RNA Binding Protein Immunoprecipitation Assay Research Articles

SLC26A4-AS1 Aggravates AngII-induced Cardiac Hypertrophy by Enhancing SLC26A4 Expression.

It has been reported that solute carrier family 26 members 4 antisense RNA 1 (SLC26A4-AS1) is highly related to cardiac hypertrophy. This research aims to investigate the role and specific mechanism of SLC26A4-AS1 in cardiac hypertrophy, providing a novel marker for cardiac hypertrophy treatment. Angiotensin II (AngII) was infused into neonatal mouse ventricular cardiomyocytes (NMVCs) to induce cardiac hypertrophy. Gene expression was detected by quantitative real-time PCR (RT-qPCR). Protein levels were evaluated via western blot. Functional assays analyzed the role of SLC26A4-AS1. The mechanism of SLC26A4-AS1 was assessed by RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and luciferase reporter assays. The P value <0.05 was identified as statistical significance. Student's t-test evaluated the two-group comparison. The difference between different groups was analyzed by one-way analysis of variance (ANOVA). SLC26A4-AS1 is upregulated in AngII-treated NMVCs and promotes AngII-induced cardiac hypertrophy. SLC26A4-AS1 regulates its nearby gene solute carrier family 26 members 4 (SLC26A4) via functioning as a competing endogenous RNA (ceRNA) to modulate the microRNA (miR)-301a-3p and miR-301b-3p in NMVCs. SLC26A4-AS1 promotes AngII-induced cardiac hypertrophy via upregulating SLC26A4 or sponging miR-301a-3p/miR-301b-3p. SLC26A4-AS1 aggravates AngII-induced cardiac hypertrophy via sponging miR-301a-3p or miR-301b-3p to enhance SLC26A4 expression.

LINC01002 Targets miR-650/FLNA Pathway to Suppress Prostate Cancer Progression

Introduction: In view of the vital implication of long noncoding RNAs in tumorigenesis, we possess the aim to determine the action effects and mechanisms of LINC01002 in prostate cancer (PCa). Methods: Expression level of LINC01002, miR-650, or filamin A (FLNA) in PCa tissues and cells was assessed using quantitative real-time PCR or Western blotting. Cell proliferative and migratory capacities were investigated by Cell Counting Kit-8 (CCK-8) and wound healing assays. Cell apoptosis was investigated by the levels of Bax and Bcl-2. Xenograft models were constructed to testify the role of LINC01002 in vivo. The anticipated binding of miR-650 to LINC01002 or FLNA was confirmed by dual-luciferase reporter or RNA binding protein immunoprecipitation assays. Results: Relatively poor expression of LINC01002 and FLNA, and high expression of miR-650 were identified in PCa tumor specimens and cells. Ectopic LINC01002 expression restrained PCa cell proliferation/migration and provoked apoptosis in vitro, and blocked solid tumor growth in Xenograft models. MiR-650 was directly targeted by LINC01002, and it also directly bound to FLNA. MiR-650 reintroduction in PCa cells overexpressing LINC01002 or FLNA partly reversed the anticancer effects of LINC01002 or FLNA overexpression, thus recovering PCa cell proliferation/migration and repressing apoptosis. Conclusion: LINC01002 deregulation was linked to PCa development. LINC01002 exerted potential anticancer effects in PCa via targeting the miR-650/FLNA pathway, which, at least in part, provided a basis for the involvement of LINC01002 as a therapeutic target in PCa.

Circular RNA hsa_circ_0007444 inhibits ovarian cancer progression through miR-23a-3p/DICER1 axis.

Ovarian cancer is the second leading cause of death in women with gynecological malignancy in China. Circular RNAs are a class of noncoding regulatory RNAs reported to be involved in cancer development and progression. Previous studies, including our own, have indicated that hsa_circ_0007444 is downregulated in ovarian cancer tissues. This study aims to elucidate the function and mechanism of hsa_circ_0007444 in ovarian cancer progression. The expression of hsa_circ_0007444 is determined by quantitative real-time PCR (qRT-PCR). Cell proliferation, invasion, migration and apoptosis are examined by cell counting-kit 8 (CCK-8), transwell and flow cytometry assays. Tumor growth and metastasis are assessed in vivo using Balb/c nude mouse xenograft model and tail vein injection model. And the mechanism of action of hsa_circ_0007444 is analysed by RNA-binding protein immunoprecipitation (RIP), luciferase reporter and rescue assays. hsa_circ_0007444 is downregulated in ovarian cancer tissues and cell lines compared with that in normal ovarian tissues and normal epithelial cell line. Gain- and loss-of-function results indicate that hsa_circ_0007444 inhibits cell proliferation, invasion, migration and increases cell apoptosis of ovarian cancer cells in vitro, and inhibits tumor growth and lung metastasis in vivo. Mechanistically, hsa_circ_0007444 can interact with AGO2 and sponge miR-23a-3p, thereby upregulating DICER1 expression, which is an important tumor suppressor in ovarian cancer. And miR-23a-3p mimics can rescue the inhibitory effect of hsa_circ_0007444 on ovarian cancer cell proliferation, invasion and migration. Therefore, hsa_circ_0007444 can inhibit ovarian cancer progression through the hsa_circ_0007444/miR-23a-3p/DICER1 axis.

MiR-199a-5P promotes osteogenic differentiation of human stem cells from apical papilla via targeting IFIT2 in apical periodontitis.

Periapical alveolar bone loss is the common consequence of apical periodontitis (AP) caused by persistent local inflammation around the apical area. Human stem cells from apical papilla (hSCAPs) play a crucial role in the restoration of bone lesions during AP. Studies have recently identified the critical role of microRNAs (miRNAs) involved in AP pathogenesis, but little is known about their function and potential molecular mechanism, especially in the osteogenesis of hSCAPs during AP. Here, we investigated the role of clinical sample-based specific miRNAs in the osteogenesis of hSCAPs. Differential expression of miRNAs were detected in the periapical tissues of normal and patients with AP via transcriptomic analysis, and the expression of miR-199a-5p was confirmed by qRT-PCR. Treatment of hSCAPs with miR-199a-5p mimics while loaded onto beta-tricalcium phosphate (β-TCP) ceramic particle scaffold to explore its effect on osteogenesis in vivo. RNA binding protein immunoprecipitation (RIP) and Luciferase reporter assay were conducted to identify the target gene of miR-199a-5p. The expression of miR-199a-5p was decreased in the periapical tissues of AP patients, and miR-199a-5p mimics markedly enhanced cell proliferation and osteogenic differentiation of hSCAPs, while miR-199a-5p antagomir dramatically attenuated hSCAPs osteogenesis. Moreover, we identified and confirmed Interferon Induced Protein with Tetratricopeptide Repeats 2 (IFIT2) as a specific target of miR-199a-5p, and silencing endogenous IFIT2 expression alleviated the inhibitory effect of miR-199a-5p antagomir on the osteogenic differentiation of hSCAPs. Furthermore, miR-199a-5p mimics transfected hSCAPs loaded onto beta-tricalcium phosphate (β-TCP) scaffolds induced robust subcutaneous ectopic bone formation in vivo. These results strengthen our understanding of predictors and facilitators of the key AP miRNAs (miR-199a-5p) in bone lesion repair under periapical inflammatory conditions. And the regulatory networks will be instrumental in exploring the underlying mechanisms of AP and lay the foundation for future regenerative medicine based on dental mesenchymal stem cells.

ETS1 regulates NDRG1 to promote the proliferation, migration, and invasion of OSCC.

The aim of this study was to investigate the molecular mechanism by which the transcription factor ETS1 regulates N-myc downstream regulatory gene 1 (NDRG1) to provide a new theoretical basis for the study of oral squamous cell carcinoma (OSCC). In this study, eight human OSCC and paraneoplastic samples were collected. The expressions of NDRG1, ETS1, and Ki67 were detected by immunohistochemistry; apoptosis was detected by tdt-mediated dUTP notched end labeling; cell migration and invasion were detected by Transwell; quantitative real-time PCR was performed to detect the expression of NDRG1; RNA-binding protein immunoprecipitation (RIP) assays detected NDRG1 expression; immunofluorescence assays detected ETS1 expression. NDRG1 and ETS1 expression was significantly upregulated in cancer tissues and CAL-27 and SCC-6 cells. Knockdown of NDRG1 and ETS1 inhibited cell proliferation, migration, invasion, cloning, and EMT while promoting apoptosis and inhibited tumor development; ETS1 positively regulated NDRG1 expression; Finally, overexpression of NDRG1 in vivo and in vitro reversed the effect of ETS1 knockdown on CAL-27 and SCC-6 cells. ETS1 positively regulates the expression of NDRG1 and promotes OSCC. Therefore, ETS1 may serve as a new target for the clinical diagnosis and treatment of OSCC.

TRIM25 inhibits HBV replication by promoting HBx degradation and the RIG-I-mediated pgRNA recognition.

The hepatitis B virus (HBV) vaccine has been efficiently used for decades. However, hepatocellular carcinoma caused by HBV is still prevalent globally. We previously reported that interferon (IFN)-induced tripartite motif-containing 25 (TRIM25) inhibited HBV replication by increasing the IFN expression, and this study aimed to further clarify the anti-HBV mechanism of TRIM25. The TRIM25-mediated degradation of hepatitis B virus X (HBx) protein was determined by detecting the expression of HBx in TRIM25-overexpressed or knocked-out HepG2 or HepG2-NTCP cells via Western blotting. Co-immunoprecipitation was performed to confirm the interaction between TRIM25 and HBx, and colocalization of TRIM25 and HBx was identified via immunofluorescence; HBV e-antigen and HBV surface antigen were qualified by using an enzyme-linked immunosorbent assay (ELISA) kit from Kehua Biotech. TRIM25 mRNA, pregenomic RNA (pgRNA), and HBV DNA were detected by quantitative real-time polymerase chain reaction. The retinoic acid-inducible gene I (RIG-I) and pgRNA interaction was verified by RNA-binding protein immunoprecipitation assay. We found that TRIM25 promoted HBx degradation, and confirmed that TRIM25 could enhance the K90-site ubiquitination of HBx as well as promote HBx degradation by the proteasome pathway. Interestingly, apart from the Really Interesting New Gene (RING) domain, the SPRY domain of TRIM25 was also indispensable for HBx degradation. In addition, we found that the expression of TRIM25 increased the recognition of HBV pgRNA by interacting with RIG-I, which further increased the IFN production, and SPRY, but not the RING domain is critical in this process. The study found that TRIM25 interacted with HBx and promoted HBx-K90-site ubiquitination, which led to HBx degradation. On the other hand, TRIM25 may function as an adaptor, which enhanced the recognition of pgRNA by RIG-I, thereby further promoting IFN production. Our study can contribute to a better understanding of host-virus interaction.

Circ-USP9X interacts with EIF4A3 to promote endothelial cell pyroptosis by regulating GSDMD stability in atherosclerosis

ABSTRACT Endothelial pyroptosis is a pathological mechanism of atherosclerosis (AS). Circular RNAs (circRNAs) are vital in AS progression by regulating endothelial cell functions. The study aimed to explore whether circ-USP9× regulated pyroptosis of endothelial cell to involve in AS development and the molecular mechanism. Pyroptosis was determined using lactate dehydrogenase (LDH) assay, enzyme linked immunosorbent assay (ELISA), flow cytometry, propidium iodide (PI) staining assay, and western blot. The mechanism of circ-USP9× was determined using RNA pull-down and RNA binding protein immunoprecipitation (RIP) assays. Results showed that circ-USP9× was upregulated in AS and oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). Knockdown of circ-USP9× suppressed ox-LDL induced pyroptosis of HUVECs. Mechanically, circ-USP9× could bind to EIF4A3 in the cytoplasm. Moreover, EIF4A3 was bound to GSDMD and further affects GSDMD stability. Overexpression of EIF4A3 rescued cell pyroptosis induced by circ-USP9× depletion. In short, circ-USP9× interacted with EIF4A3 to enhance GSDMD stability, thus further promoting ox-LDL-induced pyroptosis of HUVECs. These findings suggested that circ-USP9× participates in AS progression and may be a potential therapeutic target for AS.

CERS6 antisense RNA 1 promotes colon cancer via upregulating mitochondrial calcium uniporter.

Colon cancer (CC) belongs to a common cancer of digestive system. Long non-coding RNAs (lncRNAs) are dysregulated in numerous cancers and affect their development. The function of lncRNA CERS6 antisense RNA 1 (CERS6-AS1) in CC remains unclear. CERS6-AS1 expression in colon adenocarcinoma tissues and CC cell lines was assessed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction analysis. The function of CERS6-AS1 in CC was analysed by 5-ethynyl-2'-deoxyuridine, colony formation, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling, wound healing, Transwell and immunofluorescence assays. Mechanistic analyses including RNA pull down, RNA-binding protein immunoprecipitation and luciferase reporter assay revealed the interaction between RNAs. CERS6-AS1 expression was aberrantly upregulated in colon adenocarcinoma tissues and CC cell lines. CERS6-AS1 knockdown inhibited CC cell malignant phenotypes and in vivo tumour growth. CERS6-AS1 served as the competing endogenous RNA of microRNA-16-5p in CC, and microRNA-16-5p inhibition partly rescued the effects of CERS6-AS1 depletion on CC development. Mitochondrial calcium uniporter was targeted by microRNA-16-5p. Mitochondrial calcium uniporter upregulation completely remedied the influence of CERS6-AS1 silencing in CC progression. Moreover, CERS6-AS1 enhanced the stability of mitochondrial calcium uniporter messenger RNA via recruiting RNA-binding protein embryonic lethal abnormal vision like 1. CERS6-AS1 promotes the development of CC via upregulating mitochondrial calcium uniporter expression.

Berberine promotes IGF2BP3 ubiquitination by TRIM21 to induce G1/S phase arrest in colorectal cancer cells

The increasing incidence of colorectal cancer (CRC) has become a major global public health burden. The natural drug Berberine (BBR) has shown potential in preventing CRC, and IGF2 mRNA binding protein 3 (IGF2BP3) may be a target of BBR. This study aims to investigate the mechanisms of BBR acting on IGF2BP3 to improve CRC. The results showed that IGF2BP3 played an important role in the development of CRC. BBR down-regulated IGF2BP3 expression and inhibited CRC growth in mice. Cell thermodynamic stability analysis (CETSA) and drug affinity responsive target stability (DARTS) analysis showed BBR may bind to IGF2BP3. BBR may induce structural changes in IGF2BP3 and decrease its protein stability in cytoplasm. The results from Co-Immunoprecipitation (Co-IP) suggested that BBR promoted the ubiquitination of IGF2BP3 by tripartite motif-containing protein 21 (TRIM21). Through RNA binding protein Immunoprecipitation (RIP) assay, it was found BBR inhibited the stabilization of CDK4/CCND1 mRNA by IGF2BP3 and promoted G1/S phase arrest in CRC cells. Overexpression of IGF2BP3 in vitro and in vivo attenuated the inhibition of CRC growth by BBR. This work demonstrated the potential of BBR targeting to IGF2BP3 in improving CRC and provided a new strategy for clinical treatment on CRC as well as novel anticancer drug design based on IGF2BP3 and TRIM21.

Differential effects of the LncRNA RNF157-AS1 on epithelial ovarian cancer cells through suppression of DIRAS3- and ULK1-mediated autophagy

Analyses of several databases showed that the lncRNA RNF157 Antisense RNA 1 (RNF157-AS1) is overexpressed in epithelial ovarian cancer (EOC) tissues. In our study, suppressing RNF157-AS1 strikingly reduced the proliferation, invasion, and migration of EOC cells compared with control cells, while overexpressing RNF157-AS1 greatly increased these effects. By RNA pulldown assays, RNA binding protein immunoprecipitation (RIP) assays, and mass spectrometry, RNF157-AS1 was further found to be able to bind to the HMGA1 and EZH2 proteins. Chromatin immunoprecipitation (ChIP) assays showed that RNF157-AS1 and HMGA1 bound to the ULK1 promoter and prevented the expression of ULK1. Additionally, RNF157-AS1 interacted with EZH2 to bind to the DIRAS3 promoter and diminish DIRAS3 expression. ULK1 and DIRAS3 were found to be essential for autophagy. Combination autophagy inhibitor and RNF157-AS1 overexpression or knockdown, a change in the LC3 II/I ratio was found using immunofluorescence (IF) staining and western blot (WB) analysis. The autophagy level also was confirmed by autophagy/cytotoxicity dual staining. However, the majority of advanced EOC patients require platinum-based chemotherapy, since autophagy is a cellular catabolic response to cell stress. As a result, RNF157-AS1 increased EOC cell sensitivity to chemotherapy and death under cis-platinum (DDP) treatment by suppressing autophagy, as confirmed by cell count Kit-8 (CCK8) assays, flow cytometry, and autophagy/cytotoxicity dual staining. Therefore, the OS and PPS times were longer in EOC patients with elevated RNF157-AS1 expression. RNF157-AS1-mediated autophagy has potential clinical significance in DDP chemotherapy for EOC patients.

LncRNA LINC01094 Promotes Cells Proliferation and Metastasis through the PTEN/AKT Pathway by Targeting AZGP1 in Gastric Cancer

Long noncoding RNAs (lncRNAs) were recently reported to play an essential role in multiple cancer types. Herein, through next-generation sequencing, we screened metastasis-driving molecules by using tissues from early-stage gastric cancer (GC) patients with lymph node metastasis, and we identified a lncRNA LINC01094, which was associated with the metastasis of GC. According to the clinical data from the TCGA, GSE15459, and GSE62254 cohorts, the high expression of LINC01094 was associated with an unfavorable prognosis. Moreover, 106 clinical GC and paired normal samples were collected, and the qRT-PCR results showed that the high expression of LINC01094 was associated with high T and N stages and a poor prognosis. We found that LINC01094 promotes the proliferation and metastasis of GC in vitro and in vivo. AZGP1 was found as the protein-binding partner of LINC01094 by using RNA pulldown and RNA-binding protein immunoprecipitation (RIP) assays. LINC01094 antagonizes the function of AZGP1, downregulates the expression of PTEN, and further upregulates the AKT pathway. Collectively, our results suggested that LINC01094 might predict the prognosis of GC patients and become the therapy target for GC.

Polypyrimidine tract binding protein 1 exacerbates cardiac fibrosis by regulating fatty acid-binding protein 5.

The activation of cardiac fibroblasts (CFs) leads to overproduction of collagens and subsequently cardiac fibrosis. However, the regulatory mechanism of CF function in the process of cardiac fibrosis remains unclear. This work investigated the function of polypyrimidine tract binding protein 1 (PTBP1)/nuclear receptor NR4A1 (Nur77)/fatty acid-binding protein 5 (FABP5) axis in myocardial fibrosis. Cardiac fibrosis was induced in mice suffered left anterior descending ligation. In parallel, neonatal mouse CFs were isolated and stimulated with transforming growth factor-β1 (TGF-β1). Cardiac fibrosis was evaluated by Masson's trichrome staining. Expression of PTBP1, Nur77, FABP5, collagen I, and collagen III was measured by quantitative real-time PCR and western blotting. Proliferation of CFs was assessed by 5-ethynyl-2'-deoxyuridine assay. Molecular interaction was validated by RNA-binding protein immunoprecipitation, chromatin immunoprecipitation, and dual luciferase reporter assay. PTBP1 was up-regulated (P<0.05), whereas Nur77 (P<0.05) and FABP5 (P<0.05) were down-regulated in the fibrotic hearts of mice and TGF-β1-exposed CFs. PTBP1 overexpression facilitated proliferation (P<0.05) and collagen I (P<0.05) and collagen III (P<0.05) expression of CFs after stimulation with TGF-β1. PTBP1 reduced Nur77 stability (P<0.05) to inhibit Nur77 expression (P<0.05) in CFs. Nur77 bound to FABP5 promoter to promote the transcription (P<0.05) and expression (P<0.05) of FABP5. Silencing of Nur77 or FABP5 abolished the inhibitory effect of PTBP1 knockdown on proliferation (P<0.05) and collagen I (P<0.05) and collagen III (P<0.05) expression of CFs in vitro. PTBP1 depletion ameliorated cardiac fibrosis (P<0.05), α-smooth muscle actin (P<0.05), and collagen I (P<0.05) expression in myocardial infarction mice through regulating Nur77/FABP5 pathway (P<0.05) in vivo. PTBP1 contributed to cardiac fibrosis via promoting CF proliferation and collagen deposition through Nur77 mRNA decay and subsequent transcription inhibition of FABP5. Our findings suggest that PTBP1/Nur77/FABP5 axis may be potential targets for cardiac fibrosis therapy.

SNHG1 functions as a ceRNA in hypertrophic scar fibroblast proliferation and apoptosis through miR-320b/CTNNB1 axis.

Hypertrophic scar (HS) is a fibrotic disease caused by skin injury. Competing endogenous RNA (ceRNA) has been demonstrated to implicate in the regulation of cell malignant phenotypes. This research aims to reveal the effect of catenin beta 1 (CTNNB1) on the functions of hypertrophic scar fibroblasts (HSFBs) and its role in a ceRNA network. RNA expression level was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proliferation and apoptosis of HSFB was detected via Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis. Mechanism experiments included RNA pull down assay, luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were applied to analyze the upstream molecular mechanism of CTNNB1. CTNNB1 was highly expressed in HSFB. CTNNB1 depletion repressed malignant growth of HSFB. Mechanically, CTNNB1 was targeted by microRNA-320b (miR-320b) in HSFB. Small nucleolar RNA host gene 1 (SNHG1) aced as a ceRNA to upregulate CTNNB1 expression via sponging miR-320b in HSFB. CTNNB1 overexpression could reverse the impact of SNHG1 depletion on the proliferation and apoptosis of HSFB. SNHG1 acts as a ceRNA in modulating HSFB proliferation and apoptosis through miR-320b/CTNNB1 axis. SNHG1 act as a ceRNA to promote HSFB growth by sponging miR-320b to upregulate CTNNB1.

MetBil as a novel molecular regulator in ischemia-induced cardiac fibrosis via METTL3-mediated m6A modification.

Cardiac fibrosis is a common pathological manifestation in multiple cardiovascular diseases and often results in myocardial stiffness and cardiac dysfunctions. LncRNA (long noncoding RNA) participates in a number of pathophysiological processes. However, its role in cardiac fibrosis remains unclear. The purpose of this study was to investigate the role and molecular mechanism of MetBil in regulating cardiac fibrosis. Our data showed that METTL3 binding lncRNA (MetBil) was significantly increased both in fibrotic tissue following myocardial infarction (MI) in mice and in cardiac fibroblasts (CFs) exposed to TGF-β1 (20 ng/mL) or 20% FBS. Overexpression of MetBil augmented collagen deposition, CF proliferation and activation while silencing MetBil exhibited the opposite effects. Importantly, heterozygous knockout of MetBil alleviated cardiac fibrosis and improved cardiac function after MI. RNA pull-down and RNA-binding protein immunoprecipitation assay showed that METTL3 is a direct downstream target of MetBil; consistently, MetBil and METTL3 were co-localized in both the nucleus and cytoplasm of CFs. Interestingly, MetBil regulated METTL3 expression at protein level, but not mRNA level, in ubiquitin-proteasome pathway. Enforced expression of METTL3 canceled the antifibrotic effects of silencing MetBil reflected by increased collagen production, CF proliferation and activation. Most notably, the m6A-modified fibrosis-regulated genes mediated by METTL3 are profoundly involved in the regulation of MetBil in the cardiac fibrosis following MI. Our study reveals that MetBil as a novel regulator of fibrosis promotes cardiac fibrosis via interacting with METTL3 and regulating the expression of the methylated fibrosis-associated genes, providing a new intervening target for fibrosis-associated cardiac diseases.

NUCLEOLIN PROTECTS CARDIOMYOCYTES BY UPREGULATING PGC-1α AND PROMOTING MITOCHONDRIAL BIOGENESIS IN LPS-INDUCED MYOCARDIAL INJURY.

Background: Lipopolysaccride-induced myocardial injury was characterized by frequent mitochondrial dysfunction. Our previous studies found that nucleolin (NCL) played important protective roles in myocardial ischemia-reperfusion injury. Recently, it has been found that NCL has a protective effect on LPS-induced myocardial injury in vivo . However, the exact underlying mechanisms that how NCL protects myocardium against the LPS-induced myocardial injury remains unclear. Objective: The aim of the study is to investigate the protective role of NCL in LPS-induced myocardial injury from the aspect of mitochondrial biogenesis. Methods: The cardiac-specific NCL-knockout (NCL -/- ) or NCL f/f mice were injected with LPS (10 mg/kg) to induce LPS-induced myocardial injury. The supernatant generated after LPS stimulation of macrophages was used as the conditioned medium to stimulate H9C2 and established the injured cell model. Analysis of mRNA stability, RNA-binding protein immunoprecipitation assay, and luciferase reporter assay were performed to detect the mechanism by which NCL regulated the expression of PGC-1α. Results: The expression of NCL and PGC-1α was elevated in cardiac tissue and cardiomyocytes during LPS-induced myocardial injury. The cardiac-specific NCL-knockout decreased PGC-1α expression, inhibited mitochondrial biogenesis, and increased cardiomyocytes death during LPS-induced myocardial injury in vitro and in vivo . In contrast, the overexpression of NCL could improve mitochondrial biogenesis in H9C2 cells. Moreover, the analysis of mRNA stability and luciferase reporter assay revealed that the interaction between NCL and PGC-1α significantly promoted the stability of PGC-1α mRNA, thereby upregulating the expression of PGC-1α and exerting a cardioprotective effect. In addition, the activation of PGC-1α diminished the detrimental effects of NCL knockdown on mitochondrial biogenesis in vitro and in vivo . Conclusions: Nucleolin upregulated the gene expression of PGC-1α by directly binding to the 5'-UTR of PGC-1α mRNA and increasing its mRNA stabilities, then promoted mitochondrial biogenesis, and played protective effect on cardiomyocytes during LPS-induced myocardial injury. Taken together, all these data showed that NCL activated PGC-1α to rescue cardiomyocytes from LPS-induced myocardial injury insult, suggesting that the cardioprotective role of NCL might be a promising prospect for clinical treatment of patients with endotoxemia.

Downregulation of LncRNA SNHG7 Sensitizes Colorectal Cancer Cells to Resist Anlotinib by Regulating miR-181a-5p/GATA6.

Long noncoding RNAs are a novel class of regulators in human cancers. It has been reported that small nucleolar RNA hostgene 7 (SNHG7) can sponge microRNAs to regulate colorectal cancer (CRC) progression. Given its important regulatory role in cancer biology, we wondered whether SNHG7 is involved in drug resistance to anlotinib (ATB) in CRC. To answer this, we quantified the expression of SNHG7 by quantitative real-time PCR. We performed the Cell Counting Kit-8 and Colony formation assay, flow cytometric analysis, RNA pull-down, RNA-binding protein immunoprecipitation assay, and Luciferase reporter assay to confirm the interaction among SNHG7, miR-181a-5p, and GATA6. We found that SNHG7 was significantly upregulated in CRC tissues and cell lines and ATB-resistant cell lines, which was closely related to the poor overall survival of patients. Loss-of-function studies demonstrated that SNHG7 knockdown can inhibit CRC cell proliferation, increase apoptosis, and sensitize CRC cells to resist ATB. Mechanistic studies showed that SNHG7 acted as a competitive endogenous RNA to sponge miR-181a-5p to regulate the expression of GATA6, thereby promoting ATB resistance in ATB-resistant cell lines. In conclusion, SNHG7 plays an important role in ATB resistance, and it may be used to monitor ATB resistance in CRC.

LncRNA NEAT1 inhibits apoptosis and autophagy of ovarian granulosa cells through miR-654/STC2-mediated MAPK signaling pathway

Long non-coding RNA (lncRNA) anomalies cause early ovarian failure. LncRNA nuclear enriched abundant transcript 1 (NEAT1) was down-regulated in premature ovarian failure (POF) mice and connected to the illness, however, the mechanism remained unclear. The levels of gene and protein were measured by using quantitative real-time polymerase chain reaction, Western blot, and immunofluorescence. Follicle stimulating hormone (FSH), estradiol (E2), and luteinizing hormone (LH) levels were determined using enzyme-linked immunosorbent assay (ELISA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry were used to determine cell viability and apoptosis. The interaction of NEAT1, miR-654, and stanniocalcin-2 (STC2) was verified by dual-luciferase reporter assay or RNA binding protein immunoprecipitation (RIP) assays. The results showed NEAT1 and STC2 down-regulated, while miR-654 up-regulated in POF mice. Overexpression of NEAT1 reduced apoptosis and autophagy in cyclophosphamide (CTX)-treated ovarian granulosa cells (OGCs), and Bax, cleaved-caspase3, LC3B, LC3II/LC3I ratio were decreased and Bcl-2 and p62 were raised. NEAT1 suppressed miR-654 expression by directly targeting miR-654. The inhibition of NEAT1 overexpression on apoptosis and autophagy in OGCs was reversed by miR-654 mimics. STC2 was a target gene of miR-654, and miR-654 inhibitor reduced the apoptosis and autophagy by regulating the STC2/MAPK axis. To sum up, NEAT1 reduced miR-654 expression and modulated the STC2/MAPK pathway to decrease apoptosis and autophagy in POF, indicating a potential therapeutic target.

Long non-coding RNA NMRAL2P promotes glycolysis and reduces ROS in head and neck tumors by interacting with the ENO1 protein and promoting GPX2 transcription.

Metabolic reprogramming is a key marker in the occurrence and development of tumors. This process generates more reactive oxygen species (ROS), promoting the development of oxidative stress. To prevent ROS from harming tumor cells, tumor cells can increase the production of reducing agents to counteract excessive ROS. NMRAL2P has been shown to promote the production of reductive mRNA and plays an important role in the process of oxidative stress. In this study, the clinical data and RNA sequencing of head and neck tumors were obtained from The Cancer Genome Atlas data set. The long non-coding RNA (LncRNA) related to oxidative stress were then identified using differential and correlation analyses. The differential expression and prognosis of the identified lncRNA were then verified using samples from the library of the Second Hospital of Hebei Medical University. Only NMRAL2P was substantially expressed in cancer tissues and predicted a poor prognosis. The tumor-promoting impact of NMRAL2P was then confirmed using in vitro functional assays. The data set was then split into high- and low-expression subgroups based on the median gene expression of NMRAL2P to obtain the mRNA that had a large difference between the two groups, and examine the mechanism of NMRAL2P on GPX2 using quantitative real-time PCR, RNA binding protein immunoprecipitation assay, and chromatin immunoprecipitation. Mass spectrometry was used to identify NMRAL2P-binding proteins and western blotting was used to investigate probable mechanisms. The lncRNA NMRAL2P is associated with oxidative stress in head and neck tumors. In vitro functional assays showed that the gene has a cancer-promoting effect, increasing lactic acid and superoxide dismutase production, and reducing the production of ROS and malondialdehyde. NMRAL2P promotes the transcription of GPX2 by binding to transcription factor Nrf2. The gene also inhibits the degradation of ENO1, a crucial enzyme in glycolysis, by binding to protein ENO1. This study shows that NMRAL2P can promote glycolysis and reduce the harm to tumor cells caused by ROS. The gene can also be used as a possible target for the treatment of head and neck tumors.

Circ-JA760602 promotes the apoptosis of hypoxia-induced cardiomyocytes by transcriptionally suppressing BCL2.

Acute myocardial infarction (AMI) is myocardial necrosis caused by the complete or partial obstruction of a coronary artery. Circular RNAs (circRNAs) have been proven as regulators in the progression of various human diseases, including AMI. However, the role of novel circ-JA760602 in AMI remains unknown. Here, we investigated the role of circ-JA760602 in modulating the apoptosis of hypoxia-induced AMI cells using the AC16 cardiomyocyte in vitro cell model. The expression of circ-JA760602 in AC16 cardiomyocytes subjected to hypoxia was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was measured by cell counting kit-8 (CCK-8) assay. Apoptosis of cardiomyocytes was evaluated by TUNEL assay and flow cytometry analysis. The cellular location of circ-JA760602 was identified through fluorescence in situ hybridization (FISH) assay and subcellular fractionation assay. The downstream molecular mechanisms of circ-JA760602 were demonstrated by luciferase reporter assay, RNA binding protein immunoprecipitation (RIP) assay and chromatin immunoprecipitation (ChIP) assay. Rescue assays were performed to demonstrate the effects of BCL2 knockdown on circ-JA760602 silencing-mediated cardiomyocyte apoptosis. Circ-JA760602 expression was elevated after hypoxia treatment. Knockdown of circ-JA760602 enhanced viability and curbed apoptosis of hypoxia-treated cardiomyocytes. EGR1 and E2F1 could activate BCL2 transcription. Cytoplasmic circ-JA760602 bound with EGR1 and E2F1 to thus inhibit their nuclear translocation. BCL2 knockdown reversed the effects of circ-JA760602 silencing on the apoptosis of hypoxia-treated AC16 cells. Circ-JA760602 promotes hypoxia-induced apoptosis of cardiomyocytes by binding with EGR1 and E2F1 to inhibit the transcriptional activation of BCL2.

Circ_0008784 activates Wnt/β-catenin pathway to affect the proliferation and apoptosis of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC), as a subtype of breast tumors with aggressive nature, threatens the health of females across the globe. It's urgent to explore novel therapeutic targets for the improvement of TNBC treatments. Bioinformatics was used to identify circular RNA (circRNA) differentially expressed in TNBC tissues. The circular structure and expression in TNBC cells was subjected to analysis of quantitative polymerase chain reaction (qPCR) and PCR-agarose gel electrophoresis. Functional experiments and qPCR assays were carried out to probe the biological functions of circ_0008784 and microRNA-506-3p (miR-506-3p). It was verified by the assays that circ_0008784 propels proliferation and inhibit apoptosis of TNBC cells; and miR-506-3p was found to suppress proliferation and facilitate apoptosis of TNBC cells. TOP/FOP-Flash reporter, luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pulldown and rescue assays were implemented for exploring the underlying mechanisms of circ_0008784. It was found that circ_0008784 regulates Wnt/β-catenin signaling pathway, and augments TNBC cell progression via sponging miR-506-3p to modulate catenin beta 1 (CTNNB1). Circ_0008784 activates Wnt/β-catenin pathway to affect the proliferation and apoptosis of TNBC cells. Elucidating the mechanism of circ_0008784 underlying TNBC is of great significance to TNBC treatment.