Transcriptome-wide N6-methyladenosine methylation profile of atherosclerosis in mice.

BACKGROUND: Atherosclerosis (AS) is a critical pathological event during the progression of cardiovascular diseases. It exhibits fibrofatty lesions on the arterial wall and lacks effective treatment. N6-methyladenosine (m6A) is the most common modification of eukaryotic RNA and plays an important role in regulating the development and progression of cardiovascular diseases. However, the role of m6A modification in AS remains largely unknown. Therefore, in this study, we explored the transcriptome distribution of m6A modification in AS and its potential mechanism. METHODS: Methylation Quantification Kit was used to detect the global m6A levels in the aorta of AS mice. Western blot was used to analyze the protein level of methyltransferases. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to obtain the first transcriptome range analysis of the m6A methylene map in the aorta of AS mice, followed by bioinformatics analysis. qRT-PCR and MeRIP-qRT-PCR were used to measure the mRNA and m6A levels in target genes. RESULTS: The global m6A and protein levels of methyltransferase METTL3 were significantly increased in the aorta of AS mice. However, the protein level of demethylase ALKBH5 was significantly decreased. Through MeRIP-seq, we obtained m6A methylation maps in AS and control mice. In total, 26,918 m6A peaks associated with 13,744 genes were detected in AS group, whereas 26,157 m6A peaks associated with 13,283 genes were detected in the control group. Peaks mainly appeared in the coding sequence (CDS) regions close to the stop codon with the RRACH motif. Moreover, functional enrichment analysis demonstrated that m6A-containing genes were significantly enriched in AS-relevant pathways. Interestingly, a negative correlation between m6A methylation abundance and gene expression level was found through the integrated analysis of MeRIP-seq and RNA-seq data. Among the m6A-modified genes, a hypo-methylated but up-regulated (hypo-up) gene Fabp5 may be a potential biomarker of AS. CONCLUSIONS: Our study provides transcriptome-wide m6A methylation for the first time to determine the association between m6A modification and AS progression. Our study lays a foundation for further exploring the pathogenesis of AS and provides a new direction for the treatment of AS.

Deficiency of a novel lncRNA-HRAT protects against myocardial ischemia reperfusion injury by targeting miR-370-3p/RNF41 pathway.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) contribute to myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms by which lncRNAs modulate myocardial I/R injury have not been thoroughly examined and require further investigation. A novel lncRNA named lncRNA-hypoxia/reoxygenation (H/R)-associated transcript (lncRNA-HRAT) was identified by RNA sequencing analysis. The expression of lncRNA-HRAT exhibited a significant increase in the I/R mice hearts and cardiomyocytes treated with H/R. LncRNA-HRAT overexpression facilitates H/R-induced cardiomyocyte apoptosis. Furthermore, cardiomyocyte-specific deficiency of lncRNA-HRAT in vivo after I/R decreased creatine kinase (CK) release in the serum, reduced myocardial infarct area, and improved cardiac dysfunction. Molecular mechanistic investigations revealed that lncRNA-HRAT serves as a competing endogenous RNA (ceRNA) of miR-370-3p, thus upregulating the expression of ring finger protein 41 (RNF41), thereby aggravating apoptosis in cardiomyocytes induced by H/R. This study revealed that the lncRNA-HRAT/miR-370-3p/RNF41 pathway regulates cardiomyocyte apoptosis and myocardial injury. These findings suggest that targeted inhibition of lncRNA-HRAT may offer a novel therapeutic method to prevent myocardial I/R injury.

MicroRNA-128-1-5p attenuates myocardial ischemia/reperfusion injury by suppressing Gadd45g-mediated apoptotic signaling.

Myocardial ischemia/reperfusion (I/R) injury is a clinically fatal disease, caused by restoring myocardial blood supply after a period of ischemia or hypoxia. However, the underlying mechanism remains unclear. Recently, increasing evidence reveal that microRNAs (miRs) participate in myocardial I/R injury. This study aimed to investigate whether miR-128-1-5p contributed to cardiomyocyte apoptosis induced by myocardial I/R injury. Here, we showed that the expression of miR-128-1-5p was decreased in mice following myocardial I/R injury. Down-regulation of miR-128-1-5p was also showed in H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R), and in neonatal rat cardiomyocytes (NRCMs) with H2O2 treatment. Importantly, we found that overexpression of miR-128-1-5p ameliorates cardiomyocyte apoptosis both in H9c2 cardiomyocytes and NRCMs. Moreover, we also found that growth arrest DNA damage-inducible gene 45 gamma (Gadd45g) is identified as a direct target of miR-128-1-5p, which negatively regulated Gadd45g expression. Additionally, silencing of Gadd45g inhibits cardiomyocyte apoptosis in H9c2 cardiomyocytes and NRCMs. These results reveal a novel mechanism by which miR-128-1-5p regulates Gadd45g-mediated cardiomyocyte apoptosis in myocardial I/R injury.

Critical roles of microRNA-141-3p and CHD8 in hypoxia/reoxygenation-induced cardiomyocyte apoptosis.

BACKGROUND: Cardiovascular diseases are currently the leading cause of death in humans. The high mortality of cardiac diseases is associated with myocardial ischemia and reperfusion (I/R). Recent studies have reported that microRNAs (miRNAs) play important roles in cell apoptosis. However, it is not known yet whether miR-141-3p contributes to the regulation of cardiomyocyte apoptosis. It has been well established that in vitro hypoxia/reoxygenation (H/R) model can follow in vivo myocardial I/R injury. This study aimed to investigate the effects of miR-141-3p and CHD8 on cardiomyocyte apoptosis following H/R. RESULTS: We found that H/R remarkably reduces the expression of miR-141-3p but enhances CHD8 expression both in mRNA and protein in H9c2 cardiomyocytes. We also found either overexpression of miR-141-3p by transfection of miR-141-3p mimics or inhibition of CHD8 by transfection of small interfering RNA (siRNA) significantly decrease cardiomyocyte apoptosis induced by H/R. Moreover, miR-141-3p interacts with CHD8. Furthermore, miR-141-3p and CHD8 reduce the expression of p21. CONCLUSION: MiR-141-3p and CHD8 play critical roles in cardiomyocyte apoptosis induced by H/R. These studies suggest that miR-141-3p and CHD8 mediated cardiomyocyte apoptosis may offer a novel therapeutic strategy against myocardial I/R injury-induced cardiovascular diseases.

Bnip3 mediates doxorubicin-induced cardiomyocyte pyroptosis via caspase-3/GSDME.

AIMS: This study was aimed to investigate the role of GSDME-mediated pyroptosis in cardiac injury induced by Doxorubicin (DOX), and to evaluate the role of BH3-only protein Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) in regulation of DOX-induced pyroptosis. MAIN METHODS: HL-1 cardiomyocytes and C57BL/6J mice were treated by DOX to establish DOX-induced cardiotoxicity in vitro and in vivo models, respectively. Cell transfection was applied to regulate the expression of caspase-3, GSDME and Bnip3. Western blot was used for measuring expression of protein level. LDH-cytotoxicity assay was used to detect the LDH release. The Flow cytometry analysis was used to detect the cell death. Echocardiography was used to determine the cardiac function. HE staining was used for observing pathological feature of heart tissues. KEY FINDINGS: Our results showed that GSDME-mediated pyroptosis was involved in DOX-induced cardiotoxicity in vivo. We showed that HL-1 cardiomyocytes exposed to DOX exhibited morphological features of pyroptosis in vitro. We also showed that DOX induced activation of caspase-3 and eventually triggered GSDME-dependent pyroptosis, which was reduced by the silence or inhibitor of caspase-3. We further showed that knockdown of GSDME inhibited DOX-induced cardiomyocyte pyroptosis in vitro. Finally, DOX increased the expression of Bnip3, whereas silencing of Bnip3 blunted cardiomyocyte pyroptosis induced by DOX, which was regulated through caspase-3 activation and GSDME cleavage. SIGNIFICANCE: Our findings revealed a novel pathway that cardiomyocyte pyroptosis is regulated through Bnip3-caspase-3-GSDME pathway following DOX treatment, suggesting that Bnip3-dependent pyroptosis may offer a novel therapeutic strategy to reduce cardiotoxicity induced by DOX.

LncRNA BCAR4, targeting to miR-665/STAT3 signaling, maintains cancer stem cells stemness and promotes tumorigenicity in colorectal cancer.

BACKGROUND: Breast cancer anti-estrogen resistance 4 (BCAR4) is closely associated with colorectal cancer (CRC) initiation and propagation. However, the mechanisms underlying BCAR4 function in colon cancer remains largely unknown. In this study, we hypothesized that BCAR4 could regulate colon cancer stem/initiating cells (CSC) function and further facilitates the colon cancer progression. METHODS: qRT-PCR was used to examine the expression of BCAR4 and various CSC markers. FACS, acetaldehyde dehydrogenase (ALDH) activity and western blot assays were applicable to test the expression of CSC markers. CCK8, tumorsphere formation and transwell assays were adopted to examine the capacity of CRC cells proliferation, self-renewal and migration. Pull down assay was used to test the interaction between BCAR4 and miR-665. Luciferase reporter assay was used to examine the interaction of miR-665 and activators of transcription (STAT3). In vivo tumor xenograft study was used to verify the malignancy of CRC cells with inhibition of BCAR4. RESULTS: Breast cancer anti-estrogen resistance 4 was highly expressed in both CRC cells and stem/initiating cells. In addition, overexpression of BCAR4 facilitated the maintenance of ALDH positive cells (a type of cancer stem/initiating cells) stemness and promoted ALDH+ cells proliferation and migration. Inhibition of BCAR4 restricted ALDH+ cells proliferation and migration. We further proved that miR-665 was the target of BCAR4 and subsequently activated signal transducers and STAT3 signaling which is an important pathway in cancer stem cells self-renewal. CONCLUSIONS: Breast cancer anti-estrogen resistance 4 promotes the CRC cells stemness through targeting to miR-665/STAT3 signaling and identification of the BCAR4 in CRC stem cells provides a new insight into CRC diagnosis, treatment, prognosis and next-step translational investigations.

Critical role of Tim-3 mediated autophagy in chronic stress induced immunosuppression.

BACKGROUND: Psychological and physical stress can either enhance or suppress immune functions depending on a variety of factors such as duration and severity of stressful situation. Chronic stress exerts a significantly suppressive effect on immune functions. However, the mechanisms responsible for this phenomenon remain to be elucidated. Autophagy plays an essential role in modulating cellular homeostasis and immune responses. However, it is not known yet whether autophagy contributes to chronic stress-induced immunosuppression. T cell immunoglobulin and mucin domain 3 (Tim-3) has shown immune-suppressive effects and obviously positive regulation on cell apoptosis. Tim-3 combines with Tim-3 ligand galectin-9 to modulate apoptosis. However, its impact on autophagy and chronic stress-induced immunosuppression is not yet identified. RESULTS: We found remarkably higher autophagy level in the spleens of mice that were subjected to chronic restraint stress compared with the control group. We also found that inhibition of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated chronic stress-induced alterations of pro-inflammatory and anti-inflammatory cytokine levels. We further elucidated that 3-MA dramatically inhibited the reduction of lymphocyte numbers. Moreover, chronic stress dramatically enhanced the expression of Tim-3 and galectin-9. Inhibition of Tim-3 by small interfering RNA against Tim-3 significantly decreased the level of autophagy and immune suppression in isolated primary splenocytes from stressed mice. In addition, α-lactose, a blocker for the interaction of Tim-3 and galectin-9, also decreased the autophagy level and immune suppression. CONCLUSION: Chronic stress induces autophagy, resulting with suppression of immune system. Tim-3 and galectin-9 play a crucial regulatory role in chronic stress-induced autophagy. These studies suggest that Tim-3 mediated autophagy may offer a novel therapeutic strategy against the deleterious effects of chronic stress on the immune system.

LncRNA BCAR4 promotes colon cancer progression via activating Wnt/ß-catenin signaling.

BCAR4 (Breast Cancer Anti-Estrogen Resistance 4) is a long noncoding RNA that was identified as an oncogene in breast cancer. In our research, we found that the expression level of BCAR4 was upregulated in colon cancer tissues compared to paired normal tissues. What's more, higher BCAR4 expression was correlated with lower survival rate in patients with colon cancer. Mechanistically, we showed that BCAR4 activated Wnt/ß-catenin signaling in colon cancer by protecting ß-catenin from degradation. We also showed that BCAR4 overexpression promoted cell proliferation and migration in colon cancer. However, silencing BCAR4 inhibited cell growth and promoted apoptosis. Besides, BCAR4 knockdown decreased tumor growth in vivo. These findings indicate that BCAR4 facilitated colon cancer progression by enhancing cell proliferation and inhibiting apoptosis via BCAR4/ß-catenin axis. BCAR4 may be a useful new target for treatment of patients with colon cancer.