Inactivation of lncRNA HOTAIRM1 caused by histone methyltransferase RIZ1 accelerated the proliferation and invasion of liver cancer.

OBJECTIVE: Liver cancer is the second most common cause of cancer death, causing more than 700,000 deaths every year. It has been demonstrated that Long non-coding RNA (LncRNA) plays an important regulatory role in a series of diseases. However, the regulatory mechanism of LncRNAs in liver cancer has not been fully elucidated. The purpose of this study was to explore the interaction of lncRNA HOTAIRM1 and aberrant histone modification in liver cancer. MATERIALS AND METHODS: qRT-PCR was used to detect the expression levels of RIZ1 and miR-125b in liver cancer cells. Cell proliferation was measured using the CCK8 assay. ChIP-Real-time PCR confirmed the binding site of the promoter of HOTAIRM1 by H3K9me1. The direct target of HOTAIRM1 and miR-125b in liver cancer cells was measured by a luciferase reporter assay. Cell proliferation was detected by Cell Counting Kit-8 (CCK8). Cell invasion was measured by transwell assays and cell migration was detected by wound healing assay. RESULTS: The expression level of RIZ1 and miR-125b was upregulated, and HOTAIRM1 was downregulated in liver cancer cells. Transwell and CCK-8 assay showed that RIZ1 expression is associated with the proliferation, invasion and migration of liver cancer cells, silencing of RIZ1 inhibited cell proliferation, migration, and invasion in HEPG2 and HCC-LM3 cells. RIZ1 interference could significantly inhibit H3K9me1 expression. H3K9me1 protein can bind to HOTAIRM1 promoter directly. Furthermore, the bioinformatics prediction and luciferase assay demonstrated that miR-125b can interact with HOTAIRM1 by direct binding. HOTAIRM1 down-expression promoted HEPG2 cell growth and metastasis, which was further strengthened following the co-transfection of miR-125b. Furthermore, overexpressed HOTAIRM1 inhibited HCC-LM3 cell growth and metastasis and a complete reversal of the results seen when transfected with miR-125b. CONCLUSIONS: For the first time, we found that RIZ1 was upregulated in liver cancer cells and RIZ1-mediated H3K9me1 enrichment on the HOTAIRM1 promoter regulated the growth and metastasis of liver cancer cells by targeting miR-125b, which could further accelerate tumor proliferation, migration and invasion. It may serve as a therapeutic marker for liver cancer treatment.

Role of miR-133a in regulating TGF-ß1 signaling pathway in myocardial fibrosis after acute myocardial infarction in rats.

OBJECTIVE: The aim of this research was to explore the effect of microRNA-133a (miR-133a) on myocardial fibrosis and cardiac function after myocardial infarction in rats, and to investigate the possible regulatory mechanism. MATERIALS AND METHODS: Myocardial infarction model was successfully established in rats by ligation of the left anterior descending coronary artery. After miR-133a overexpression in rats myocardium, cardiac function was examined by echocardiography. Meanwhile, the degree of myocardial fibrosis was detected by Masson staining. In addition, the expression of α-smooth muscle actin (α-SMA) in cardiomyocytes was detected by immunohistochemistry. Quantitative Real-time polymerase chain reaction (qRT-PCR) was performed to analyze the expression level of miR-133a in the junction of myocardial infarction. The mRNA expressions of transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), collagen type 1 (col 1), collagen type 3 (col 3) and α-SMA were measured by qRT-PCR as well. Furthermore, the protein levels of the above genes were detected by Western blotting. RESULTS: MiR-133a expression in the infarct border zone of myocardial tissue was significantly decreased on the 28th day after myocardial infarction surgery (p<0.05). In addition, up-regulation of miRNA-133a in myocardial tissue of rats with myocardial infarction could remarkably improve cardiac function and reduce collagen volume fraction. Furthermore, the mRNA and protein expression levels of TGF-ß1, CTGF, col1, col3, α-SMA in myocardial tissue were obviously decreased after miRNA-133a up-regulation (p<0.001). CONCLUSIONS: Overexpression of miR-133a down-regulates the mRNA and protein levels of TGF-ß1 and CTGF after myocardial infarction. Moreover, this may eventually reduce myocardial collagen deposition, inhibit myocardial fibrosis and improve cardiac function.

Hypoxic preconditioning BMSCs-exosomes inhibit cardiomyocyte apoptosis after acute myocardial infarction by upregulating microRNA-24.

OBJECTIVE: To elucidate the regulatory effect of hypoxic preconditioning bone marrow mesenchymal stem cells (BMSCs)-exosomes on cardiomyocyte apoptosis in acute myocardial infarction (AMI) rats. MATERIALS AND METHODS: BMSCs-derived exosomes were extracted by Exoquick method. Expressions of exosome surface markers were determined by Western blot. The AMI model in rats was established by LAD ligation. Rats were randomly assigned into sham group, AMI group, AMI+H-exo group and AMI+N-exo group. MicroRNA-24 expression in rat myocardium was detected at different time points. Subsequently, hypoxic preconditioning or normoxic preconditioning BMSCs-exosomes were intramyocardially injected into rats. Infarct size was calculated through TTC (triphenyltetrazolium chloride) staining. Cardiomyocyte apoptosis was accessed with Terminal Deoxynucleotidyl Transferase dUTP Nick-end Labeling (TUNEL). Heart function of AMI rats was evaluated by echocardiography. Protein expressions of apoptotic genes in rat myocardium were detected by Western blot. RESULTS: The mRNA level of microRNA-24 was higher in H-exo group than N-exo group. Injection of hypoxic preconditioning BMSCs-exosomes markedly upregulated microRNA-24 level, reduced infarct size and improved cardiac function in AMI rats. Protein expressions of Bax, caspase-3 and cleaved-caspase-3 were downregulated by BMSCs-exosomes treatment. H9c2 cells showed upregulated microRNA-24 level and decreased apoptotic rate after incubation with hypoxic preconditioning BMSCs-exosomes. The above cellular performances were partially reversed by transfection of microRNA-24 inhibitor. CONCLUSIONS: Hypoxic preconditioning BMSCs-exosomes inhibit cardiomyocyte apoptosis in AMI rats by upregulating microRNA-24.

miR-409 down-regulates Jak-Stat pathway to inhibit progression of liver cancer.

OBJECTIVE: To elucidate the influence of microRNA-409 and the Jak-Stat pathway on the development of liver cancer. PATIENTS AND METHODS: The quantitative Real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression of microRNA-409 in hepatocarcinoma, paracancerous tissues and normal liver tissues, and the correlation between its expression and clinicopathological parameters of patients was analyzed, with the area under the microRNA-409 curve (AUC) being detected. The level of microRNA-409 in different liver cancer cells was detected by qPCR. Then it was overexpressed or knock-downed in the liver cancer cells by cell transfection technique. The cell apoptosis and viability after inhibition or overexpression of microRNA-409 were evaluated by propidium iodide (PI) staining and cell counting kit-8 (CCK-8) assay. Subsequently, Jak2 and Stat3 mRNA levels were detected by qPCR in hepatocarcinoma and paracancerous tissues, with their protein levels analyzed by Western blot after microRNA-409 was inhibited or up-regulated. At last, CCK-8 assay was performed to evaluate the effect of Jak2 on cell viability. RESULTS: Compared with paracancerous and normal liver tissues, the level of microRNA-409 was remarkably reduced in hepatocarcinoma tissues and was negatively correlated with tumor stage, tumor size and overall survival time of patients with liver cancer. Meanwhile, microRNA-409 expression in hepatoma cell lines was also strikingly lower than that in normal liver cells. After overexpression of microRNA-409 in HHCC, cell viability significantly decreased while apoptosis increased, and opposite results were shown in HepG2 cells after miR409 was knock-downed. In liver cancer tissues, the levels of Jak2 and Stat3 were significantly higher than those in adjacent tissues. Additionally, up-regulating microRNA-409 reduced the level of Jak2 and Stat3 protein, while down-regulating it elevated them. In addition, Jak2 could reverse the inhibitory effect of microRNA-409 on the proliferation of hepatoma cells. CONCLUSIONS: Highly-expressed microRNA-409 can down-regulate the Jak-Stat signaling pathway and inhibit cell proliferation to slow down the progression of liver cancer.

MiR-210 knockdown promotes the development of pancreatic cancer via upregulating E2F3 expression.

OBJECTIVE: The aim of this study was to explore the role of microRNA-210 (miR-210) and E2F3 in the development of pancreatic cancer and to investigate the possible underlying mechanism. PATIENTS AND METHODS: The expression level of miR-210 in pancreatic cancer tissues, para-cancerous tissues, and normal pancreatic tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between miR-210 expression and pathological indicators of pancreatic cancer was analyzed. Meanwhile, the expression of miR-210 in pancreatic cancer cells and normal pancreatic ductal epithelial cells was detected by qRT-PCR. After transfection with miR-210 mimics and inhibitor, the viability and cell cycle of pancreatic cancer cells were detected by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The binding condition of miR-210 and E2F3 was verified by Dual-Luciferase reporter gene assay. RESULTS: MiR-210 was lowly expressed in pancreatic cancer tissues than that of para-cancerous tissues. The expression of miR-210 was negatively correlated with TNM stage and tumor size of pancreatic cancer. In vitro experiments showed that the miR-210 was downregulated in pancreatic cancer cells than that of normal pancreatic ductal epithelial cells. Meanwhile, overexpression of miR-210 arrested cell cycle decreased cell viability and downregulated E2F3 expression in pancreatic cancer cells. Dual-Luciferase reporter gene assay indicated that E2F3 bound to mi-210. Further experiments confirmed that E2F3 was negatively regulated by miR-210. CONCLUSIONS: MiR-210 knockdown promotes cell proliferation by upregulating E2F3 expression, thereby promoting the progression of pancreatic cancer.

LncRNA LOC554202 promotes proliferation and migration of gastric cancer cells through regulating p21 and E-cadherin.

OBJECTIVE: To explore whether long noncoding RNA (lncRNA) LOC554202 could regulate proliferative and migratory abilities of gastric cancer (GC) cells. MATERIALS AND METHODS: Expression level of LOC554202 in GC cell lines HGC-27 and MGC-803, as well as normal gastric mucosal cell line GES-1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). LOC554202 knockdown or overexpression in HGC-27 and MGC-803 cells was achieved by transfection of LOC554202-siRNA or pcDNA-LOC554202, respectively. Cell cycle in GC cells was accessed by flow cytometry. Migratory ability of GC cells was determined by wound healing assay and transwell assay. Finally, protein expressions of p21 and E-cadherin in GC cells were detected by Western blot. RESULTS: LOC554202 expression was higher in GC cells than that of gastric mucosal cells (p<0.01). Overexpression of LOC554202 in MGC-803 cells enhanced proliferative and migratory abilities, but decreased protein expressions of p21 and E-cadherin (p<0.01). On the contrary, LOC554202 overexpression in HGC-27 cells decreased proliferative and migratory abilities, but increased protein expressions of p21 and E-cadherin (p<0.01). CONCLUSIONS: LncRNA LOC554202 is highly expressed in GC cells. It could promote proliferative and migratory abilities by downregulating expression levels of p21 and E-cadherin in GC cells.

Methods to Study Lysosomal AMPK Activation.

The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis. It is activated by the upstream kinase LKB1 (liver kinase B1) when the AMP/ATP ratio is increased during starvation or heightened exercises. Based on reconstitution experiments using purified individual proteins, AMPK was demonstrated to be directly phosphorylated on its conserved residue Thr172 by LKB1, which was promoted by increased levels of AMP. However, recent studies have engendered a paradigm shift for how AMPK is activated inside the cell or animal tissues, unraveling that AXIN binds to LKB1 and tethers it to AMPK located on the surface of late endosome and lysosome (hereafter, only lysosome is discussed) in response to glucose starvation. Moreover, AXIN extends its interaction with the v-ATPase-Ragulator complex, which is paradoxically also required for activation of mTORC1 despite the fact that the two kinases AMPK and mTORC1 are inversely activated. Here, we summarize the experimental procedures of the assays for translocation of AXIN/LKB1 to the detergent-resistant lipid fractions of lysosomal membrane and the assembly of AMPK-activating complexes thereon. These methods will be useful for determining whether AMPK activation induced by various metabolic stresses or by pharmacological stimuli is mediated by the v-ATPase-Ragulator-AXIN/LKB1 axis.

Targeted drug regulation on methylation of p53-BAX mitochondrial apoptosis pathway affects the growth of cholangiocarcinoma cells.

OBJECTIVE: To study the mechanism of 5-aza-2-deoxycytidine (DAC; a methylation inhibitor) on growth of the human cholangiocarcinoma QBC939 cell line. METHODS: A colourimetric assay was used to detect growth of QBC939 cells treated with DAC (0.1-100 µmol/l) over 24 h, 48 h and 72 h. Cell morphology was observed by transmission electron microscopy (TEM). The cell cycle and apoptosis were analysed by flow cytometry. Hypermethylation of the promoters of the p53-BAX mitochondrial apoptosis genes cyclin-dependent kinase inhibitor 2A (CDKN2A), death-associated protein kinase 1 (DAPK1) and PYD and CARD domain containing (PYCARD) was detected by methylation-specific polymerase chain reaction, with and without DAC treatment. RESULTS: DAC inhibited QBC939 cell growth with a half maximal inhibitory concentration of 5 µmol/l at 72 h. After DAC treatment, apoptosis was observed by TEM. Flow cytometric analysis of propidium iodide-positive cells demonstrated increased apoptosis of DAC-treated QBC939 cells (43.04%) compared with untreated cells (4.31%). DAC treatment resulted in demethylation of the gene promoters of CDKN2A and DAPK1 in QBC939 cells. CONCLUSIONS: DAC induces apoptosis of QBC939 cells by reactivation of hypermethylated p53-BAX mitchondrial apoptosis genes in cholangiocarcinoma cells.