RXRγ attenuates cerebral ischemia-reperfusion induced ferroptosis in neurons in mice through transcriptionally promoting the expression of GPX4.

Cerebral ischemia is a common cerebrovascular disease with high mortality and disability rate. Exploring its mechanism is essential for developing effective treatment for cerebral ischemia. Therefore, this study aims to explore the regulatory effect and mechanism of retinoid X receptor γ (RXRγ) on cerebral ischemia-reperfusion (I/R) injury. A mouse intraluminal middle cerebral artery occlusion model was established, and PC12 cells were exposed to anaerobic/reoxygenation (A/R) as an in vitro model in this study. Cerebral I/R surgery or A/R treatment induced ferroptosis, downregulated RXRγ and GPX4 (glutathione peroxidase 4) levels, upregulated cyclooxygenase-2 (COX-2) level and increased ROS (reactive oxygen species) level in A/R induced cells or I/R brain tissues in vivo or PC12 cells in vitro. Knockdown of RXRγ downregulated GPX4 and increased COX-2 and ROS levels in A/R induced cells. RXRγ overexpression has the opposite effect. GPX4 knockdown reversed the improvement of RXRγ overexpression on COX-2 downregulation, GPX4 upregulation and ferroptosis in PC12 cells. Furthermore, chromatin immunoprecipitation (ChIP) and luciferase reporter gene assays revealed that RXRγ bound to GPX4 promoter region and activated its transcription. Overexpression of RXRγ or GPX4 alleviated brain damage and inhibited ferroptosis in I/R mice. In conclusion, RXRγ-mediated transcriptional activation of GPX4 might inhibit ferroptosis during I/R-induced brain injury.

[Down-regulation of lncRNA TTTY15 targeting miR-4500 to inhibit the biological characteristics of A172 glioma cells].

OBJECTIVE: To explore the effect and mechanism of down-regulating lncRNA TTTY15 targeting miR-4500 on the proliferation, apoptosis, migration and invasion of A172 glioma cells. METHODS: The difference in TTTY15 expression between the glioma cells and tissue was determined with a qRT-PCR method. Complementary binding sites of TTTY15 and miR-4500 were predicted with Starbase software, and the targeting relationship was validated with a luciferase reporter system. A172 glioma cells were divided into Control, si-NC (transfected with control siRNA), si-TTTY15 (transfected with TTTY15 siRNA), si-TTTY15+Anti-miR-NC (co-transfected with TTTY15 siRNA and inhibitor control) and si-TTTY15+Anti-miR-4500 (co-transfected with TTTY15 siRNA and miR-4500 inhibitor) groups. Proliferation, apoptosis, migration and invasion, and the expression of Bax, Bcl-2, MMP-2 and MMP-9 proteins of the A172 glioma cells were respectively detected with CCK-8, flow cytometry, Transwell chamber and Western blotting assays. RESULTS: The expression of TTTY15 in glioma cells and glioma tissues have both increased. The expression levels of TTTY15 and miR-4500 in glioma tissues were inversely correlated. TTTY15 and miR-4500 are mutually targeted. Compared with those of the Control and si-NC groups, the glioma cells in the si-TTTY15 group showed increased level of miR-4500, decreased survival rate, increased apoptosis rate, enhanced cell migration and invasion, increased expression of Bax protein, and decreased expression of Bcl-2, MMP-2 and MMP-9 proteins (P<0.05). Compared with those of the si-TTTY15+Anti-miR-NC group, the A172 glioma cells in the si-TTTY15+Anti-miR-4500 group showed decreased level of miR-4500, increased cell survival rate, decreased apoptosis rate, enhanced cell migration and invasion, decreased expression of Bax protein, and increased expression of Bcl-2, MMP-2, and MMP-9 proteins (P<0.05). CONCLUSION: Down-regulating TTTY15 targeting miR-4500 can inhibit the proliferation, migration, invasion and induce apoptosis of the A172 glioma cells.

PAICS is related to glioma grade and can promote glioma growth and migration.

Glioma is a common malignant tumour of the brain. In this study, we aimed to investigate diagnostic biomarkers and its role in glioma. Weighted gene co-expression network analysis (WGCNA) and Cytoscape software were used to screen the marker genes in glioma. RT-qPCR and Western blotting methods were performed to determine the expression of PAICS, ERCC1 and XPA genes in glioma tissues. Expression level of PAICS in different grades of glioma was examined by immunohistochemistry. CCK8 and Colony formation assays were used to detect cell proliferation. Cell adhesion assay was used to detect adhesion ability. Wound healing and transwell tests were used to detect cell migration ability. Flow cytometry was used to detect cell cycle and apoptosis. According to the predicted co-expression network, we identified the hub gene PAICS. Furthermore, we observed that PAICS expression level was up-regulated in glioma tissues compared with normal tissues, and the expression level was correlated with the grade of glioma. Moreover, we found PAICS can promote glioma cells proliferation and migration in vitro. Flow cytometry results showed that si-PAICS cells were stalled at the G1 phase compared with the si-NC cells and knocking down PAICS expression can increase apoptotic rate. PAICS can regulate the mRNA and protein levels of nucleotide excision repair pathway core genes ERCC1 and XPA. l-aspartic acid can affect the expression of PAICS and then inhibit glioma cell proliferation. Our results indicated that PAICS can promote glioma proliferation and migration. PAICS may act as a potential diagnostic marker and a therapeutic target for glioma.

Identification and validation of core genes associated with intracranial aneurysms through bioinformatics analysis and Mendelian randomization.

Intracranial aneurysms (IAs) often go undetected until rupture, leading to significant morbidity and mortality. Identifying biomarkers for early detection of IAs is crucial. The current study attempted to identify core genes linked with IAs and determine their relevance through Mendelian randomization. Limma helped identify differentially expressed genes between IAs and control superficial temporal artery samples. WGCNA was utilized to find IA-related modules and associated genes, which were further evaluated using KEGG and GO analyses to ascertain their potential roles. Five highly associated genes were screened with the CytoHubba plugin of Cytoscape software. ROC curves assessed the diagnostic efficacy of these genes. A two-sample Mendelian randomization evaluated the causal relationship between the core gene PTRPC and IAs, along with its correlation with immune infiltration. WGCNA and differential expression analysis depicted 584 related genes involved in cellular metabolism and chemokine activity. PTPRC was among the top highly associated genes identified through Cytoscape. It showed significant diagnostic value for IAs. Moreover, mendelian randomization depicted that PTPRC in CD4+ T cells is related to IA risk, with an OR of 0.63538 (95 % CI = 0.41636-0.96959, p = 0.03545). No reverse causal relationship was observed between PTPRC and IAs, with an OR of 0.99947 (95 % CI = 0.99719-1.00176, p = 0.65022). Additionally, immune cell infiltration results indicated a positive correlation between PTPRC in IAs with neutrophils and unactivated dendritic cells and a negative association with regulatory T cells (Tregs). PTPRC was identified as a core gene linked with IAs, providing evidence for IA diagnosis and studying molecular mechanisms.

Micropeptide AF127577.4-ORF hidden in a lncRNA diminishes glioblastoma cell proliferation via the modulation of ERK2/METTL3 interaction.

Micropeptides hidden in long non-coding RNAs (lncRNAs) have been uncovered to program various cell-biological changes associated with malignant transformation-glioblastoma (GBM) cascade. Here, we identified and characterized a novel hidden micropeptide implicated in GBM. We screened potential candidate lncRNAs by establishing a workflow involving ribosome-bound lncRNAs, publicly available MS/MS data, and prognosis-related lncRNAs. Micropeptide expression was detected by western blot (WB), immunofluorescence (IF), and immunohistochemistry (IHC). Cell proliferation rate was assessed by calcein/PI staining and EdU assay. Proteins interacted with the micropeptide were analyzed by proteomics after co-immunoprecipitation (Co-IP). We discovered that lncRNA AF127577.4 indeed encoded an endogenous micropeptide, named AF127577.4-ORF. AF127577.4-ORF was associated with GBM clinical grade. In vitro, AF127577.4-ORF could suppress GBM cell proliferation. Moreover, AF127577.4-ORF reduced m6A methylation level of GBM cells. Mechanistically, AF127577.4-ORF diminished ERK2 interaction with m6A reader methyltransferase like 3 (METTL3) and downregulated phosphorylated ERK (p-ERK) level. The ERK inhibitor reduced p-ERK level and downregulated METTL3 protein expression. AF127577.4-ORF weakened the stability of METTL3 protein by ERK. Also, AF127577.4-ORF suppressed GBM cell proliferation via METTL3. Our study identifies a novel micropeptide AF127577.4-ORF hidden in a lncRNA, with a potent anti-proliferating function in GBM by diminishing METTL3 protein stability by reducing the ERK2/METTL3 interaction. This micropeptide may be beneficial for development of therapeutic strategies against GBM.

FAM3A mediates the phenotypic switch of human aortic smooth muscle cells stimulated with oxidised low-density lipoprotein by influencing the PI3K-AKT pathway.

Family with sequence similarity 3 member A (FAM3A) is a multifunctional protein that is related to the pathological process of various disorders. FAM3A is reportedly able to affect the phenotypic change of vascular smooth muscle cells under a hypertensive state. Whether FAM3A mediates the phenotypic switch of vascular smooth muscle cells under an atherosclerotic state remains unaddressed. This work investigated the roles and mechanisms of FAM3A in mediating the phenotypic switch of human aortic smooth muscle cells (HASMCs) stimulated with oxidised low-density lipoprotein (ox-LDL) in vitro. FAM3A expression was elevated in HASMCs following ox-LDL treatment. FAM3A silencing led to a suppressive effect on ox-LDL-provoked proliferation, migration and inflammation of HASMCs, whereas FAM3A overexpression had an opposite effect. Ox-LDL elicited a change in HASMCs from a contractile phenotype to a synthetic phenotype, which was inhibited by FAM3A silencing or enhanced by FAM3A overexpression. Further investigation elucidated that FAM3A silencing repressed and FAM3A overexpression promoted ox-LDL-induced activation of the PI3K-AKT pathway in HASMCs. Reactivation of AKT reversed the suppressive effect of FAM3A silencing on the ox-LDL-induced phenotypic switch of HASMCs. Restraining AKT blocked the promoting effect of FAM3A overexpression on the ox-LDL-induced phenotypic switch of HASMCs. In summary, this work elucidates that FAM3A mediates the ox-LDL-induced phenotypic switch of HASMCs by influencing the PI3K-AKT pathway, indicating a potential role for FAM3A in atherosclerosis.

Hsa_Circ_0000826 inhibits the proliferation of colorectal cancer by targeting AUF1.

Many circular RNAs (circRNAs) are reported to be abnormally expressed during the progression of various tumors, and these circRNAs can be used as anti-tumor targets. Therefore, it is important to identify circRNAs that can be used effectively for the clinical diagnosis and treatment of colorectal cancer (CRC). Here, we report that hsa_Circ_0000826 (Circ_0000826), a circRNA with significantly reduced expression level in CRC tissues, is associated with a poor prognosis in patients. The silencing of Circ_0000826 promotes the proliferation of CRC cells. Conversely, the overexpression of Circ_0000826 restricted CRC cell proliferation both in vitro and in vivo. Furthermore, Circ_0000826 could target AU-rich element RNA-binding protein 1 (AUF1). AUF1, known as heterogeneous nuclear ribonucleoprotein D (hnRNP D), could bind to the c-MYC 3'-UTR and promote c-MYC expression. When Circ_0000826 binds to AUF1, it competitively inhibits the binding of AUF1 to the c-MYC 3'-UTR, which inhibits the c-MYC expression and cell proliferation. These results provide novel insights into the functional mechanism of Circ_0000826 action in CRC progression and indicate its potential use as a therapeutic target in CRC.

Study on the role of transcription factor SPI1 in the development of glioma.

BACKGROUND: Glioma is a common malignant brain tumor. The purpose of this study was to investigate the role of the transcription factor SPI1 in glioma. METHODS: SPI1 expression in glioma was identified using qRT-PCR and Western blotting. Cell proliferation was assessed using the CCK8 assay. Transwell and wound healing assays were utilized to evaluate cell migration. Additionally, cell cycle and apoptosis were detected using flow cytometry. RESULTS: We observed that the expression level of SPI1 was up-regulated in glioma tissues, compared to normal tissues. Furthermore, we found that SPI1 is able to promote proliferation and migration of glioma cells in vitro. Flow cytometry results demonstrate that, compared to si-NC cells, si-SPI1 cells stagnated in the G1 phase, and down-regulation of SPI1 expression is able to increase rates of apoptosis. Double luciferase activity and chromatin immunoprecipitation assay results indicated that SPI1 can bind to the promoter sites and promote the proliferation and migration of glioma cells by regulating the expression of oncogenic PAICS. CONCLUSIONS: Our results suggest that SPI1 can promote proliferation and migration of glioma. Furthermore, SPI1 can be utilized as a potential diagnostic marker and therapeutic target for glioma.

lncRNA SNHG17 is Downregulated in Gestational Diabetes Mellitus (GDM) and Has Predictive Values.

AIM: Long non-coding RNA (lncRNA) SNHG17 has been shown to participate in type 2 diabetes mellitus, while its role in gestational diabetes mellitus (GDM) is unknown. METHODS: Quantitative real-time PCR (qRT-PCR) assays were conducted to compare the differential expression of SNHG17 among 60 GDM patients and 60 healthy pregnant female controls. In addition, peripheral blood samples from 240 pregnant females were collected to evaluate the predictive value of SNHG17 for GDM patients. All females were followed-up until delivery to record the occurrence of GDM and perinatal outcomes. GDM-free curves were plotted to compare the occurrence of GDM between high- and low- SNHG17 expression groups. The diagnostic value of plasma SNHG17 for GDM was analyzed by ROC curve analysis. Moreover, the cell counting kit (CCK-8) assay was performed to evaluate the impact of SNHG17 on cell viability of INS-1, and the level of insulin secretion was detected by enzyme linked immunosorbent assay (ELISA) after overexpression or knockdown of SNHG17. RESULTS: SNHG17 was downregulated in GDM patients compared to normal pregnant females. Low plasma expression levels of SNHG17 were closely correlated with the high incidence rate of GDM (GDM-free curve). Remarkably, plasma expression levels of SNHG17 at 4 weeks before the diagnosis of GDM (diagnosed by standard method) can be used to distinguish (ROC curve) GDM patients (diagnosed during follow-up) from normal pregnant females (GDM was not diagnosed during follow-up). CONCLUSION: Plasma circulating SNHG17 is downregulated in GDM and has predictive values.

Association of MACC1 expression with lymphatic metastasis in colorectal cancer: A nested case-control study.

MACC1 gene is a newly discovered gene and plays an important role in the metastasis of colorectal cancer (CRC). The objective of this study was to investigate whether MACC1 is an independent factor associated with lymphatic metastasis in CRC patients. We analyzed the association between MACC1 expression and lymphatic metastasis in a nested case-control study including 99 cases and 198 matched controls in CRC patients, assessed from August 2001 to March 2015. Cases were defined as lymphatic metastasis and non-lymphatic metastasis according to AJCC TNM stages; for each case, two age-matched control without lymphatic and distant metastasis was randomly selected from the study participants. Demographic, variables about metastasis and MACC1 expression were collected. In multivariate analysis, the OR (95% CI) of MACC1 expression was 1.5 (1.1 to 2.0) in patients with lymphatic metastasis versus non-lymphatic metastasis after adjusting all variables. After adjustment for all variables and age stratification, MACC1 expression was found to be an independent risk factor for lymph node metastasis in the middle-aged group (OR 2.1, 95%CI 1.1-4.0). A nonlinear relationship between MACC1 expression and 64-75 age group was observed. The probability of metastasis slightly increased with the MACC1 level lower than turning point 1.4. At the same time, the probability of lymphatic metastasis was obviously increased even after adjusting all variables when MACC1 level higher than 1.4 (OR 11.2, 95% CI 1.5-81.5; p = 0.017) in the middle age group. The expression of MACC1 was not associated with lymphatic metastasis in populations younger than 64 or older than 75. The results demonstrates that increased MACC1 level in 64-75 age group might be associated with lymphatic metastasis in CRC patients.

Caveolin-1 regulates the expression of miR-183 and inhibits the invasion and migration of invasive pituitary adenomas by affecting early growth response 1 (EGR1)/Krueppel-like factor 5 (KLF5) interaction.

BACKGROUND: This study aims to investigate the mechanism through which Caveolin-1 (CAV-1) regulates the expression of micro ribonucleic acid (miR)-183 in invasive pituitary adenoma (IPA) tissues and GH3 cells, and explore the effects of CAV-1 and miR-183 on the invasion and migration ability of GH3 cells. METHODS: Western blotting was used to detect the expression level of CAV-1, early growth response 1 (EGR1) and Krueppel-like factor 5 (KLF5). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-183. The mechanisms of interaction between CAV-1, EGR1, and KLF5 were studied by immunoprecipitation experiments. Transwell and cell scratch tests were used to determine the invasion and migration ability of GH3 cells. The dual-luciferase reporter gene experiment was used to detect the effects of EGR1 and KLF5 on miR-183 luciferase activity and verify the targeting relationship between miR-183 and ezrin. RESULTS: The expression of CAV-1 was up-regulated. However, following the knockdown of CAV-1, the invasion and migration ability of GH3 cells was significantly inhibited (P<0.05). The expression of miR-183 was down-regulated, but the expression level of miR-183 was markedly increased following the knockdown of CAV-1 (P<0.05). The knockdown of CAV-1 inhibited the nuclear ectopic of the EGR1 protein in GH3 cells. At the same time, the interaction between EGR1 and KLF5 in GH3 cells was significantly inhibited (P<0.05). The luciferase activity of miR-183 increased significantly after overexpression of KLF5 while overexpression of EGR1 and KLF5 had no significant effect on intracellular luciferase activity. Overexpression of miR-183 markedly inhibited the luciferase activity of wild-type EZR and the expression of the EZR protein in GH3 cells. Furthermore, the overexpression of miR-183 or the inhibition of EZR can reduce the invasion and migration ability of GH3 cells. The simultaneous overexpression or inhibition of miR-183 and EZR expression has no obvious effect on the invasion and migration ability of GH3 cells. CONCLUSIONS: CAV-1 up-regulates the expression of miR-183 by inhibiting the nuclear ectopic of EGR1 and the interaction between EGR1 and KLF5 in GH3 cells. Also, miR-183 negatively regulates the expression of EZR and inhibits the invasion and migration of GH3 cells.

Madecassoside prevents Aß(25-35)-induced inflammatory responses and autophagy in neuronal cells through the class III PI3K/Beclin-1/Bcl-2 pathway.

Inflammatory responses and autophagy have been implicated in the amyloid-ß (Aß) aggregation in Alzheimer's disease (AD) due to recycling cellular waste and eliminating toxic protein aggregates. Madecassoside (Mad), a triterpenoid saponin compound, has been found to improve impaired cognitive function. However, little was known about the protection of Mad nerve cells against inflammation response and autophagy, as well as their underlying mechanism. In the present study, we investigated whether Mad could prevent Aß25-35-induced inflammatory responses and autophagy, as well as the possible mechanism. Transmission electron microscopy results showed that Mad could significantly reduce Aß25-35-induced autophagosomes in neural cells. Mad could also increase cell viability whereas decrease remarkably LDH leakage in Aß25-35-induced neural cells. Both ELISA assay and western blot showed that Mad attenuated inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-6 and COX-2 production. Moreover, western blot results showed that Mad could block the conversion of light chain3-I (LC3-I) to light chain3-II (LC3-II), reduce Beclin-1, whereas increase anti-apoptotic protein Bcl-2 level. The levels of Beclin-1 and hVps34 in control vector-transfected NG108-15 neural cells but not in Bcl-2 transfected NG108-15 neural cells were reduced by Mad. The levels of inflammatory cytokines including TNF-α and IL-6 productions decreased significantly by the treatment with Mad. These results demonstrated that Mad protected neural cells against inflammation and autophagy induced by Aß25-35 through the class III PI3K/Beclin-1/Bcl-2 pathway. Our findings provide evidences for the beneficial effect of Mad on the treatment of AD.

Expression and antioxidation of Nrf2/ARE pathway in traumatic brain injury.

OBJECTIVE: To explore the expression of Nrf2/ARE pathway in hindbrain tissue after the traumatic brain injury (TBI) and its anti-oxidative stress effect in the secondary nerve injury. METHODS: The mice with Nrf2 gene knockout were used for the establishment of brain injury model. The experimental animals were divided into four groups: (Nrf2(+)/(+)) sham-operation group, (Nrf2(+)/(+)) brain injury group, (Nrf2(-)/(-)) sham-operation group and (Nrf2(-)/(-)) brain injury group. The specimen 24 h after cerebral trauma was selected. Then RT-PCR method was adopted to detect the expression of Nrf2 mRNA in brain; Western blotting method was adopted to detect the levels of Nrf2, HO-1 and NQO1 proteins in brain; ELISA method was adopted to detect the oxidative stress indicators: protein carbonyls, 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). RESULTS: The Nrf2 mRNA and protein of Nrf2(-)/(-) mice were not expressed, and the difference of the relative amount of Nrf2 mRNA between Nrf2(+)/(+) TBI group and Nrf2(+)/(+) sham-operation group was not statistically significant (P>0.05); the level of Nrf2 protein in Nrf2(+)/(+) TBI group increased significantly compared with the Nrf2(+)/(+) sham-operation group (P<0.01); in the sham-operation groups, the levels of HO-1 and NQO1 proteins of Nrf2(-)/(-) mice decreased obviously compared with the Nrf2(+)/(+) mice (P<0.01); after brain injury, the levels of HO-1 and NQO1 proteins of Nrf2(+)/(+) mice increased obviously compared with the corresponding sham-operation group (P<0.01); the levels of HO-1 and NQO1 proteins of Nrf2(-)/(-) mice in TBI group had no obvious change compared with the corresponding sham-operation group (P>0.05); there was only a little amount of expression of protein carbonyls, 4-HNE and 8-OHdG proteins in brain tissues in the Nrf2(+)/(+) and Nrf2(-)/(-) sham-operation groups, and the difference was not statistically significant (P>0.05); after brain injury, the three oxidative stress indicators were significantly up-regulated in the Nrf2(+)/(+) and Nrf2(-)/(-) groups, and the up-regulation of the latter group was more significant (P<0.01). CONCLUSIONS: After TBI the Nrf2/ARE pathway is activated and the activity of Nrf2 transcription regulation increases. However, the regulation dose not occur in the gene transcription level and only could increase the Nrf2 protein level, while the mRNA expression level has no obvious change. The nerve cell protective effect of Nrf2/ARE pathway in TBI achieves through inhibiting the oxidative stress injuries.