat regions had different changes in the liver tissue of large yellow croaker in the response to the 12‰, 24‰, 36‰ salinity stress of 4 w (weeks). In the combined analysis of WGBS and transcriptome, the complement and coagulation cascade pathways were significantly enriched, in which the complement-related genes C7, C3, C5, C4, C1R, MASP1, and CD59 were mainly changed in response to salinity stress. In the studied area of MASP1 gene promoter, the methylation levels of many CpG sites as well as total cytosine were strongly negatively correlated with mRNA expression level. Methylation function analysis of MASP1 promoter further proved that DNA methylation could inhibit the activity of MASP1 promoter, indicating that salinity may affect the expressions of complement-related genes by DNA methylation of gene promoter region.

MiR-424-5p targets HSP90AA1 to facilitate proliferation and restrain differentiation in skeletal muscle development

MiR-424-5p was found to be a potential regulator in the proliferation, migration, and invasion of various cancer cells. However, the effects and functional mechanism of miR-424-5p in the process of myogenesis are still unclear. Previously, using microRNA (miRNA) sequencing and expression analysis, we discovered that miR-424-5p was expressed differentially in the different skeletal muscle growth periods of Xuhuai goats. We hypothesized that miR-424-5p might play an important role in skeletal muscle myogenesis. Then, we found that the proliferation ability of the mouse myoblast cell (C2C12 myoblast cell line) was significantly augmented, whereas the C2C12 differentiation was repressed after increasing the expression of miR-424-5p. Mechanistically, HSP90AA1 presented a close interrelation with miR-424-5p, which was predicted as a target gene in the progression of skeletal muscle myogenesis, using transcriptome sequencing, dual luciferase reporter gene detection, and qRT-PCR. The silencing of HSP90AA1 obviously increased C2C12 proliferation and diminished differentiation, which is consistent with the ability of miR-424-5p in C2C12. Altogether, our findings indicated the role of miR-424-5p as a novel potential regulator via HSP90AA1 during muscle myogenesis progression.

MiR-129-5p Participates in Hair Follicle Growth by Targeting HOXC13 in Rabbit.

Mammalian hair formation is critically determined by the growth of hair follicles (HF). MiRNAs are crucial in the periodic development of hair follicles; they maintain epidermal homeostasis by targeting genes and influencing the activity of signaling pathways and related regulators. Our study discovered miR-129-5p to be overexpressed in the skin of Angora rabbits during catagen, and was negatively correlated with HOXC13 expression (Pearson’s R = −0.313, p < 0.05). The dual-Luciferase reporter gene detection system and Western blotting confirmed that miR-129-5p targeted HOXC13. In addition, miR-129-5p overexpression was found to significantly inhibit the expression of hair follicle development-related genes (HFDRGs), such as BCL2, WNT2, CCND1, and LEF1 (p < 0.01), and promoted the expression of SFRP2, TGF-β1, and FGF2 (p < 0.01), which was the same as the knockdown of HOXC13. In contrast, the knockout of miR-129-5p was the opposite, and it demonstrated similar results to the overexpression of HOXC13. CCK8 and flow cytometry demonstrated that miR-129-5p mimics significantly promoted the apoptosis of dermal papilla cells (DPCs) and inhibited proliferation (p < 0.01), while the inhibitor was found to reduce the apoptosis of DPCs and promote proliferation (p < 0.01). These results showed that miR-129-5p can participate in the periodic development of HF by targeting HOXC13, and it can induce apoptosis and inhibit proliferation of DPCs. These results will help to understand the role and mechanism of miR-129-5p in the periodic development of HF, and will provide support for subsequent studies, not only providing a theoretical basis for genetically improving the quality of hair in animals in the future, but also a new theory and method for diagnosing and treating hair loss in humans.

LncRNA ANRIL Promotes Autophagy Activation Through miR-16-5p/TLR4 Axis in Allergic Rhinitis.

Allergic rhinitis (AR) is an allergic disease of nasal mucosa. LncRNAs are key modulators affecting AR development. Neverthelss, the impact of lncRNA ANRIL in AR is not clear. This work decided to study the mechanism underlying the impact of ANRIL on TLR4 expression through targeting miR-16-5p during autophagy and epithelial barrier dysfunction in the progression of AR. Human nasal epithelial cells were exposed to TNF-α to establish AR cell model, AR mice model was constructed by ovalbumin (OVA) treatment. QRT-PCR or western blot assays were applied to measure the levels of mRNA and proteins. Dual-luciferase reporter gene detection and RIP assay were conducted to verify the association between ANRIL and miR-16-5p. Autophagy flux assessment by mRFP-GFP-LC3 method was performed to detect autophagy level. AR progression could induce the autophagy, and the expressions of tight junction proteins were downregulated in AR cell model. Moreover, knockdown of ANRIL reversed the effect of AR on autophagy-related protein and tight junction proteins MiR-16-5p was found to be bound with ANRIL and miR-16-5p inhibitor could reverse ANRIL knockdown-induced downregulation of autophagy-related proteins and epithelial barrier dysfunction. In addition, miR-16-5p directly targeted TLR4. Furthermore, knockdown of ANRIL reversed miR-16-5p and TLR4 expression, autophagy level, and tight junction protein levels in nasal mucosa of AR mice. This study illustrated that ANRIL acted as a promotion factor in AR induced autophagy and epithelial barrier dysfunction by enhancing the expression of TLR4 via interacting with miR-16-5p.

Exosomal miR-142-3p secreted by hepatitis B virus (HBV)-hepatocellular carcinoma (HCC) cells promotes ferroptosis of M1-type macrophages through SLC3A2 and the mechanism of HCC progression.

Most patients with hepatitis B virus (HBV) infection will develop hepatocellular carcinoma (HCC). This study aimed to explore the potential mechanism of miR-142-3p in HCC caused by HBV infection. HepG2 cells and M1 macrophages were cocultured and then infected with HBV to establish an in vitro model. MicroRNA (miRNA) and messenger RNA (mRNA) expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The protein expressions of COX2, ACSL4, PTGS2, GPX4, and NOX1 were analyzed by Western blot. Flow cytometry and TUNEL assays were used to assess cell reactive oxygen species (ROS) and ferroptosis, respectively. Cell invasion and migration were measured by Transwell assay. To evaluate the ferroptosis of M1-type macrophages, glutathione (GSH), malondialdehyde (MDA), and Fe2+ content was detected by corresponding kits. Dual luciferase reporter gene detection verified the targeting relationship between miR-142-3p and SLC3A2. MiR-142-3p was highly expressed in HBV-infected HCC patients and HBV-infected M1-type macrophages. Inhibition of miR-142-3p or overexpression of SLC3A2 reversed ferroptosis and inhibited the proliferation, migration, and invasion of HCC cells. Our findings indicated that miR-142-3p promoted HBV-infected M1-type macrophage ferroptosis through SLC3A2, affecting the production of GSH, MDA, and Fe2+ and accelerating the development of HCC. The regulation of miR-142-3p and its target genes will help to clarify the pathogenesis of HCC induced by HBV infection and provide new theoretical foundations and therapeutic targets.

MiR-1-3p targets CENPF to repress tumor-relevant functions of gastric cancer cells

Here we noted significantly downregulated miR-1-3p in gastric cancer (GC) tissue compared with adjacent normal tissue through qRT-PCR. Lowly expressed miR-1-3p correlated GC progression. Overexpressing miR-1-3p could restrain tumor-relevant cell behaviors in GC, while miR-1-3p inhibitor treatment triggered the opposite results. Moreover, dual-luciferase reporter gene detection identified specific binding sites of miR-1-3p in CENPF 3’untranslated region. Upregulating miR-1-3p constrained cell progression of GC via CENPF downregulation. Western blot, qRT-PCR and dual-luciferase detections manifested that miR-1-3p negatively mediated CENPF expression in GC cells. Thus, we demonstrated that miR-1-3p negatively mediated CENPF to hamper GC progression. CENPF may be an underlying target for GC therapy.

MicroRNA-181a Regulates the Proliferation and Differentiation of Hu Sheep Skeletal Muscle Satellite Cells and Targets the YAP1 Gene.

MicroRNA (miRNA) is of great importance to muscle growth and development, including the regulation of the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). In our research group’s previous study, we found that miR-181a is differentially expressed in the longissimus dorsi muscle of Hu sheep at different stages. We speculated that miR-181a may participate in the growth and development process of Hu sheep. To understand the mechanism of miR-181a regulating the growth and development of Hu sheep skeletal muscle, we extracted skeletal muscle satellite cells from the longissimus dorsi muscle of 3-month-old Hu sheep fetuses and performed a series of experiments. Our results showed that miR-181a suppressed SMSCs’ proliferation using QRT-PCR, Western blot, CCK-8, EDU, and Flow cytometry cycle tests. In addition, QRT-PCR, Western blot, and immunofluorescence indicated that miR-181a facilitated the differentiation of SMSCs. Then, we used dual-luciferase reporter gene detection, QRT-PCR, and Western blot to find that the Yes1-related transcription regulator (YAP1) is the target gene of miR-181a. Our study supplies a research basis for understanding the regulation mechanism of miR-181a on the growth of Hu sheep skeletal muscle.

Upregulation of microRNA-34a enhances myocardial ischemia-reperfusion injury via the mitochondrial apoptotic pathway

Mitochondrial oxidative injury can result in many cardiovascular diseases including cardiac ischemia-reperfusion (I/R) injury. This study was designed to investigate whether microRNA-34a (miR-34a) influences cardiac I/R or hypoxia/reoxygenation (H/R) injury by regulating the mitochondrial apoptotic pathway from oxidative injury.In vivo, myocardial infarction size was examined by Evan blue/TTC staining. Apoptosis was assessed by TUNEL assay. Heart function was measured by echocardiography. Lactate dehydrogenase (LDH) and creatine kinase (CK) were evaluated. In vitro, H9c2 cardiomyocytes were exposed to H/R stimulation. Cell viability was assessed by the CCK-8 assay and apoptosis was detected by Annexin V/PI staining. Mitochondrial superoxide, mitochondrial membrane potential (MMP) and ATP production was evaluated by detection kits, and related proteins were detected by western blotting analysis. We observed that the level of miR-34a was significantly upregulated in I/R rats compared to the sham group. Injection of adenovirus inhibiting miR-34a into the left ventricular anterior wall improved heart function and decreased I/R injury. H9c2 cardiomyocytes exposed to H/R stimulation displayed an obvious increase in miR-34a expression. In addition, miR-34a inhibitor alleviated, whereas miR-34a mimic aggravated H/R-induced mitochondrial injury. Bcl-2 was identified as a target gene of miR-34a by dual-luciferase reporter gene detection. Knockdown of Bcl-2 abolished the cardioprotection of the miR-34a inhibitor in H9c2 cells. In summary,our study demonstrates that inhibition of miR-34a exhibits therapeutic potential in treatment of myocardial I/R injury by restraining mitochondrial apoptosis.

Interaction between CASP8AP2 and ZEB2-CtBP2 Regulates the Expression of LEF1

Low expression of CTBP2 and CASP8AP2 correlated with poor outcome and predicted risk of relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to investigate the molecular mechanism by which CASP8AP2 regulates LEF1 expression by interacting with CtBP2 and ZEB2 in Acute lymphoblastic lymphoma (ALL). There was an interaction between CASP8AP2, ZEB2, and CtBP2, and then the interaction between CtBP2 and ZEB2 was observed after downregulating the expression of CASP8AP2. The wild type (containing the ZEB2 binding site) or mutant (containing a mutant binding site) LEF1 gene promoter sequence was inserted into the pGL3-basic plasmid, and a dual-luciferase reporter gene detection system was used to observe how CASP8AP2, ZEB2, and CtBP2 regulate the transcription of the LEF1 gene. We conclude that CASP8AP2, CtBP2, and ZEB2 can all bind to the LEF1 gene promoter region and reduce the luciferase activity of the LEF1 promoter. Meanwhile, the interaction of ZEB2 and the LEF1 promoter was significantly weakened after downregulation of CASP8AP2. Knockdown of CASP8AP2 in the 697 cell lines resulted in the significant upregulation of the mRNA expression levels of the stemness-related genes CD44, JAG1, and SALL4. In conclusion, CASP8AP2 is vital for the interaction between CtBP2 and ZEB2, inhibiting LEF1 and stemness-related genes expression ALL. Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2022.2033369 .

MiR-505 inhibits replication of Borna disease virus 1 via inhibition of HMGB1-mediated autophagy.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus which was recently demonstrated to cause deadly human encephalitis. Viruses can modulate microRNA expression, in turn modulating cellular immune responses and regulating viral replication. A previous study indicated that BoDV-1 infection down-regulated the expression of miR-505 in rats. However, the underlying mechanism of miR-505 during BoDV-1 infection remains unknown. In this study, we found that miR-505 can inhibit autophagy activation by down-regulating the expression of its target gene HMGB1, and ultimately inhibit the replication of BoDV-1. Specifically, we found that the expression of miR-505 was significantly down-regulated in rat primary neurons stably infected with BoDV-1. Overexpression of miR-505 can inhibit the replication of BoDV-1 in cells. Bioinformatics analysis and dual luciferase reporter gene detection confirmed that during BoDV-1 infection, the high-mobility group protein B1 (HMGB1) that mediates autophagy is the direct target gene of miR-505. The expression of HMGB1 was up-regulated after BoDV-1 infection, and overexpression of miR-505 could inhibit the expression of HMGB1. Autophagy-related detection found that after infection with BoDV-1, the expression of autophagy-related proteins and autophagy-related marker LC3 in neuronal cells was significantly up-regulated. Autophagy flow experiments and transmission electron microscopy also further confirmed that BoDV-1 infection activated HMGB1-mediated autophagy. Further regulating the expression of miR-505 found that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy. The discovery of this mechanism may provide new ideas and directions for the prevention and treatment of BoDV-1 infection in the future.

Knockdown of circRNA-Memo1 Reduces Hypoxia/Reoxygenation Injury in Human Brain Endothelial Cells Through miRNA-17-5p/SOS1 Axis.

Circ-Memo1 has been proved to be upregulated in ischemia-reperfusion induced acute injury of kidney tissues. However, the potential role of circ-Memo1 in cerebral hypoxia/reoxygenation (H/R) injury is still unclear.Blood samples were collected from 25 ischemic stroke patients and 25 healthy controls. To construct the H/R model, human brain microvascular endothelial cells (HBMVECs) were cultured under the hypoxic condition, followed by reoxygenation. Cell viability was analyzed by MTT assay. Flow cytometry was carried out to examine cell apoptosis. The level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured by MDA and SOD assay kits, respectively. The levels of TNF-α, IL-1β, and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter gene detection was employed to verify the binding relationships between circ-Memo1, miR-17-5p, and SOS1.Circ-Memo1 and SOS1 expressions were increased, and miR-17-5p expression was reduced in ischemic stroke patients. Circ-Memo1 silencing promoted cell viability, inhibited the activation of ERK/NF-κB signaling pathway, reduced oxidative stress and inflammatory response, and inhibited cell apoptosis. Moreover, miR-17-5p functioned as the sponge of circ-Memo1, and SOS1 was identified as the target of miR-17-5p. The protective effect of circ-Memo1 knockdown on cell injury after H/R treatment was weakened by miR-17-5p inhibition.Knockdown of circ-Memo1 alleviated H/R injury of HBMVEC cells by regulating the miR-17-5p/SOS1 axis, indicating that circ-Memo1 might be a potential treatment target for cerebral H/R injury.

MiR-937-3p promotes metastasis and angiogenesis and is activated by MYC in lung adenocarcinoma

BackgroundNon-small cell lung cancer (NSCLC) is still one of the diseases with the highest mortality and morbidity, and lung adenocarcinoma (LUAD) accounts for more than half of all NSCLC cases in most countries. miRNA can be used as a potential biological marker and treatment for lung adenocarcinoma. However, the effect of miR-937-3p to the invasion and metastasis of LUAD cells is not clear.MethodsmiRNA microarray is used to analyze the expression of miRNA in lung adenocarcinoma tissue. Transwell migration, Wound-healing assay and Western blot analysis are used to analyze cell migration, invasion and epithelial-mesenchymal transition (EMT) capabilities. Tube formation is used to assess angiogenesis ability. In addition, dual luciferase reporter gene detection is used to identify the potential binding between miRNA and target mRNA. In vivo experiments were performed on male NOD/SCID nude mice by tail vein injection to establish a transplanted tumor model. The CHIP experiment is used to verify the transcription factors of miRNA.ResultIn our study, miR-937-3p was high-regulated in LUAD cell lines and tissues, and its expression level was related to tumor progression. We found that miR-937-3p high-expression has an effect on cell invasion and metastasis. In molecular mechanism, miR-937-3p causes SOX11 reduction by directly binding to the 3′-UTR of SOX11.In addition, MYC affects miR-937-3p transcription by binding to its promoter region.ConclusionsOur research shows that miR-937-3p is mediated by MYC and can control the angiogenesis, invasion and metastasis of LUAD by regulating SOX11, thereby promoting the progress of LUAD. We speculate that miR-937-3p can be used as a therapeutic target and potential biomarker for LUAD.