miR-18a-3p and Its Target Protein HuR May Regulate Myogenic Differentiation in Immune-Mediated Necrotizing Myopathy

Immune-mediated necrotizing myopathy (IMNM) is characterized by manifestation of myonecrosis and regeneration of muscle fibers; however, the underlying pathogenesis remains unclear. This study aimed to investigate the role and mechanism of miR-18a-3p and its target RNA-binding protein HuR in IMNM. HuR and miR-18a-3p levels were detected in the skeletal muscles of 18 patients with IMNM using quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) and western blotting analysis. Human myoblasts were transfected with small interfering RNA targeting HuR and miR-18a-3p mimic or inhibitor. Myogenic differentiation markers, myogenin and myosin heavy chain, were analyzed by qRT-PCR, western blotting analysis, and immunofluorescence staining. The results showed that miR-18a-3p was upregulated (p=0.0002), whereas HuR was downregulated (p=0.002) in the skeletal muscles of patients with IMNM. The expression of miR-18a-3p in patients with IMNM was negatively correlated with those of HuR (r = -0.512, p = 0.029). We also found that disease activity was positively correlated with HuR expression (r = 0.576, p = 0.012) but muscle activity was negatively correlated with miR-18a-3p expression (r = -0.550, p = 0.017). Besides, bioinformatics analysis and dual-luciferase reporter assays suggested that miR-18a-3p could directly target HuR. Cellular experiments showed that overexpression of miR-18a-3p inhibited myogenic differentiation by targeting HuR, whereas inhibition of miR-18a-3p led to opposite results. Therefore, miR-18a-3p and its target protein HuR may be responsible for modulating the myogenic process in IMNM and can thus be therapeutic targets for the same.

Bak instead of Bax plays a key role in metformin-induced apoptosis s in HCT116 cells

Metformin (Met) exhibits anticancer ability in various cancer cell lines. This report aims to explore the exact molecular mechanism of Met-induced apoptosis in HCT116 cells, a human colorectal cancer cell line. Met-induced reactive oxygen species (ROS) increase and ROS-dependent cell death accompanied by plasma membrane blistering, mitochondrial swelling, loss of mitochondrial membrane potential, and release of cytochrome c. Western blotting analysis showed that Met upregulated Bak expression but downregulated Bax expression. Most importantly, silencing Bak instead of Bax inhibited Met-induced loss of mitochondrial membrane potential, indicating the key role of Bak in Met-induced apoptosis. Live-cell fluorescence resonance energy transfer (FRET) analysis showed that Met unlocked the binding of Mcl-1 to Bak, and enhanced the binding of Bim to Bak and subsequent Bak homo-oligomerization. Western blotting analysis showed that Met enhanced AMPK phosphorylation and Bim expression, and compound C, an inhibitor of AMPK, inhibited Met-induced Bim upregulation. Although Met increased the expression of Bcl-xL, overexpression of Bcl-xL did not prevent Met-induced apoptosis. In summary, our data demonstrate for the first time that Met promotes ROS-dependent apoptosis by regulating the Mcl-1-Bim-Bak axis.

Release of HMGB1 in Podocytes Exacerbates Lipopolysaccharide-Induced Acute Kidney Injury

Objective. Acute kidney injury (AKI) usually occurs during sepsis. Inflammation factors, such as high-mobility group box 1 (HMGB1), are dramatically upregulated under septic conditions. In our current work, the functions of HMGB1 in AKI were explored. Methods. An AKI model was induced by the lipopolysaccharide (LPS) challenge in C57 mice. Podocytes were challenged by LPS for different durations. Subsequently, podocytes transfected with HMGB1 siRNA were exposed to LPS for 24 h. The expressions of supernatant HMGB1 and cellular active caspase-3 were examined by Western blotting analysis. To explore the effect of HMGB1 on tubular epithelial cells (TECs), HK-2 cells were exposed to HMGB1 at various concentrations for 24 h. Epithelial-mesenchymal transition (EMT) of HK-2 cells was evaluated by Western blotting analysis. Mitochondrial division and apoptosis of HK-2 cells were assessed by MitoTracker Red and Western blotting analysis, respectively. Results. Compared with the sham control group, the expression of HMGB1 was increased in the kidney of AKI mice. Moreover, the expression of supernatant HMGB1 was increased in LPS-challenged podocytes compared with the control group. Knockdown of HMGB1 attenuated LPS-induced podocyte injury. Besides, EMT in TECs was triggered by HMGB1. Mitochondrial damage and apoptosis of HK-2 cells exposed to HMGB1 were markedly elevated compared with the control group. Conclusions. Collectively, HMGB1 release in podocytes was induced by LPS, subsequently leading to exacerbated AKI.

Mechanisms involved in the anti-tumor effects of Toosendanin in glioma cells

Background Toosendanin (TSN) is a triterpenoid compound mainly used as an ascaris repellant. Recent studies have shown that it possesses antitumor effects in many types of tumor cells. However, the effects of TSN on glioma cells have rarely been reported. Methods Different assays were performed to investigate the effects of TSN on the different glioma cell lines including U87MG and LN18. The assays included colony formation, wound healing, and transwell assays. Furthermore, Hoechst 33342 staining, flow cytometry, and western blotting analysis were performed to investigate the apoptotic activities of TSN. Finally, the results were confirmed using a xenograft tumor model that comprised of nude mice. Results In vitro, the CCK-8 and colony formation assays showed that TSN effectively inhibited glioma cell proliferation. Moreover, the inhibitory effects on glioma cell migration and invasion were demonstrated through the wound healing and transwell assays, respectively. Hoechst 33342 staining, flow cytometry, and western blotting assays demonstrated the significant effect of TSN in the apoptosis induction of glioma cells. Furthermore, the anti-glioma effect of TSN was exerted through the inhibition of the PI3K/Akt/mTOR signaling pathways as demonstrated by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model confirmed the suppressive effect of TSN on tumor growth in vivo. Conclusion Our results suggest that TSN is a promising chemotherapeutic drug for patients with glioma.

Mechanisms Involved in the Anti-Tumor Effects of Toosendanin in Glioma Cells

Background: Toosendanin (TSN) is a triterpenoid compound mainly used as an ascaris repellant. Recent studies have shown that it possesses antitumor effects in many types of tumor cells. However, the effects of TSN on glioma cells have rarely been reported. Methods: Different assays were performed to investigate the effects of TSN on the different glioma cell lines including U87MG and LN18. The assays included colony formation, wound healing, and transwell assays. Furthermore, Hoechst 3342 staining, flow cytometry, and western blotting analysis were performed to investigate the apoptotic activities of TSN. Finally, the results were confirmed using a xenograft tumor model that comprised of nude mice. Results: In vitro, the CCK-8 and colony formation assays showed that TSN effectively inhibited glioma cell proliferation. Moreover, the inhibitory effects on glioma cell migration and invasion were demonstrated through the wound healing and transwell assays, respectively. Hoechst 33342 staining, flow cytometry, and western blotting assays demonstrated the significant effect of TSN in the apoptosis induction of glioma cells. Furthermore, the anti-glioma effect of TSN was exerted through the inhibition of the PI3K/Akt/mTOR signaling pathways as demonstrated by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model confirmed the suppressive effect of TSN on tumor growth in vivo. Conclusion: Our results suggest that TSN is a promising chemotherapeutic drug for patients with glioma.

Puerarin Ameliorates Caerulein-Induced Chronic Pancreatitis via Inhibition of MAPK Signaling Pathway

Pancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP), and pancreatic stellate cells (PSCs) are considered to be the key cells. Puerarin is the most important flavonoid active component in Chinese herb Radix Puerariae, and it exhibited anti-fibrotic effect in various fibrous diseases recently. However, the impact and molecular mechanism of puerarin on CP and pancreatic fibrosis remain unknown. This study systematically investigated the effect of puerarin on CP and pancreatic fibrosis in vivo and in vitro. H&E staining, Sirius Red staining, qRT-PCR and Western blotting analysis of fibrosis and inflammation related genes of pancreatic tissues showed that puerarin notably ameliorated pancreatic atrophy, inflammation and fibrosis in a model of caerulein-induced murine CP. Western blotting analysis of pancreatic tissues showed the phosphorylation level of MAPK family proteins (JNK1/2, ERK1/2 and p38 MAPK) significantly increased after modeling of cerulein, while puerarin could inhibit their phosphorylation levels to a certain extent. We found that puerarin exerted a marked inhibition on the proliferation, migration and activation of PSCs, determined by CCK-8 assay, transwell migration assay, scratch wound-healing assay and expression levels of α-SMA, Fibronectin, Col1α1 and GFAP. Western blotting result demonstrated that puerarin markedly inhibited the phosphorylation of MAPK family proteins (JNK1/2, ERK1/2 and p38 MAPK) of PSCs in a dose-dependent manner whether or not stimulated by platelet-activating factor. In conclusion, the present study showed that puerarin could be a potential therapeutic candidate in the treatment of CP, and the MAPK pathway might be its important target.

Long Non-coding RNA LINC02474 Affects Metastasis and Apoptosis of Colorectal Cancer by Inhibiting the Expression of GZMB

BackgroundColorectal cancer (CRC) is one of the most frequently diagnosed malignancies. Metastasis is the main event that impedes the therapeutic effect on CRC, and its underlying mechanisms remain largely unclear. LINC02474 is a novel long noncoding RNA (lncRNA) associated with metastasis of CRC, while little is known about how LINC02474 regulates these malignant characteristics.MethodsExpressions of LINC02474 and granzyme B (GZMB) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blotting analysis. Cell metastasis was detected by transwell assay and metastatic nude mouse model, and apoptosis was determined by Western blotting analysis and flow cytometry. Besides, the interaction between LINC02474 and GZMB was detected by dual-luciferase reporter assays.ResultsThe expression of LINC02474 was significantly up-regulated in CRC tissues. Moreover, depletion of LINC02474 damaged the metastatic abilities of CRC cells in vivo and in vitro while boosting apoptosis. Besides, up-regulation of LINC02474 could promote migration and invasion, while apoptosis was inhibited in CRC cells. Besides, down-regulation of LINC02474 promoted the expression of GZMB, and interference of GZMB could increase the metastatic abilities of CRC cells while reducing apoptosis. Furthermore, LINC02474 was related to the transcriptional repression of GZMB in CRC cells determined by the dual-luciferase reporter assay.ConclusionsThe findings revealed that a novel lncRNA, LINC02474, as an oncogene, could promote metastasis, but limit apoptosis partly by impeding GZMB expression in CRC. Besides, LINC02474 had the potential to be used as a biomarker in the prognosis of CRC.

Integrative Analysis of HMMR as a Potential Target of Prognosis and Therapy in Hepatocellular Carcinoma

Background: Previous work has indicated Hyaluronic acid-mediated motor receptor (HMMR) plays an important role in regulating tumor metastasis. However, few researchers address the clinical significance of HMMR and its underlying mechanisms for regulating hepatocellular carcinoma (HCC). This study focuses on the underlying effect of HMMR in the development and prognosis of HCC. Materials and Methods: In the present study, data of RNA and miRNA sequencing array were obtained from Oncomine dataset or The Cancer Genome Atlas (TCGA) dataset, the distinctive genomic patterns associated with HMMR expression and its correlation with prognosis were analysed by using R package. Gene set enrichment analysis (GSEA) were performed on genes expressed aberrantly. We also performed Reverse Transcription-polymerase Chain Reaction (RT-PCR), Immunohistochemical (IHC) staining and Western blotting analysis to evaluate the expression of HMMR in liver cancer cell lines or 12 HCC samples from The Affiliated Hospital of Southwest Medical University. Results: A total of 407 tumor tissue samples in TCGA dataset were evaluated, combined with analysis in Oncomine dataset, we found HMMR expression was increased in HCC compared to normal tissues. Higher expression of HMMR was correlated with poorer overall survival and disease-free survival outcomes. Moreover, multivariate Cox regression analysis revealed that HMMR expression was an independent risk factor for overall survival (HMMR: hazard ratio [HR] = 1.154, 95% confidence interval [CI] = 1.080-1.233, p<0.001). Consistently, RT-PCR, IHC staining and Western blotting analysis further confirmed that HMMR expression was increased in HCC compared with patient-matched adjacent normal liver tissues. Notably, GSEA analysis revealed that differential gene expression in HMMR-high patients (compared with HMMRlow patients) were enriched in cell proliferation and p53 signaling pathway. Moreover, comprehensive analysis showed a strong correlation between HMMR upregulation and miRNA changes. Conclusion: The high expression of HMMR is a poor prognostic factor in HCC and might serve as a potential target of therapy in patients with HCC.

Extracellular vesicle microRNA cargoes from intermittent hypoxia-exposed cardiomyocytes and their effect on endothelium

Background Intermittent hypoxia (IH), as the crucial pathophysiological feature of obstructive sleep apnea syndrome (OSAS), is an independent risk factor initiating the progression of cardiovascular complications. However, how IH cause cardiovascular injury and initiate inter-organ communication remains unclear. Extracellular vesicle (EV) are reported to be involved in cell-to-cell and organ-to-organ communications through selectively carrying RNA and protein cargos from donor cells and delivering them to recipient cells. By utilizing an unbiased miRNA microarray approach, current study attempted to determine whether IH may alter miRNA profiles in cardiomyocyte-derived extracellular vesicles and whether EVs from IH-treated cardiomyocytes could affect endothelial function. Methods EVs were isolated from culture medium of normoxia- or intermittent hypoxia-treated C57BL/6 mouse primary cardiomyocytes. miRNA array assay was used to identify myocardial EV miRNA. The expression of target miRNAs was validated by qPCR and the enriched function of the target miRNAs were predicted by bioinformatics and western blotting analysis. Moreover, vascular functional study validated whether EVs from IH-exposed cardiomyocytes could influence endothelial function. Results 63 differentially expressed miRNAs, including 32 up-regulated and 31 down-regulated miRNAs were identified in EVs from IH-exposed cardiomyocytes. Among them, 16 miRNAs with homologous sequence in mouse and human were selected and verified by qPCR assay and 11 miRNAs were proved with the same tendency as miRNA array identified. KEGG pathway and western blotting analysis showed the main enriched pathway of differentially expressed miRNA was associated with PI3K/Akt signaling pathway. Of note, EVs from IH-exposed cardiomyocytes dramatically impaired endothelium-dependent relaxation and inhibited Akt/eNOS pathway in endothelial cells. Conclusions This study provides the first evidence that IH causes significant alteration in myocardial EV miRNA composition, which may contribute to IH or OSAS-triggered cardiovascular injury and organ-to-organ communication.