Rats ; Animals ; Myocardial Reperfusion Injury/metabolism* ; Luteolin/metabolism* ; Down-Regulation ; Rats, Sprague-Dawley ; MicroRNAs/metabolism* ; Reperfusion Injury ; Apoptosis

BACKGROUND: Dysfunction of the gap junction channel protein connexin 43 (Cx43) contributes to myocardial ischemia/reperfusion (I/R)-induced ventricular arrhythmias. Cx43 can be regulated by small ubiquitin-like modifier (SUMO) modification. Protein inhibitor of activated STAT Y (PIASy) is an E3 SUMO ligase for its target proteins. However, whether Cx43 is a target protein of PIASy and whether Cx43 SUMOylation plays a role in I/R-induced arrhythmias are largely unknown. METHODS: Male Sprague-Dawley rats were infected with PIASy short hairpin ribonucleic acid (shRNA) using recombinant adeno-associated virus subtype 9 (rAAV9). Two weeks later, the rats were subjected to 45 min of left coronary artery occlusion followed by 2 h reperfusion. Electrocardiogram was recorded to assess arrhythmias. Rat ventricular tissues were collected for molecular biological measurements. RESULTS: Following 45 min of ischemia, QRS duration and QTc intervals statistically significantly increased, but these values decreased after transfecting PIASy shRNA. PIASy downregulation ameliorated ventricular arrhythmias induced by myocardial I/R, as evidenced by the decreased incidence of ventricular tachycardia and ventricular fibrillation, and reduced arrythmia score. In addition, myocardial I/R statistically significantly induced PIASy expression and Cx43 SUMOylation, accompanied by reduced Cx43 phosphorylation and plakophilin 2 (PKP2) expression. Moreover, PIASy downregulation remarkably reduced Cx43 SUMOylation, accompanied by increased Cx43 phosphorylation and PKP2 expression after I/R. CONCLUSION PIASy downregulation inhibited Cx43 SUMOylation and increased PKP2 expression, thereby improving ventricular arrhythmias in ischemic/reperfused rats heart.
Rats ; Male ; Animals ; Myocardial Reperfusion Injury/metabolism* ; Connexin 43/genetics* ; Sumoylation ; Down-Regulation ; Rats, Sprague-Dawley ; Arrhythmias, Cardiac/drug therapy* ; Myocardial Ischemia/metabolism* ; RNA, Small Interfering/metabolism*

Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans ; Gefitinib/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Lung Neoplasms/pathology* ; Down-Regulation ; Quinazolines/therapeutic use* ; Drug Resistance, Neoplasm/genetics* ; ErbB Receptors/metabolism* ; Adenocarcinoma of Lung ; Protein Kinase Inhibitors/therapeutic use* ; RNA, Small Interfering/genetics* ; Cell Line, Tumor ; Amino Acid Transport System y+/metabolism*

BACKGROUND: Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are the main causes of restenosis (RS) in diabetic lower extremity arterial disease (LEAD). However, the relevant pathogenic mechanisms are poorly understood. METHODS: In this study, we introduced a "two-step injury protocol" rat RS model, which started with the induction of atherosclerosis (AS) and was followed by percutaneous transluminal angioplasty (PTA). Hematoxylin-eosin (HE) staining and immunohistochemistry staining were used to verify the form of RS. Two-step transfection was performed, with the first transfection of Lin28a followed by a second transfection of let-7c and let-7g, to explore the possible mechanism by which Lin28a exerted effects. 5-ethynyl-2΄-deoxyuridine (EdU) and Transwell assay were performed to evaluate the ability of proliferation and migration of VSMCs. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression of Lin28a protein and let-7 family members. RESULTS: Using a combination of in vitro and in vivo experiments, we discovered that let-7c, let-7g, and microRNA98 (miR98) were downstream targets of Lin28a. More importantly, decreased expression of let-7c/let-7g increased Lin28a, leading to further inhibition of let-7c/let-7g. We also found an increased level of let-7d in the RS pathological condition, suggesting that it may function as a protective regulator of the Lin28a/let-7 loop by inhibiting the proliferation and migration of VSMCs. CONCLUSION These findings indicated the presence of a double-negative feedback loop consisting of Lin28a and let-7c/let-7g, which may be responsible for the vicious behavior of VSMCs in RS.
Rats ; Animals ; Down-Regulation ; MicroRNAs/metabolism* ; Feedback ; Cell Proliferation/genetics* ; Atherosclerosis

OBJECTIVE: Circular RNAs (circRNAs) participate in several important pathological processes and have been used in the diagnosis and treatment of various diseases. This study aimed to investigate the role of circRNAs in neural tube defects (NTDs). METHOD: We characterized circRNA-associated competitive endogenous RNA (ceRNA) networks in brain tissue of low folate -induced NTDs mouse at embryonic day 13.5 by high-throughput sequencing. The expression levels of Circzfp644, miR-20-5p and Gas7 were detected by RT-PCR. Gas7 and Circzfp644 functions were determined by miRNA-mimics and inhibitors in mouse teratocarcinoma cells (F9 cells), and luciferase gene reporter assay was assessed in the F9 cells. In addition, the expression levels of Circzfp644, miR-20-5p and Gas7 were determined by Nanostring in human NTDs tissues. RESULTS: We detected 57 circRNA transcripts, 16 miRNAs, and 148 mRNAs that were significantly dysregulated in NTDs brain tissues compared with their expression levels in control (normal) tissues. Circzfp644 shared miRNA response elements with the growth arrest specific 7 ( Gas7) gene and competitively bound with miR-20-5p to increase the expression of Gas7. Downregulation of Circzfp644 and Gas7 and upregulation of miR-20-5p were found in human NTD tissue. CONCLUSION This study provides new perspectives on the role of circRNAs in nervous system development and the pathogenesis of NTDs.
Humans ; Animals ; Mice ; RNA, Circular/genetics* ; MicroRNAs/metabolism* ; Down-Regulation ; Neural Tube Defects/genetics* ; Folic Acid

OBJECTIVE: To explore the diagnosis, treatment and genetic analysis of an infant with Schaaf-Yang syndrome (SYS). METHODS: An infant suspected for SYS at the Hunan Provincial Children's Hospital on June 10, 2022 was subjected to trio-whole exome sequencing, and Sanger sequencing was used to verify the candidate variant. Structure of the wild-type and mutant proteins was constructed to analyze the potential hazard. RESULTS: The infant was found to harbor a heterozygous frameshifting variant of c.1908delG (p.R637Gfs*65) of the MAGEL2 gene, which was found in neither of his parents. The variant has not been recorded by the public databases, and no relevant literature was retrieved. As the result of the variant, the MAGEL2 protein only retained part of its proline domain, which may lead to destruction and/or down-regulation of its function. CONCLUSION The c.1908delG (p.R637Gfs*65) variant of the MAGEL2 gene probably underlay the pathogenesis in this child. Combined with his clinical characteristics, the child was diagnosed with SYS. Above finding has also enriched the mutational spectrum of the MAGEL2 gene.
Humans ; Infant ; Down-Regulation ; Heterozygote ; Mutation ; Parents ; Proteins

OBJECTIVES: Graves' ophthalmopathy (GO) is a multifactorial disease, and the mechanism of non coding RNA interactions and inflammatory cell infiltration patterns are not fully understood. This study aims to construct a competing endogenous RNA (ceRNA) network for this disease and clarify the infiltration patterns of inflammatory cells in orbital tissue to further explore the pathogenesis of GO. METHODS: The differentially expressed genes were identified using the GEO2R analysis tool. The Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology analysis were used to analyze differential genes. RNA interaction relationships were extracted from the RNA interactome database. Protein-protein interactions were identified using the STRING database and were visualized using Cytoscape. StarBase, miRcode, and DIANA-LncBase Experimental v.2 were used to construct ceRNA networks together with their interacted non-coding RNA. The CIBERSORT algorithm was used to detect the patterns of infiltrating immune cells in GO using R software. RESULTS: A total of 114 differentially expressed genes for GO and 121 pathways were detected using both the KEGG and gene ontology enrichment analysis. Four hub genes (SRSF6, DDX5, HNRNPC,and HNRNPM) were extracted from protein-protein interaction using cytoHubba in Cytoscape, 104 nodes and 142 edges were extracted, and a ceRNA network was identified (MALAT1-MIR21-DDX5). The results of immune cell analysis showed that in GO, the proportions of CD8⁺ T cells and CD4⁺ memory resting T cells were upregulated and downregulated, respectively. The proportion of CD4 memory resting T cells was positively correlated with the expression of MALAT1, MIR21, and DDX5. CONCLUSIONS This study has constructed a ceRNA regulatory network (MALAT1-MIR21-DDX5) in GO orbital tissue, clarifying the downregulation of the proportion of CD4⁺ stationary memory T cells and their positive regulatory relationship with ceRNA components, further revealing the pathogenesis of GO.
Humans ; CD8-Positive T-Lymphocytes ; RNA, Long Noncoding/genetics* ; Algorithms ; CD4-Positive T-Lymphocytes ; Down-Regulation ; Graves Ophthalmopathy/genetics* ; Gene Regulatory Networks ; MicroRNAs/genetics* ; Serine-Arginine Splicing Factors ; Phosphoproteins

OBJECTIVE: To observe the clinical effect of acupuncture for delayed sleep-wake phase disorder (DSWPD). METHODS: A total of 84 patients with DSWPD were randomized into an observation group (42 cases, 2 cases dropped off) and a control group (42 cases, 3 cases dropped off). On the basis of sleep hygiene education, acupuncture was applied at Shenmai (BL 62), Zhaohai (KI 6), Hegu (LI 4), Taichong (LR 3), Zusanli (ST 36) and Sanyinjiao (SP 6) in the observation group, while placebo acupuncture was applied at the same acupoints in the control group. The treatment lasted for 8 weeks, once every other day, 3 times a week in the 1st to 4th weeks; once every 3 days, 2 times a week in the 5th to 8th weeks. Before and after treatment, the actigraphy (ACT) indexes of objective sleep (total time of stay in bed, total sleep time, sleep efficiency, the number of awakenings and the wake time after falling asleep) and plasma cortisol (CORT) level were observed; before and after treatment and in follow-up of 1, 3 months after treatment, the scores of morningness-eveningness questionnaire (MEQ), insomnia severity index (ISI), fatigue severity scale (FSS) and Epworth sleepiness scale (ESS) were observed in the two groups. RESULTS: Compared before treatment, the total sleep time was prolonged, the sleep efficiency was improved, the number of awakenings was reduced, and the wake time after falling asleep was shortened after treatment in the observation group (P<0.01, P<0.05), and those in the observation group after treatment were superior to the control group (P<0.01, P<0.05). Compared before treatment, the MEQ scores after treatment in both groups and in the follow-up of 1, 3 months after treatment in the observation group were increased (P<0.01), and the MEQ score of each time point after treatment in the observation group was higher than the control group (P<0.01). The scores of ISI, FSS and ESS after treatment, and the scores of ISI、ESS in follow-up of 1, 3 months after treatment in the observation group were decreased compared with those before treatment (P<0.01, P<0.05), and in the observation group, the scores of ISI, FSS and ESS of each time point after treatment were lower than those in the control group (P<0.01, P<0.05). After treatment, the plasma CORT level in the observation group was decreased compared with that before treatment and that in the control group (P<0.01, P<0.05). CONCLUSION Acupuncture can improve the sleep and wake phase of patients with DSWPD, improve sleep quality and daytime function, and its mechanism may be related to the down-regulation of plasma CORT level.
Humans ; Acupuncture Therapy ; Sleep ; Acupuncture Points ; Down-Regulation ; Sleep Duration

OBJECTIVE: To investigate the efficacy of Bushen Huoxue Fang (BSHXF, a traditional Chinese medicine formula) for improving recurrent spontaneous abortion (RSA) in mice and the role of tyrosine kinase (JAK2) and transcriptional activator (STAT3) signaling pathway in its therapeutic mechanism. METHODS: Female CBA/J mice were caged with male DBA/2 mice to establish RSA mouse models, which were randomly divided into model group, dydrogesterone group and BSHXF group, with the female mice caged with male BALB/c mice as the control group (n=6). From the first day of pregnancy, the mice were subjected to daily intragastric administration of BSHXF, dydrogesterone, or distilled water (in control and model groups) for 12 days. After the treatments, serum levels of antithrombin III (AT-III), activated protein C (APC), tissue plasminogen activator (t-PA), progesterone, human chorionic gonadotropin (HCG), and estradiol (E2) were detected in each group using ELISA. HE staining was used to observe the morphological changes of the endometrium of the mice. Western blotting was performed to determine the expressions of p-JAK2, p-Stat3 and Bcl-2 in the placenta of the mice. RESULTS: Compared with the control mice, the mouse models of RSA showed a significantly increased embryo loss rate with decreased serum levels of AT-III, T-PA, progesterone, APC and HCG, increased placental expressions of p-JAK2, p-STAT3 and Bax, and decreased expression of Bcl-2 (P < 0.05). Treatments with BSHXF and dydrogesterone both increased serum levels of AT-III, t-PA and HCG in the mouse models; Serum APC level was significantly reduced in BSHXF group and serum progesterone level was significantly increased in dydrogesterone group (P < 0.05). CONCLUSION BSHXF can improve the prethrombotic state and inhibit cell apoptosis by downregulating the JAK2/STAT3 pathway to increase the pregnancy rate in mouse models of RSA.
Animals ; Mice ; Abortion, Habitual/prevention & control* ; Signal Transduction ; Down-Regulation ; Disease Models, Animal

Objective: To analyze the expression levels of the F9 gene and F9 protein in hepatocellular carcinoma by combining multiple gene chip data, real-time fluorescence quantitative PCR (RT qPCR), and immunohistochemistry. Additionally, explore their correlation with the occurrence and development of hepatocellular carcinoma, as well as with various clinical indicators and prognosis. Methods: The mRNA microarray dataset from the GEO database was analyzed to identify the F9 gene with significant expression differences associated with hepatocellular carcinoma. Liver cancer and adjacent tissues were collected from 18 cases of hepatocellular carcinoma. RT-qPCR method was used to detect the F9 gene expression level. Immunohistochemistry was used to detect the F9 protein level. Combined with the TCGA database information, the correlation between F9 gene expression level and prognostic and clinicopathological parameters was analyzed. The biological function of F9 co-expressed genes associated with hepatocellular carcinoma was analyzed by the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Statistical analysis was performed using Graphpad Prism software. Results: Meta-analysis results showed that the expression of the F9 gene was lower in HCC tissues than in non-cancerous tissues. Immunohistochemistry results were basically consistent with those of RT-qPCR. The data obtained from TCGA showed that the F9 gene had lower expression values in stages III-IV, T3-T4, and patients with vascular invasion. A total of 127 genes were selected for bioinformatics analysis as co-expressed genes of F9, which were highly enriched in redox processes and metabolic pathways. Conclusion: This study validates that the F9 gene and F9 protein are lower in HCC. The down-regulation of the F9 gene predicts adverse outcomes, which may provide a new therapeutic target for HCC.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Down-Regulation ; Prognosis ; Gene Expression ; Gene Expression Regulation, Neoplastic